WorldWideScience

Sample records for antigen-presenting cells determine

  1. Presentation of lipid antigens to T cells.

    Science.gov (United States)

    Mori, Lucia; De Libero, Gennaro

    2008-04-15

    T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

  2. Effective antigen presentation to helper T cells by human eosinophils.

    Science.gov (United States)

    Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

    2016-12-01

    Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

  3. Bone marrow-derived thymic antigen-presenting cells determine self-recognition of Ia-restricted T lymphocytes

    International Nuclear Information System (INIS)

    Longo, D.L.; Kruisbeek, A.M.; Davis, M.L.; Matis, L.A.

    1985-01-01

    The authors previously have demonstrated that in radiation-induced bone marrow chimeras, T-cell self-Ia restriction specificity appeared to correlate with the phenotype of the bone marrow-derived antigen-presenting (or dendritic) cell in the thymus during T-cell development. However, these correlations were necessarily indirect because of the difficulty in assaying thymic function directly by adult thymus transplant, which has in the past been uniformly unsuccessful. They now report success in obtaining functional T cells from nude mice grafted with adult thymuses reduced in size by treatment of the thymus donor with anti-thymocyte globulin and cortisone. When (B10 Scn X B10.D2)F1 nude mice (I-Ab,d) are given parental B10.D2 (I-Ad) thymus grafts subcutaneously, their T cells are restricted to antigen recognition in association with I-Ad gene products but not I-Ab gene products. Furthermore, thymuses from (B10 X B10.D2)F1 (I-Ab,d)----B10 (I-Ab) chimeras transplanted 6 months or longer after radiation (a time at which antigen-presenting cell function is of donor bone marrow phenotype) into (B10 X B10.D2)F1 nude mice generate T cells restricted to antigen recognition in association with both I-Ad and I-Ab gene products. Thymuses from totally allogeneic bone marrow chimeras appear to generate T cells of bone marrow donor and thymic host restriction specificity. Thus, when thymus donors are radiation-induced bone marrow chimeras, the T-cell I-region restriction of the nude mice recipients is determined at least in part by the phenotype of the bone marrow-derived thymic antigen presenting cells or dendritic cells in the chimeric thymus

  4. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  5. The antigen presenting cells instruct plasma cell differentiation.

    Science.gov (United States)

    Xu, Wei; Banchereau, Jacques

    2014-01-06

    The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  6. Can resting B cells present antigen to T cells

    International Nuclear Information System (INIS)

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1985-01-01

    Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

  7. Antigen presentation by resting B cells. Radiosensitivity of the antigen-presentation function and two distinct pathways of T cell activation

    International Nuclear Information System (INIS)

    Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

    1984-01-01

    In this report we have examined the ability of small resting B cells to act as antigen-presenting cells (APC) to antigen-specific MHC-restricted T cells as assessed by either T cell proliferation or T cell-dependent B cell stimulation. We found that 10 of 14 in vitro antigen-specific MHC-restricted T cell clones and lines and three of four T cell hybridomas could be induced to either proliferate or secrete IL-2 in the presence of lightly irradiated (1,000 rads) purified B cells and the appropriate foreign antigen. All T cell lines and hybridomas were stimulated to proliferate or make IL-2 by macrophage- and dendritic cell-enriched populations and all T cells tested except one hybridoma caused B cell activation when stimulated with B cells as APC. Furthermore, lightly irradiated, highly purified syngeneic B cells were as potent a source of APC for inducing B cell activation as were low density dendritic and macrophage-enriched cells. Lymph node T cells freshly taken from antigen-primed animals were also found to proliferate when cultured with purified B cells and the appropriate antigen. This APC function was easily measured when the cells were irradiated with 1,000 rads, but was greatly diminished or absent when they were irradiated with 3,300 rads. In addition, this radiosensitivity allowed us to easily distinguish B cell antigen presentation from presentation by the dendritic cell and macrophage, as the latter was resistant to 3,300 rads. Finally, one T cell clone that failed to proliferate when B cells were used as APC was able to recruit allogeneic B cells to proliferate in the presence of syngeneic B cells and the appropriate antigen. This result suggests that there are at least two distinct pathways of activation in T cells, one that leads to T cell proliferation and one that leads to the secretion of B cell recruitment factor(s)

  8. Harnessing Dendritic Cells for Tumor Antigen Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Nierkens, Stefan [Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, Nijmegen 6525 GA (Netherlands); Janssen, Edith M., E-mail: edith.janssen@cchmc.org [Division of Molecular Immunology, Cincinnati Children' s Hospital Research Foundation, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 (United States)

    2011-04-26

    Dendritic cells (DC) are professional antigen presenting cells that are crucial for the induction of anti-tumor T cell responses. As a consequence, research has focused on the harnessing of DCs for therapeutic interventions. Although current strategies employing ex vivo-generated and tumor-antigen loaded DCs have been proven feasible, there are still many obstacles to overcome in order to improve clinical trial successes and offset the cost and complexity of customized cell therapy. This review focuses on one of these obstacles and a pivotal step for the priming of tumor-specific CD8{sup +} and CD4{sup +} T cells; the in vitro loading of DCs with tumor antigens.

  9. Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

    International Nuclear Information System (INIS)

    Rivera, Julie A.; McGuire, Travis C.

    2005-01-01

    To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV WSU5 infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixtures of CD8+ and CD4+ cells as detected with 51 Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection

  10. Chloroquine inhibits accessory cell presentation of soluble natural and synthetic protein antigens

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...... was time- and dose-dependent. A brief treatment solely of the accessory cells with the drug compromised their ability to stimulate primed T cells in a subsequent culture provided the accessory cells were treated with chloroquine before their exposure to the antigen. These results suggest that chloroquine...... acts on an early event in the antigen handling by accessory cells. Chloroquine is a well known inhibitor of lysosomal proteolysis, and it is likely that its effect on antigen presentation is caused by an inhibition of antigen degradation....

  11. Viral sequestration of antigen subverts cross presentation to CD8(+ T cells.

    Directory of Open Access Journals (Sweden)

    Eric F Tewalt

    2009-05-01

    Full Text Available Virus-specific CD8(+ T cells (T(CD8+ are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC. Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T(CD8+. Direct presentation of vaccinia virus (VACV antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T(CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T(CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation

  12. Impact of aging on antigen presentation cell function of dendritic cells.

    Science.gov (United States)

    Wong, Christine; Goldstein, Daniel R

    2013-08-01

    Older people exhibit increased mortality to infections and cancer as compared to younger people, indicating that aging impairs immunity. Dendritic cells (DCs) are key for bridging the innate and adaptive arms of the immune system by priming antigen specific T cells. Discerning how aging impacts DC function to initiate adaptive immune responses is of great biomedical importance as this could lead to the development of novel therapeutics to enhance immunity with aging. This review details reports indicating that aging impairs the antigen presenting function of DCs but highlights other studies indicating preserved DC function with aging. How aging impacts antigen presentation by DCs is complex and without a clear unifying biological underpinning. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

    International Nuclear Information System (INIS)

    Abbas, A.K.; Haber, S.; Rock, K.L.

    1985-01-01

    Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

  14. Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes.

    Directory of Open Access Journals (Sweden)

    Carlos Alfaro

    Full Text Available Dendritic cells (DC are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs as a result of being co-attracted by interleukin-8 (IL-8, for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA protein, were able to cross-present the antigen to CD8 (OT-1 and CD4 (OT-2 TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d are coinjected in the footpad of mice with autologous DC (H-2(b. In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

  15. A sub-population of circulating porcine gammadelta T cells can act as professional antigen presenting cells.

    Science.gov (United States)

    Takamatsu, H-H; Denyer, M S; Wileman, T E

    2002-09-10

    A sub-population of circulating porcine gammadelta T cells express cell surface antigens associated with antigen presenting cells (APCs), and are able to take up soluble antigen very effectively. Functional antigen presentation by gammadelta T cells to memory helper T cells was studied by inbred pig lymphocytes immunised with ovalbumin (OVA). After removing all conventional APCs from the peripheral blood of immunised pigs, the remaining lymphocytes still proliferated when stimulated with OVA. When gammadelta T cells were further depleted, OVA specific proliferation was abolished, but reconstitution with gammadelta T cells restored proliferation. The proliferation was blocked by monoclonal antibodies (mAb) against MHC class II or CD4, and by pre-treatment of gammadelta T cells with chloroquine. These results indicate that a sub-population of circulating porcine gammadelta T cells act as APCs and present antigen via MHC class II.

  16. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...... molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has...

  17. Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells

    DEFF Research Database (Denmark)

    Kirkin, Alexei F.; Dzhandzhugazyan, Karine N.; Guldberg, Per

    2018-01-01

    In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT...... antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25...... patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can...

  18. Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

    Science.gov (United States)

    Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

    2018-02-21

    Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

  19. Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines

    Directory of Open Access Journals (Sweden)

    Cleo Goyvaerts

    2015-01-01

    Full Text Available In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

  20. Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

    Science.gov (United States)

    Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

    2012-01-01

    Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

  1. A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

    Science.gov (United States)

    Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

    2016-06-01

    The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

  2. Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

    Science.gov (United States)

    Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

    2001-10-01

    Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

  3. Activation of nickel-specific CD4+ T lymphocytes in the absence of professional antigen-presenting cells.

    Science.gov (United States)

    Nasorri, Francesca; Sebastiani, Silvia; Mariani, Valentina; De Pità, Ornella; Puddu, Pietro; Girolomoni, Giampiero; Cavani, Andrea

    2002-01-01

    Allergic contact dermatitis ensues from exaggerated T cell responses to haptens. Dendritic cells are required for the initiation of hapten sensitization, but they may not be necessary for disease expression. Here we investigated the antigen-presenting cell requirement of nickel-specific CD4+ lymphocytes isolated from the blood of six allergic individuals. A significant proportion (42 out of 121; 35%) of the T cell clones proliferated in vitro to nickel also in the absence of professional antigen-presenting cells, suggesting a direct T-T hapten presentation. Antigen-presenting-cell-independent T cells showed a predominant T helper 1 phenotype. Nickel recognition by these T cells was major histocompatibility complex class II restricted, not influenced by CD28 triggering, independent from their state of activation, and did not require processing. The capacity of this T cell subset to be directly stimulated by nickel was not due to unique antigen-presenting properties, as both antigen-presenting-cell-dependent and antigen-presenting-cell-independent clones displayed comparable levels of HLA-DR, CD80, and CD86, and were equally capable of presenting nickel to antigen-presenting-cell-independent clones. In contrast, neither T cell types activated antigen-presenting-cell-dependent T lymphocytes. T-T presentation induced T cell receptor downregulation, CD25, CD80, CD86, and HLA-DR upregulation, and interferon-gamma release, although to a lesser extent compared to those induced by dendritic cell-T presentation. Following T-T presentation, the clones did not undergo unresponsiveness and maintained the capacity to respond to dendritic cells pulsed with antigen. In aggregate, our data suggest that antigen-presenting-cell-independent T cell activation can effectively amplify hapten- specific immune responses.

  4. Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen

    DEFF Research Database (Denmark)

    Rasmussen, A B; Zocca, M-B; Bonefeld, C M

    2004-01-01

    Melanoma antigen recognized by T cell 1 (MART-1) is regarded as a candidate peptide for vaccination against malignant melanoma, and it is of importance to develop strategies to improve the vaccine-elicited T-cell activation towards MART-1. T-cell activation is, among other determinants, dependent...... on the density of specific major histocompatibility complex-peptide complexes on the surface of the antigen-presenting cell. In this study, we explored the cell-surface presentation of a substituted MART-1 peptide encoded by transfected minigenes. We investigated the potential of proteasomal targeting compared...... to non-proteasomal targeting of the epitope to increase its cell-surface presentation. Furthermore, we explored the potential of incorporating multiple minigenes instead of one to increase cell-surface presentation. We show that both proteasomal targeting and repetition of the minigene increase cell...

  5. The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells.

    Science.gov (United States)

    den Haan, Joke M M; Arens, Ramon; van Zelm, Menno C

    2014-12-01

    The adaptive immune system consists of T and B cells that express clonally distributed antigen receptors. To achieve functional adaptive immune responses, antigen-specific T cell populations are stimulated by professional antigen-presenting cells like dendritic cells (DCs), which provide crucial stimulatory signals for efficient expansion and development of effector functions. Antigen-specific B cells receive costimulatory signals from helper T cells to stimulate affinity maturation and isotype switching. Here we elaborate on the interactions between DCs, T cells and B cells, and on the important signals for efficient induction of adaptive immune responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Nadja Kettern

    Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

  7. Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

    Science.gov (United States)

    Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

    2017-11-17

    Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

  8. The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

    DEFF Research Database (Denmark)

    Møller, Morten; Rath, Martin F; Klein, David C

    2006-01-01

    The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal g...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

  9. Antigen presenting cells costimulatory signaling during pre-implantation pregnancy 

    Directory of Open Access Journals (Sweden)

    Anna Sławek

    2012-09-01

    Full Text Available  Success of pregnancy depends on many factors. Three phenomena inducing immune tolerance against semi-allogeneic conceptus may play a crucial role in the pre-implantation period of pregnancy: influence of sex hormones in sex cycle, presence of oocyte or embryo and the presence of semen in the female reproductive tract. On the other hand dendritic cells are the most effective antigen-presenting cells in regulation of immune phenomena and also are considered as potent participants in inducing immune tolerance in the pregnancy. They communicate with T cells in cell contact-dependent manner or via cytokines. During cell-cell contacts, costimulatory molecules play a key role and their expression is often dependent on cytokines milieu. Both costimulatory molecules and cytokines influence generation of T regulatory cells. Interactions of these molecules are closely related. In this paper we would like to pay attention to the importance of antigen presenting cells costimulatory potency in immune regulation during a pre-implantation period of pregnancy.

  10. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Science.gov (United States)

    Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

    2012-01-01

    Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  11. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Directory of Open Access Journals (Sweden)

    Ewoud Bernardus Compeer

    2012-03-01

    Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  12. Endothelial cells present antigens in vivo

    Directory of Open Access Journals (Sweden)

    Tellides George

    2004-03-01

    Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

  13. Antigen Cross-Presentation of Immune Complexes

    Science.gov (United States)

    Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

    2014-01-01

    The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

  14. Dissecting antigen processing and presentation routes in dermal vaccination strategies

    NARCIS (Netherlands)

    Platteel, Anouk C M; Henri, Sandrine; Zaiss, Dietmar M; Sijts, Alice J A M

    2017-01-01

    The skin is an attractive site for vaccination due to its accessibility and presence of immune cells surveilling this barrier. However, knowledge of antigen processing and presentation upon dermal vaccination is sparse. In this study we determined antigen processing routes that lead to CD8(+) T cell

  15. Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

    Directory of Open Access Journals (Sweden)

    Charlotte F Inman

    Full Text Available Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+ antigen-presenting cell subset, whilst SIRPα(-CD11R1(+ antigen-presenting cells (APCs are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+ antigen-presenting cells as orchestrators of early-life mucosal immune development.

  16. Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

    Science.gov (United States)

    Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

    2017-07-28

    Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll

  17. Granulocytes: New Members of the Antigen-Presenting Cell Family

    Directory of Open Access Journals (Sweden)

    Ang Lin

    2017-12-01

    Full Text Available Granulocytes, the most abundant types of leukocytes, are the first line of defense against pathogen invasion. However, the plasticity and diversity of granulocytes have been increasingly revealed, especially with regard to their versatile functions in orchestrating adaptive immune responses. A substantial body of recent evidence demonstrates that granulocytes can acquire the function as antigen-presenting cells under pathological or inflammatory conditions. In addition, they can acquire surface expression of MHC class II and costimulatory molecules as well as T cell stimulatory behavior when cultured with selected cytokines. The classic view of granulocytes as terminally differentiated, short-lived phagocytes is therefore changing to phenotypically and functionally heterogeneous cells that are engaged in cross-talk with other leukocyte populations and provide an additional link between innate and adaptive immunity. In this brief review, we summarize the current knowledge on the antigen-presenting capacity of granulocyte subsets (neutrophils, eosinophils, and basophils. Underlying mechanisms, relevant physiological significance and potential controversies are also discussed.

  18. Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

    Science.gov (United States)

    Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

    2017-06-01

    We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

  19. Ultraviolet light-induced suppression of antigen presentation

    International Nuclear Information System (INIS)

    Spellman, C.W.; Tomasi, T.B.

    1983-01-01

    Ultraviolet (UV) light irradiation of animals results in the development of specific T suppressor cells that inhibit antitumor immune responses. It is thought that suppression may arise as a consequence of altered antigen presentation by UV-irradiated epidermal cells. This hypothesis is based on evidence demonstrating that specific lymphoid tissues from UV-irradiated hosts exhibit impaired antigen-presenting function and that animals cannot be contact sensitized when antigens are applied to a UV-irradiated skin site. Langerhans cells of the skin are likely candidates as targets of UV-induced defects in antigen presentation as they bear Fc and C3b receptors, express Ia antigens, are of bone marrow origin, and are capable of presenting antigen in vitro. We speculate on the possible clinical usefulness of UV-induced tolerance to specific antigens such as those encountered in monoclonal antibody therapy and tissue transplantation

  20. Identification of a peptide binding protein that plays a role in antigen presentation

    International Nuclear Information System (INIS)

    Lakey, E.K.; Margoliash, E.; Pierce, S.K.

    1987-01-01

    The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

  1. Cationic liposomes promote antigen cross-presentation in dendritic cells by alkalizing the lysosomal pH and limiting the degradation of antigens

    Directory of Open Access Journals (Sweden)

    Gao J

    2017-02-01

    Full Text Available Jie Gao,1–3 Lukasz J Ochyl,1,3 Ellen Yang,4 James J Moon1,3,5 1Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA; 2Department of Pharmaceutical Sciences, School of Pharmacy, Second Military Medical University, Shanghai, People’s Republic of China; 3Biointerfaces Institute, 4Department of Chemistry, 5Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA Abstract: Cationic liposomes (CLs have been widely examined as vaccine delivery nanoparticles since they can form complexes with biomacromolecules, promote delivery of antigens and adjuvant molecules to antigen-presenting cells (APCs, and mediate cellular uptake of vaccine components. CLs are also known to trigger antigen cross-presentation – the process by which APCs internalize extracellular protein antigens, degrade them into minimal CD8+ T-cell epitopes, and present them in the context of major histocompatibility complex-I (MHC-I. However, the precise mechanisms behind CL-mediated induction of cross-presentation and cross-priming of CD8+ T-cells remain to be elucidated. In this study, we have developed two distinct CL systems and examined their impact on the lysosomal pH in dendritic cells (DCs, antigen degradation, and presentation of peptide:MHC-I complexes to antigen-specific CD8+ T-cells. To achieve this, we have used 3β-[N-(N',N'-dimethylaminoethane-carbamoyl] cholesterol (DC-Chol and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP as the prototypical components of CLs with tertiary amine groups and compared the effect of CLs and anionic liposomes on lysosomal pH, antigen degradation, and cross-presentation by DCs. Our results showed that CLs, but not anionic liposomes, elevated the lysosomal pH in DCs and reduced antigen degradation, thereby promoting cross-presentation and cross-priming of CD8+ T-cell responses. These studies shed new light on CL-mediated cross-presentation and suggest that intracellular fate of vaccine

  2. A population dynamics analysis of the interaction between adaptive regulatory T cells and antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    David Fouchet

    Full Text Available BACKGROUND: Regulatory T cells are central actors in the maintenance of tolerance of self-antigens or allergens and in the regulation of the intensity of the immune response during infections by pathogens. An understanding of the network of the interaction between regulatory T cells, antigen presenting cells and effector T cells is starting to emerge. Dynamical systems analysis can help to understand the dynamical properties of an interaction network and can shed light on the different tasks that can be accomplished by a network. METHODOLOGY AND PRINCIPAL FINDINGS: We used a mathematical model to describe a interaction network of adaptive regulatory T cells, in which mature precursor T cells may differentiate into either adaptive regulatory T cells or effector T cells, depending on the activation state of the cell by which the antigen was presented. Using an equilibrium analysis of the mathematical model we show that, for some parameters, the network has two stable equilibrium states: one in which effector T cells are strongly regulated by regulatory T cells and another in which effector T cells are not regulated because the regulatory T cell population is vanishingly small. We then simulate different types of perturbations, such as the introduction of an antigen into a virgin system, and look at the state into which the system falls. We find that whether or not the interaction network switches from the regulated (tolerant state to the unregulated state depends on the strength of the antigenic stimulus and the state from which the network has been perturbed. CONCLUSION/SIGNIFICANCE: Our findings suggest that the interaction network studied in this paper plays an essential part in generating and maintaining tolerance against allergens and self-antigens.

  3. Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Lynette Beattie

    2010-03-01

    Full Text Available Kupffer cells (KCs represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+ T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+ T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

  4. Modulation of Immune Responses by Exosomes Derived from Antigen-Presenting Cells

    Directory of Open Access Journals (Sweden)

    Botros B. Shenoda

    2016-01-01

    Full Text Available Exosome-mediated signaling is important in mediating the inflammatory response. To exert their biological or pathophysiological functions in the recipient cells, exosomes deliver a diverse array of biomacromolecules including long and short coding and non-coding RNAs, proteins, and lipids. Exosomes secreted by antigen-presenting cells can confer therapeutic benefits by attenuating or stimulating the immune response. Exosomes play a crucial role in carrying and presenting functional major histocompatibility peptide complexes to modulate antigen-specific T cell responses. Exosomes from Dendritic Cells (DCs can activate T and B cells and have been explored for their immunostimulatory properties in cancer therapy. The immunosuppressive properties of exosomes derived from macrophages and DCs can reduce inflammation in animal models for several inflammatory disorders. This review focuses on the protective role of exosomes in attenuating inflammation or augmenting immune response, emphasizing studies on exosomes derived from DCs and macrophages.

  5. Modulation of antigen presenting cell functions during chronic HPV infection

    Directory of Open Access Journals (Sweden)

    Abate Assefa Bashaw

    2017-12-01

    Full Text Available High-risk human papillomaviruses (HR-HPV infect basal keratinocytes, where in some individuals they evade host immune responses and persist. Persistent HR-HPV infection of the cervix causes precancerous neoplasia that can eventuate in cervical cancer. Dendritic cells (DCs are efficient in priming/cross-priming antigen-specific T cells and generating antiviral and antitumor cytotoxic CD8+ T cells. However, HR-HPV have adopted various immunosuppressive strategies, with modulation of DC function crucial to escape from the host adaptive immune response. HPV E6 and E7 oncoproteins alter recruitment and localization of epidermal DCs, while soluble regulatory factors derived from HPV-induced hyperplastic epithelium change DC development and influence initiation of specific cellular immune responses. This review focuses on current evidence for HR-HPV manipulation of antigen presentation in dendritic cells and escape from host immunity.

  6. Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

    Science.gov (United States)

    Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

    2011-02-01

    Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  7. Activation of professional antigen presenting cells by acharan sulfate isolated from giant African snail, Achatina fulica.

    Science.gov (United States)

    Kim, Hyun-Sun; Lee, Young-Hee; Lee, Young-Ran; Im, Sun-A; Lee, Jae-Kwon; Kim, Yeong Shik; Sim, Joon-Soo; Choi, Hyung Seok; Lee, Chong-Kil

    2007-07-01

    Acharan sulfate isolated from the giant African snail, Achatina fulica, has been reported to have antitumor activity in vivo. In an effort to determine the mechanisms of its antitumor activity, we examined the effects of acharan sulfate on professional antigen presenting cells (APCs). Acharan sulfate increased the phagocytic activity, the production of cytokines such as TNF-alpha and IL-1beta, and the release of nitric oxide on a macrophage cell line, Raw 264.7 cells. In addition, acharan sulfate induced phenotypic and functional maturation of immature dendritic cells (DCs). Immature DCs cultured with acharan sulfate expressed higher levels of class II MHC molecules and major co-stimulatory molecules such as B7-1, B7-2, and CD40. Functional maturation of immature DCs cultured in the presence of acharan sulfate was confirmed by the increased allostimulatory capacity and IL-12 production. These results suggest that the antitumor activity of acharan sulfate is partly due to the activation of professional antigen presenting cells.

  8. B lymphocytes as natural antigen-presenting cells (APC) of their own Ig receptor determinants

    International Nuclear Information System (INIS)

    Yurin, V.L.; Rudensky, A.Yu.; Rabinovich, O.R.; Kulakova, O.G.; Bobreneva, R.A.

    1986-01-01

    The authors use Igk-lb allotype-specific rat T cell proliferation(Pr) in vitro as a model of natural Ig determinants B cell presentation in Ig-specific T-B cell interactions. As shown before Igk-lb-specific responsiveness of AUG(RT-l/sup c/, Igk-la) and WAG (RT-l, Igk-la) rats is controlled by dominant Ir gene, linked to RT-l/sup c/. Only IgG(Igk-lb)-pulsed splenic APC of AUG(responder) but not WAG(non-responder) origin induce specific F 1 (WAGxAUG) T cell Pr. The same restriction was observed if purified B cells from Igk-l congeneic AUG-lb and WAG-lb rats were used as APC. B cell presentation was found to be sensitive to high irradiation dose(2000 rad). Anti-RT-l monoclonal antibody inhibition studies suggested RT-lB(I-A) molecule as a main restricting element of Igk-lb T cell recognition. B cell and splenic APC presentation of Igk-lb allotype was not inhibited by poly- and monoclonal anti-Igk-lb antibodies. Allelic exclusion of Igk-lb presentation by B cells from heterozygous F 1 (WAG-lbx AUG) rats was demonstrated by panning with antiallotypic reagents. Important, that irradiated anti-Igk-lb T cells induce specific Pr of normal Igk-lb-positive B cells. The data demonstrate MHC-restricted B cell presentation of their own receptor determinants, distinct from serologically-defined epitopes. T cell recognition of these determinants induce specific Pr of Ig-recognizing T cells and Ig-presenting B lymphocytes

  9. Protein-scaffold Directed Nanoscale Assembly of T Cell Ligands: Artificial Antigen Presentation with Defined Valency, Density and Ratio.

    Science.gov (United States)

    Smith, Mason R; Tolbert, Stephanie V; Wen, Fei

    2018-05-07

    Tuning antigen presentation to T cells is a critical step in investigating key aspects of T cell activation. However, existing technologies have limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. Here, we developed an artificial antigen presentation platform based on protein-scaffold directed assembly that allows fine control over the spatial and stoichiometric organization of T cell ligands on a 3D yeast-cell surface. Using this system, we observed that the T cell activation threshold on a 3D surface is independent of peptide-major histocompatibility complex (pMHC) valency, but instead determined by the overall pMHC surface density. When intercellular adhesion molecule 1 (ICAM-1) was co-assembled with pMHC, it enhanced antigen recognition sensitivity by 6-fold. Further, T cells responded with different magnitudes to varying ratios of pMHC and ICAM-1 and exhibited a maximum response at a ratio of 15% pMHC and 85% ICAM-1, introducing an additional parameter for tuning T cell activation. This protein-scaffold directed assembly technology is readily transferrable to acellular surfaces for translational research as well as large-scale T-cell manufacturing.

  10. Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

    Science.gov (United States)

    Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael

    2017-06-08

    Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

  11. Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

    2012-09-21

    In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

  12. Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

    Directory of Open Access Journals (Sweden)

    Ian A Cockburn

    2010-05-01

    Full Text Available Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

  13. Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

    Directory of Open Access Journals (Sweden)

    Bacellar O.

    1998-01-01

    Full Text Available It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC mAb (W6/32 suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

  14. Human Parvovirus B19 Induced Apoptotic Bodies Contain Altered Self-Antigens that are Phagocytosed by Antigen Presenting Cells

    Science.gov (United States)

    Thammasri, Kanoktip; Rauhamäki, Sanna; Wang, Liping; Filippou, Artemis; Kivovich, Violetta; Marjomäki, Varpu; Naides, Stanley J.; Gilbert, Leona

    2013-01-01

    Human parvovirus B19 (B19V) from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease) commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic disease and persistent infection suggests B19V can serve as a model for viral host interactions and the role of viruses in the pathogenesis of autoimmune diseases. Here we investigate the involvement of B19V in the breakdown of immune tolerance. Previously, we demonstrated that the non-structural protein 1 (NS 1) of B19V induces apoptosis in non-permissive cells lines and that this protein can cleave host DNA as well as form NS1-DNA adducts. Here we provide evidence that through programmed cell death, apoptotic bodies (ApoBods) are generated by B19V NS1 expression in a non-permissive cell line. Characterization of purified ApoBods identified potential self-antigens within them. In particular, signature self-antigens such as Smith, ApoH, DNA, histone H4 and phosphatidylserine associated with autoimmunity were present in these ApoBods. In addition, when purified ApoBods were introduced to differentiated macrophages, recognition, engulfment and uptake occurred. This suggests that B19V can produce a source of self-antigens for immune cell processing. The results support our hypothesis that B19V NS1-DNA adducts, and nucleosomal and lysosomal antigens present in ApoBods created in non-permissive cell lines, are a source of self-antigens. PMID:23776709

  15. Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

    Science.gov (United States)

    Gardell, Jennifer L; Parker, David C

    2017-01-01

    Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

  16. Use of immunoradiometric analysis to determine Rickettsia antigens in cell cultures and chick embryos

    Energy Technology Data Exchange (ETDEWEB)

    Prozorovskiy, S.V.; Alekseeva, N.V.; Knyazeva, E.N.; Ignatovich, V.F.; Barkhatova, O.T.

    1984-02-01

    A modification of an immunoradiometric analysis to determine Rickettsia antigens in various biological substrates was studied, using rickettsious diagnostricums, egg and cell cultures of Rickettsia. The method was highly sensitive for the determination of minimal quantities of antigens in these substrates. The method appears to be promising for studies related to the detection of microorganisms and their antigens. 5 references.

  17. ONCOLYTIC VIRUS-MEDIATED REVERSAL OF IMPAIRED TUMOR ANTIGEN PRESENTATION

    Directory of Open Access Journals (Sweden)

    Shashi Ashok Gujar

    2014-04-01

    Full Text Available Anti-tumor immunity can eliminate existing cancer cells and also maintain a constant surveillance against possible relapse. Such an antigen-specific adaptive response begins when tumor-specific T cells become activated. T cell activation requires two signals on antigen presenting cells (APCs: antigen presentation through MHC molecules and co-stimulation. In the absence of one or both of these signals, T cells remain inactivated or can even become tolerized. Cancer cells and their associated microenvironment strategically hinder the processing and presentation of tumor antigens and consequently prevent the development of anti-tumor immunity. Many studies, however, demonstrate that interventions that overturn tumor-associated immune evasion mechanisms can establish anti-tumor immune responses of therapeutic potential. One such intervention is oncolytic virus (OV-based anti-cancer therapy. Here we discuss how OV-induced immunological events override tumor-associated antigen presentation impairment and promote appropriate T cell:APC interaction. Detailed understanding of this phenomenon is pivotal for devising the strategies that will enhance the efficacy of OV-based anti-cancer therapy by complementing its inherent oncolytic

  18. Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression.

    Directory of Open Access Journals (Sweden)

    Chikara Furusawa

    Full Text Available The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.

  19. Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression.

    Science.gov (United States)

    Furusawa, Chikara; Yamaguchi, Tomoyuki

    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification.

  20. Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

    Directory of Open Access Journals (Sweden)

    Schendel Dolores J

    2010-09-01

    Full Text Available Abstract Background Antigen-loaded dendritic cells (DC are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. Methods In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC compared with conventional DC prepared in seven days (7d mDC, which represent the most common form of DC used for vaccines to date. Results Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivtRNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. Conclusions This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

  1. Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs.

    Science.gov (United States)

    Wang, Chao; Sun, Wujin; Ye, Yanqi; Bomba, Hunter N; Gu, Zhen

    2017-01-01

    The immune system protects the body against a wide range of infectious diseases and cancer by leveraging the efficiency of immune cells and lymphoid organs. Over the past decade, immune cell/organ therapies based on the manipulation, infusion, and implantation of autologous or allogeneic immune cells/organs into patients have been widely tested and have made great progress in clinical applications. Despite these advances, therapy with natural immune cells or lymphoid organs is relatively expensive and time-consuming. Alternatively, biomimetic materials and strategies have been applied to develop artificial immune cells and lymphoid organs, which have attracted considerable attentions. In this review, we survey the latest studies on engineering biomimetic materials for immunotherapy, focusing on the perspectives of bioengineering artificial antigen presenting cells and lymphoid organs. The opportunities and challenges of this field are also discussed.

  2. CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

    Science.gov (United States)

    Hardman, Clare S; Chen, Yi-Ling; Salimi, Maryam; Jarrett, Rachael; Johnson, David; Järvinen, Valtteri J; Owens, Raymond J; Repapi, Emmanouela; Cousins, David J; Barlow, Jillian L; McKenzie, Andrew N J; Ogg, Graham

    2017-12-22

    Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  3. Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes, and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease.

    Science.gov (United States)

    Kawasaki, Yasufumi; Sato, Kazuya; Hayakawa, Hiroko; Takayama, Norihito; Nakano, Hirofumi; Ito, Ryoji; Mashima, Kiyomi; Oh, Iekuni; Minakata, Daisuke; Yamasaki, Ryoko; Morita, Kaoru; Ashizawa, Masahiro; Yamamoto, Chihiro; Hatano, Kaoru; Fujiwara, Shin-Ichiro; Ohmine, Ken; Muroi, Kazuo; Kanda, Yoshinobu

    2018-04-17

    Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4 + and CD8 + T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg null mice. CD4 + T cells and, to a lesser extent, CD8 + T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4 + T cells also supported the activation and proliferation of CD8 + T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4 + T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4 + T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC) +/+ mice but not in MHC -/- mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD. Copyright © 2018. Published by Elsevier Inc.

  4. ImmunoChip Study Implicates Antigen Presentation to T Cells in Narcolepsy

    DEFF Research Database (Denmark)

    Faraco, Juliette; Lin, Ling; Kornum, Birgitte Rahbek

    2013-01-01

    receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells...

  5. Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells

    Science.gov (United States)

    Boivin, Nicolas; Baillargeon, Joanie; Doss, Prenitha Mercy Ignatius Arokia; Roy, Andrée-Pascale; Rangachari, Manu

    2015-01-01

    Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals. PMID:25885435

  6. Relative Efficacy of Uptake and Presentation of Mycobacterium bovis BCG Antigens by Type I Mouse Lung Epithelial Cells and Peritoneal Macrophages ▿

    Science.gov (United States)

    Kumari, Mandavi; Saxena, Rajiv K.

    2011-01-01

    Flow cytometric studies indicated that both peritoneal macrophages (PMs) and primary lung epithelial (PLE) cells isolated from mouse lungs could take up fluorescence-tagged Mycobacterium bovis BCG. BCG uptake in both cases was significantly inhibited by cytochalasin D, indicating active internalization of BCG by these cells. Confocal microscopy data further confirmed that BCG was internalized by PLE cells. BCG sonicate antigen (sBCG) had marked toxicity toward PMs but was relatively nontoxic to PLE cells. Accordingly, BCG sonicate antigen induced a significantly higher apoptotic and necrotic response in PMs compared to that in PLE cells. Both PMs and PLE cells exposed to BCG antigens and fixed thereafter could efficiently present antigens to purified BCG-sensitized T helper cells, as assessed by the release of interleukin-2 (IL-2) and gamma interferon (IFN-γ). If, however, PLE cells were fixed before exposure to BCG, antigen presentation was abrogated, indicating that the PLE cells may in some way process the BCG antigen. A comparison of efficacies of BCG-pulsed PLE cells and PMs to present antigen at various antigen-presenting cell (APC)/T cell ratios indicated that PMs had only marginally greater APC function than that of PLE cells. Staining with specific monoclonal antibodies indicated that the cultured PLE cells used for antigen presentation essentially comprised type I epithelial cells. Our results suggest that type I lung epithelial cells may present BCG antigens to sensitized T helper cells and that their performance as APCs is comparable with that of PMs. PMID:21646448

  7. Interleukin production by neonatal spleen cells during and as a result of antigen presentation: The effect of ultraviolet light

    International Nuclear Information System (INIS)

    Levin, D.; Gershon, H.

    1989-01-01

    Antigen presentation by neonatal murine spleen cells and the production of lymphokines and interleukins involved in the stimulation of a T-helper-2 (TH2) cell line (D10-G4.1) were studied as were the effects of ultra violet (UV)-irradiation on this system. Neonatal spleen cells are less capable than adult cells of performing the initial steps of the immune response required for antigen dependent activation of TH2 cells. These steps include soluble antigen processing and presentation and as a result reduced production of IL-4 and IL-1-Inducer Factor (IL-1-IF) by the T-helper cells and reduced production of IL-1 and IL-2 by the antigen presenting cell population. Spontaneous membrane IL-1 activity is low in the neonate, however, when exposed to IL-1-IF they can express adult levels. Ultraviolet (UV) irradiation of the antigen presenting population has a damaging effect on all the above mentioned processes. Antigen processing and presentation, induction of D10 IL-4 production and proliferation, and IL-2 production demonstrate two different age related patterns of UV-irradiation induced damage: a dose dependent inhibition when adult cells are irradiated and an inverse effect in which low doses of irradiation were more inhibitory than higher doses when neonatal cells are irradiated. However, the secretion and membrane expression of IL-1 by both age groups are directly and totally inhibited by the range of UV-irradiation doses used and cannot be reinduced with a supplement of a crude IL-1-IF. While the capacity to produced IL-1 is totally destroyed by UV-irradiation, the ability to produce IL-2 remains intact and remains responsive to an IL-2-Inducer activity during proper antigen presentation. The low responses of neonatal antigen presenting spleen cell populations and the damaging effect of UV on both neonatal and adult responses are not due to the induction of suppressor factors

  8. ImmunoChip study implicates antigen presentation to T cells in narcolepsy.

    Directory of Open Access Journals (Sweden)

    Juliette Faraco

    Full Text Available Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip. Three loci located outside the Human Leukocyte Antigen (HLA region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@, variants in two additional narcolepsy loci, Cathepsin H (CTSH and Tumor necrosis factor (ligand superfamily member 4 (TNFSF4, also called OX40L, attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

  9. Skewing to the LFA-3 adhesion pathway by influenza infection of antigen-presenting cells

    NARCIS (Netherlands)

    van Kemenade, F. J.; Kuijpers, K. C.; de Waal-Malefijt, R.; van Lier, R. A.; Miedema, F.

    1993-01-01

    The effect of influenza (FLU) infection on heterotypic conjugate formation between antigen-presenting cells and T lymphocytes has been studied with FLU-specific T cell clones and FLU-infected B-lymphoblastoid cells (B-LCL). Conjugate formation between FLU-infected B-LCL (FLU+ B-LCL) and T cells was

  10. Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

    Directory of Open Access Journals (Sweden)

    Kennedy Colleen

    2011-12-01

    Full Text Available Abstract Background Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

  11. The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

    Science.gov (United States)

    Torreno-Pina, Juan A.; Manzo, Carlo; Salio, Mariolina; Aichinger, Michael C.; Oddone, Anna; Lakadamyali, Melike; Shepherd, Dawn; Besra, Gurdyal S.; Cerundolo, Vincenzo

    2016-01-01

    Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters. PMID:26798067

  12. Deletion of Batf3-dependent antigen-presenting cells does not affect atherosclerotic lesion formation in mice.

    Directory of Open Access Journals (Sweden)

    Jesus Gil-Pulido

    Full Text Available Atherosclerosis is the main underlying cause for cardiovascular events such as myocardial infarction and stroke and its development might be influenced by immune cells. Dendritic cells (DCs bridge innate and adaptive immune responses by presenting antigens to T cells and releasing a variety of cytokines. Several subsets of DCs can be discriminated that engage specific transcriptional pathways for their development. Basic leucine zipper transcription factor ATF-like 3 (Batf3 is required for the development of classical CD8α+ and CD103+ DCs. By crossing mice deficient in Batf3 with atherosclerosis-prone low density lipoprotein receptor (Ldlr-/--deficient mice we here aimed to further address the contribution of Batf3-dependent CD8α+ and CD103+ antigen-presenting cells to atherosclerosis. We demonstrate that deficiency in Batf3 entailed mild effects on the immune response in the spleen but did not alter atherosclerotic lesion formation in the aorta or aortic root, nor affected plaque phenotype in low density lipoprotein receptor-deficient mice fed a high fat diet. We thus provide evidence that Batf3-dependent antigen-presenting cells do not have a prominent role in atherosclerosis.

  13. Engineering tolerance using biomaterials to target and control antigen presenting cells.

    Science.gov (United States)

    Tostanoski, Lisa H; Gosselin, Emily A; Jewell, Christopher M

    2016-05-01

    Autoimmune diseases occur when cells of the adaptive immune system incorrectly recognize and attack "self" tissues. Importantly, the proliferation and differentiation of these cells is triggered and controlled by interactions with antigen presenting cells (APCs), such as dendritic cells. Thus, modulating the signals transduced by APCs (e.g., cytokines, costimulatory surface proteins) has emerged as a promising strategy to promote tolerance for diseases such as multiple sclerosis, type 1 diabetes, and lupus. However, many approaches have been hindered by non-specific activity of immunosuppressive or immunoregulatory cues, following systemic administration of soluble factors via traditional injections routes (e.g., subcutaneous, intravenous). Biomaterials offer a unique opportunity to control the delivery of tolerogenic signals in vivo via properties such as controlled particle size, tunable release kinetics, and co-delivery of multiple classes of cargo. In this review, we highlight recent reports that exploit these properties of biomaterials to target APCs and promote tolerance via three strategies, i) passive or active targeting of particulate carriers to APCs, ii) biomaterial-mediated control over antigen localization and processing, and iii) targeted delivery of encapsulated or adsorbed immunomodulatory signals. These reports represent exciting advances toward the goal of more effective therapies for autoimmune diseases, without the broad suppressive effects associated with current clinically-approved therapies.

  14. Comparison of microglia and infiltrating CD11c+ cells as antigen presenting cells for T cell proliferation and cytokine response

    DEFF Research Database (Denmark)

    Wlodarczyk, Agnieszka; Løbner, Morten; Cédile, Oriane

    2014-01-01

    BACKGROUND: Tissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells...... (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages. METHODS: In this work we compared the antigen presenting capacity of CD11c+ and CD11c- microglia subsets with infiltrating CD11c......+ APC, which include DC. The microglial subpopulations (CD11c- CD45dim CD11b+ and CD11c+ CD45dim CD11b+) as well as infiltrating CD11c+ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR...

  15. Probiotic metabolites from Bacillus coagulans GanedenBC30TM support maturation of antigen-presenting cells in vitro

    Science.gov (United States)

    Benson, Kathleen F; Redman, Kimberlee A; Carter, Steve G; Keller, David; Farmer, Sean; Endres, John R; Jensen, Gitte S

    2012-01-01

    AIM: To study the effects of probiotic metabolites on maturation stage of antigen-presenting immune cells. METHODS: Ganeden Bacillus coagulans 30 (GBC30) bacterial cultures in log phase were used to isolate the secreted metabolite (MET) fraction. A second fraction was made to generate a crude cell-wall-enriched fraction, by centrifugation and lysis, followed by washing. A preparation of MET was subjected to size exclusion centrifugation, generating three fractions: < 3 kDa, 3-30 kDa, and 30-200 kDa and activities were tested in comparison to crude MET and cell wall in primary cultures of human peripheral blood mononuclear cell (PBMC) as a source of antigen-presenting mononuclear phagocytes. The maturation status of mononuclear phagocytes was evaluated by staining with monoclonal antibodies towards CD14, CD16, CD80 and CD86 and analyzed by flow cytometry. RESULTS: Treatment of PBMC with MET supported maturation of mononuclear phagocytes toward both macrophage and dendritic cell phenotypes. The biological activity unique to the metabolites included a reduction of CD14+ CD16+ pro-inflammatory cells, and this property was associated with the high molecular weight metabolite fraction. Changes were also seen for the dendritic cell maturation markers CD80 and CD86. On CD14dim cells, an increase in both CD80 and CD86 expression was seen, in contrast to a selective increase in CD86 expression on CD14bright cells. The co-expression of CD80 and CD86 indicates effective antigen presentation to T cells and support of T helper cell differentiation. The selective expression of CD86 in the absence of CD80 points to a role in generating T regulatory cells. CONCLUSION: The data show that a primary mechanism of action of GBC30 metabolites involves support of more mature phenotypes of antigen-presenting cells, important for immunological decision-making. PMID:22563167

  16. CD4+ T cell-mediated rejection of MHC class II-positive tumor cells is dependent on antigen secretion and indirect presentation on host APCs.

    Science.gov (United States)

    Haabeth, Ole Audun Werner; Fauskanger, Marte; Manzke, Melanie; Lundin, Katrin U; Corthay, Alexandre; Bogen, Bjarne; Tveita, Anders Aune

    2018-05-11

    Tumor-specific CD4+ T cells have been shown to mediate efficient anti-tumor immune responses against cancer. Such responses can occur through direct binding to MHC class II (MHC II)-expressing tumor cells or indirectly via activation of professional antigen-presenting cells (APC) that take up and present the tumor antigen. We have previously shown that CD4+ T cells reactive against an epitope within the Ig light chain variable region of a murine B cell lymphoma can reject established tumors. Given the presence of MHC II molecules at the surface of lymphoma cells, we investigated whether MHC II-restricted antigen presentation on tumor cells alone was required for rejection. Variants of the A20 B lymphoma cell line that either secreted or intracellularly retained different versions of the tumor-specific antigen revealed that antigen secretion by the MHC II-expressing tumor cells was essential both for the priming and effector phase of CD4+ T cell-driven anti-tumor immune responses. Consistent with this, genetic ablation of MHC II in tumor cells, both in the case of B lymphoma and B16 melanoma, did not preclude rejection of tumors by tumor antigen-specific CD4+ T cells in vivo. These findings demonstrate that MHC class II expression on tumor cells themselves is not required for CD4+ T cell-mediated rejection, and that indirect display on host APC is sufficient for effective tumor elimination. These results support the importance of tumor-infiltrating APC as mediators of tumor cell killing by CD4+ T cells. Copyright ©2018, American Association for Cancer Research.

  17. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    International Nuclear Information System (INIS)

    Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

    2011-01-01

    Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  18. A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

    2011-08-05

    Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

  19. Immunologic effects of whole body ultraviolet (uv) irradiation. II. Defect in splenic adherent cell antigen presentation for stimulation of T cell proliferation

    International Nuclear Information System (INIS)

    Letvin, N.L.; Fox, I.J.; Greene, M.I.; Benacerraf, B.; Germain, R.N.

    1980-01-01

    Ultraviolet (uv) irradiation has been shown to alter many parameters of the immunologic reactivity of mice. The altered responsiveness of uv-irradiated mice, as measured by delayed-type hypersensitivity (DTH) and primary in vitro plaque-forming cell (PFC) responses to T-dependent antigens, has recently been correlated with a functional defect in the splenic adherent cell population of these animals. The present studies describe a model of this altered responsiveness, which allows further clarification of the effects of external uv irradiation on the splenic antigen-presenting cell (APC) in its interactions with T cells

  20. Selection of restriction specificities of virus-specific cytotoxic T cells in the thymus: no evidence for a crucial role of antigen-presenting cells

    International Nuclear Information System (INIS)

    Zinkernagel, R.M.

    1982-01-01

    The proposal was tested that (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras expressed predominantly P1-restricted T cells because donor derived stem cells were exposed to recipient derived antigen-presenting cells in the thymus. Because P1 recipient-derived antigen-presenting cells are replaced only slowly after 6-8 wk by (P1 X P2) donor-derived antigen-presenting cells in the thymus and because replenished pools of mature T cells may by then prevent substantial numbers of P2-restricted T cells to be generated, a large portion of thymus cells and mature T cells were eliminated using the following treatments of 12-20-wk-old (P1 X P2) F1 leads to P1 irradiation bone marrow chimeras: (a) cortisone plus antilymphocyte serum, (b) Cytoxan, (c) three doses of sublethal irradiation (300 rad) 2d apart, and (d) lethal irradiation (850 rad) and reconstitution with T cell-depleted (P1 X P2) F1 stem cells. 12-20 wk after this second treatment, (P1 X P2) leads to P1 chimeras were infected with vaccinia-virus. Virus-specific cytotoxic T cell reactivity was expressed by chimeric T cells of (P1 X P[2) F1 origin and was restricted predominantly to P1. Virus-specific cytotoxic T cells, therefore, do not seem to be selected to measurable extent by the immigrating donor-derived antigen-presenting cells in the thymus; their selection depends apparently from the recipient-derived radioresistant thymus cells

  1. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

    Science.gov (United States)

    Kang, Jung-Ok; Lee, Jee-Boong; Chang, Jun

    2016-01-01

    Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines.

  2. Native IgG2a(b) is barely antigenic to major histocompatibility complex class II-restricted T cells owing to inefficient internalization by professional antigen-presenting cells.

    Science.gov (United States)

    Bartnes, K; Hannestad, K

    2000-04-01

    Peptide epitopes derived from immunoglobulin variable regions represent tumour-specific antigens on B-cell neoplasms and can be recognized by syngeneic, major histocompatibility complex (MHC) class II-restricted T cells. Immunoglobulin peptide/MHC class II complexes may also be involved in autoimmunity and CD4+ T-cell-mediated B-cell regulation. Thus, the IgG2a(b) H-chain allopeptide gamma2a(b) 435-451 presented on I-Ad mimics the epitope implicated in herpes simplex virus-induced autoimmune stromal keratitis and is the target of T helper 1 (Th1) clones that suppress IgG2a(b) production in vivo. We here report that spleen and thymus cells constitutively present the autologous gamma2a(b) epitope to a gamma2a(b) 435-451/I-A(d) reactive T-cell hybridoma as a function of the animal housing conditions (specific pathogen-free or not) and the serum levels of IgG2a(b). Constitutive presentation in the spleen was predominantly performed by dendritic cells. Whereas spleen cells poorly presented native IgG2a(b) to a gamma2a(b) 435-451/I-A(d) reactive T-cell hybridoma, IgG2a(b) in the form of immune complexes were presented > 200-fold more efficiently owing to internalization via low-affinity FcgammaR on macrophages. The antigenicity could also be improved by homotypic aggregation and by targeting IgG2a(b) to complement receptors on the A20 B-cell lymphoma. Mice without detectable IgG2a(b)-containing immune complexes typically exhibited minimal constitutive presentation. Nevertheless, native IgG2a(b) can sensitize antigen-presenting cells in vivo, as mice that were devoid of immune complexes and carried an IgG2a(b)-producing tumour did present constitutively, even at physiological IgG2a(b) serum levels. Whereas the amounts of IgG released from most B-cell lymphomas may be too low to allow spontaneous priming of tumour-specific MHC class II-restricted T cells, administration of tumour immunoglobulin in aggregated form might improve the efficacy of idiotype vaccination.

  3. Intersection of autophagy with pathways of antigen presentation.

    Science.gov (United States)

    Patterson, Natalie L; Mintern, Justine D

    2012-12-01

    Traditionally, macroautophagy (autophagy) is viewed as a pathway of cell survival. Autophagy ensures the elimination of damaged or unwanted cytosolic components and provides a source of cellular nutrients during periods of stress. Interestingly, autophagy can also directly intersect with, and impact, other major pathways of cellular function. Here, we will review the contribution of autophagy to pathways of antigen presentation. The autophagy machinery acts to modulate both MHCI and MHCII antigen presentation. As such autophagy is an important participant in pathways that elicit host cell immunity and the elimination of infectious pathogens.

  4. T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation

    Directory of Open Access Journals (Sweden)

    Andrew W. Woodham

    2016-12-01

    Full Text Available Human papillomavirus type 16 (HPV16 infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs, the resident epithelial antigen-presenting cells (APCs. The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1, but without costimulation (signal 2. We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8+ T cells receiving signal 1+2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals. Keywords: T cell anergy, T cell ignorance, Immune tolerance, Human papillomavirus, HPV16, Langerhans cells

  5. Autophagy-related protein Vps34 controls the homeostasis and function of antigen cross-presenting CD8α+ dendritic cells.

    Science.gov (United States)

    Parekh, Vrajesh V; Pabbisetty, Sudheer K; Wu, Lan; Sebzda, Eric; Martinez, Jennifer; Zhang, Jianhua; Van Kaer, Luc

    2017-08-01

    The class III PI3K Vacuolar protein sorting 34 (Vps34) plays a role in both canonical and noncanonical autophagy, key processes that control the presentation of antigens by dendritic cells (DCs) to naive T lymphocytes. We generated DC-specific Vps34 -deficient mice to assess the contribution of Vps34 to DC functions. We found that DCs from these animals have a partially activated phenotype, spontaneously produce cytokines, and exhibit enhanced activity of the classic MHC class I and class II antigen-presentation pathways. Surprisingly, these animals displayed a defect in the homeostatic maintenance of splenic CD8α + DCs and in the capacity of these cells to cross-present cell corpse-associated antigens to MHC class I-restricted T cells, a property that was associated with defective expression of the T-cell Ig mucin (TIM)-4 receptor. Importantly, mice deficient in the Vps34-associated protein Rubicon, which is critical for a noncanonical form of autophagy called "Light-chain 3 (LC3)-associated phagocytosis" (LAP), lacked such defects. Finally, consistent with their defect in the cross-presentation of apoptotic cells, DC-specific Vps34 -deficient animals developed increased metastases in response to challenge with B16 melanoma cells. Collectively, our studies have revealed a critical role of Vps34 in the regulation of CD8α + DC homeostasis and in the capacity of these cells to process and present antigens associated with apoptotic cells to MHC class I-restricted T cells. Our findings also have important implications for the development of small-molecule inhibitors of Vps34 for therapeutic purposes.

  6. Role of interleukin-1 in antigen presentation by normal articular chondrocytes

    International Nuclear Information System (INIS)

    Tiku, M.L.; Liu, S.; Tiku, K.

    1986-01-01

    Recently the authors have described that normal articular chondrocytes of rabbits present antigen to immune T cells. In the present study the authors investigated the role of interleukin-1 (IL-1) on antigen presentation by chondrocytes. For these experiments the antigen pulsed chondroyctes were either untreated or fixed with paraformaldehyde and then co-cultured with immune T cells. T cell proliferation was measured by 3 H-thymidine incorporation. Pulsed non-fixed chondrocytes presented antigen, as expected, but pulsed and fixed cells failed to present antigen to T cells. The 3 H-TdR incorporation was partially restored by addition of purified human IL-1. Next, IL-1 activity was measured in primary chondrocyte culture supernatants stimulated with or without lipopolysaccharide (LPS) in comitogen thymocyte assay. No activity was detected in chondrocyte supernatants. Propagated chondrocyte culture supernatants also lacked IL-1 activity when stimulated with LPS in the presence of increasing concentration of indomethacin. On the other hand the authors observed that chondrocyte culture supernatants in a dose dependent manner inhibited human IL-1 induced 3 H-TdR incorporation of murine thymocytes. This suggested that these cells may produce an inhibitor of IL-1 and IL-1 production by chondrocytes may be essential for T cell proliferation by these cells. Inability to detect IL-1 in chondrocyte supernatants may be due to the presence of an inhibitor to IL-1. These findings may help in elucidating the immunological mechanisms in situations where chondrocytes and T cell interact, such as in arthritis

  7. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

    Directory of Open Access Journals (Sweden)

    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  8. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Directory of Open Access Journals (Sweden)

    Isabel Correa

    2018-03-01

    Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  9. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

  10. Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

    Science.gov (United States)

    Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

    2018-01-01

    Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

  11. Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

    Science.gov (United States)

    Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

    2018-01-01

    Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty

  12. Loss of Proliferation and Antigen Presentation Activity following Internalization of Polydispersed Carbon Nanotubes by Primary Lung Epithelial Cells

    Science.gov (United States)

    Kumari, Mandavi; Sachar, Sumedha; Saxena, Rajiv K.

    2012-01-01

    Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells. PMID:22384094

  13. The role and mechanics of dendritic cells in tumor antigen acquisition and presentation following laser immunotherapy

    Science.gov (United States)

    Laverty, Sean M.; Dawkins, Bryan A.; Chen, Wei R.

    2018-02-01

    We extend our model of the antitumor immune response initiated by laser-immunotherapy treatment to more closely examine key steps in the immune response 1) tumor antigen acquisition by antigen-presenting dendritic cells (DCs) and 2) cytotoxic T cell (CTL) priming by lymphatic DCs. Specifically we explore the formation of DC-CTL complexes that lead to CTL priming. We find that the bias in the dissociation rate of the complex influences the outcome of treatment. In particular, a bias towards priming favors a rapid activated CTL response and the clearance of tumors.

  14. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Harjeet Singh

    Full Text Available Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28 that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC in the presence of interleukin (IL-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT. We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

  15. Dynamics of antigen presentation to transgene product-specific CD4+ T cells and of Treg induction upon hepatic AAV gene transfer

    Directory of Open Access Journals (Sweden)

    George Q Perrin

    2016-01-01

    Full Text Available The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4+ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8 vector expressing cytoplasmic ovalbumin (OVA into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4+ T cells (requiring professional antigen-presenting cells, e.g., macrophages and CD4+CD25+FoxP3+ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4+ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

  16. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer.

    Science.gov (United States)

    Priceman, Saul J; Gerdts, Ethan A; Tilakawardane, Dileshni; Kennewick, Kelly T; Murad, John P; Park, Anthony K; Jeang, Brook; Yamaguchi, Yukiko; Yang, Xin; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E; Wu, Anna M; Brown, Christine E; Forman, Stephen J

    2018-01-01

    Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with "on-target off-tumor" activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies.

  17. Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

    Science.gov (United States)

    Priceman, Saul J.; Gerdts, Ethan A.; Tilakawardane, Dileshni; Kennewick, Kelly T.; Murad, John P.; Park, Anthony K.; Jeang, Brook; Yamaguchi, Yukiko; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E.; Brown, Christine E.; Forman, Stephen J.

    2018-01-01

    ABSTRACT Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. PMID:29308300

  18. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4+ intestinal intraepithelial lymphocytes

    International Nuclear Information System (INIS)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru

    2013-01-01

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4 + IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4 + IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4 + IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4 + IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4 + LPLs and primed splenic CD4 + T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4 + IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo

  19. Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response

    Directory of Open Access Journals (Sweden)

    Song C

    2016-08-01

    Full Text Available Chanyoung Song,* Young-Woock Noh,* Yong Taik Lim SKKU Advanced Institute of Nanotechnology (SAINT, School of Chemical Engineering, Sungkyunkwan University, Suwon, South Korea *These authors contributed equally to this work Abstract: Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI-coated polymer nanoparticles (NPs as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide (PLGA NPs containing ovalbumin (OVA by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA NPs for antigen delivery and cross-presentation on dendritic cells (DCs were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA NPs. Therefore, the PEI-coated PLGA (OVA NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy. Keywords: antigen delivery, dendritic cells, polymer NPs, vaccine, cross-presentation

  20. A Francisella tularensis live vaccine strain that improves stimulation of antigen-presenting cells does not enhance vaccine efficacy.

    Directory of Open Access Journals (Sweden)

    Deanna M Schmitt

    Full Text Available Vaccination is a proven strategy to mitigate morbidity and mortality of infectious diseases. The methodology of identifying and testing new vaccine candidates could be improved with rational design and in vitro testing prior to animal experimentation. The tularemia vaccine, Francisella tularensis live vaccine strain (LVS, does not elicit complete protection against lethal challenge with a virulent type A Francisella strain. One factor that may contribute to this poor performance is limited stimulation of antigen-presenting cells. In this study, we examined whether the interaction of genetically modified LVS strains with human antigen-presenting cells correlated with effectiveness as tularemia vaccine candidates. Human dendritic cells infected with wild-type LVS secrete low levels of proinflammatory cytokines, fail to upregulate costimulatory molecules, and activate human T cells poorly in vitro. One LVS mutant, strain 13B47, stimulated higher levels of proinflammatory cytokines from dendritic cells and macrophages and increased costimulatory molecule expression on dendritic cells compared to wild type. Additionally, 13B47-infected dendritic cells activated T cells more efficiently than LVS-infected cells. A deletion allele of the same gene in LVS displayed similar in vitro characteristics, but vaccination with this strain did not improve survival after challenge with a virulent Francisella strain. In vivo, this mutant was attenuated for growth and did not stimulate T cell responses in the lung comparable to wild type. Therefore, stimulation of antigen-presenting cells in vitro was improved by genetic modification of LVS, but did not correlate with efficacy against challenge in vivo within this model system.

  1. The Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation

    Science.gov (United States)

    Mou, Yongbin; Xing, Yun; Ren, Hongyan; Cui, Zhihua; Zhang, Yu; Yu, Guangjie; Urba, Walter J.; Hu, Qingang; Hu, Hongming

    2017-01-01

    Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

  2. Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus

    International Nuclear Information System (INIS)

    Salfeld, J.; Pfaff, E.; Noah, M.; Schaller, H.

    1989-01-01

    The precore/core gene of hepatitis B virus directs the synthesis of two polypeptides, the 21-kilodalton subunit (p21c) forming the viral nucleocapsid (serologically defined as core antigen [HBcAg]) and a secreted processed protein (p17e, serologically defined as HBe antigen [HBeAg]). Although most of their primary amino acid sequences are identical, HBcAg and HBeAg display different antigenic properties that are widely used in hepatitis B virus diagnosis. To locate and to characterize the corresponding determinants, segments of the core gene were expressed in Escherichia coli and probed with a panel of polyclonal or monoclonal antibodies in radioimmunoassays or enzyme-linked immunosorbent assays, Western blots, and competition assays. Three distinct major determinants were characterized. It is postulated that HBcAg and HBeAg share common basic three-dimensional structure exposing the common linear determinant HBe1 but that they differ in the presentation of two conformational determinants that are either introduced (HBc) or masked (HBe2) in the assembled core. The simultaneous presentation of HBe1 and HBc, two distinctly different antigenic determinants with overlapping amino acid sequences, is interpreted to indicate the presence of slightly differently folded, stable conformational states of p21c in the hepatitis virus nucleocapsid

  3. Specificity of antigens on UV radiation-induced antigenic tumor cell variants measured in vitro and in vivo

    International Nuclear Information System (INIS)

    Hostetler, L.W.; Romerdahl, C.A.; Kripke, M.L.

    1989-01-01

    The purpose of this study was to determine whether antigenic variants cross-react immunologically with the parental tumor and whether the UVR-associated antigen unique to UVR-induced tumors is also present on the variants. Antigenic (regressor) variants and nonimmunogenic (progressor) clones derived from UV-irradiated cultures of the C3H K1735 melanoma and SF19 spontaneous fibrosarcoma cell lines were used to address these questions. In an in vivo immunization and challenge assay, the antigenic variants did not induce cross-protection among themselves, but each induced immunity against the immunizing variant, the parent tumor cells, and nonimmunogenic clones derived from UV-irradiated parent cultures. Therefore, the variants can be used to induce in mice a protective immunity that prevents the growth of the parent tumor and nonimmunogenic clones, but not other antigenic variants. In contrast, immunization with cells of the parental tumor or the nonimmunogenic clones induced no protective immunity against challenge with any of the cell lines. Utilizing the K1735 melanoma-derived cell lines in vitro, T-helper (Th) cells isolated from tumor-immunized mice were tested for cross-reactivity by their ability to collaborate with trinitrophenyl-primed B-cells in the presence of trinitrophenyl-conjugated tumor cells. Also, the cross-reactivity of cytotoxic T-lymphocytes from tumor-immunized mice was assessed by a 4-h 51Cr-release assay. Antigenic variants induced cytotoxic T-lymphocytes and Th activity that was higher than that induced by the parent tumor and nonimmunogenic clones from the UVR-exposed parent tumor and cross-reacted with the parental tumor cells and nonimmunogenic clones, but not with other antigenic variants

  4. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

    International Nuclear Information System (INIS)

    Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

  5. Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

    International Nuclear Information System (INIS)

    Myers, C.D.; Vitetta, E.S.

    1989-01-01

    B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells

  6. Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

    Science.gov (United States)

    Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard

    2017-01-01

    Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8+ and CD4+ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8+ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. PMID:28555838

  7. Robust and Accurate Discrimination of Self/Non-Self Antigen Presentations by Regulatory T Cell Suppression

    OpenAIRE

    Furusawa, Chikara; Yamaguchi, Tomoyuki

    2016-01-01

    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self ...

  8. Repopulated antigen presenting cells induced an imbalanced differentiation of the helper T cells in whole body gamma irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hae Ran; Jo, Sung Kee [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Paik, Sang Kee [Chungnam National University, Taejon (Korea, Republic of)

    2004-07-01

    Therapeutic irradiation of cancer patients, although it may be protected by several antioxidant agents against free radicals, often induces chronic sequelae such as inflammation (allergic inflammation). This is a limiting factor for radiotherapy. Following radiotherapy, the inflammation or injury can occur in any organ with a high radiosensitivity such as the lung, bladder, kidney, liver, stomach and intestine. The mechanism by which ionizing radiation initiates inflammation is, however, poorly understood. In recent studies, it was suggested that a factor for irradiation-induced inflammation might be the over production of IL-4 that enhances fibroblast proliferation and collagen synthesis. During the early stages after irradiation, type 2 of the helper T cells might be the major source of IL-4, and later on there seems to be an activation of the other IL-4 producing cell types, e.q. macrophages or mast cells. This is interesting because inflammation is classically seen to be dominated by Th1 cells secreting IFN-{gamma}. In the previous study, we were interested in the enhancement of the IL-4 and the IgE production during the development of immune cells after {gamma}-irradiation. We were able to deduce that IL-4 production was increased because of the shifted differentiation of the naive Th cells by the repopulated antigen presenting cells after irradiation. The aim of the present study was to precisely define whether antigen-presenting cells (APCs) of whole body irradiation-treated mice could influence the shifted differentiation of the Th cells. This view can be demonstrated by confirming that the shifted functional status of the Th cells is induced by the altered function of the repopulated macrophages after whole body irradiation (WBI)

  9. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  10. Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner.

    Science.gov (United States)

    Riedl, Petra; Reiser, Michael; Stifter, Katja; Krieger, Jana; Schirmbeck, Reinhold

    2014-07-01

    Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Spontaneous CD4+ and CD8+ T-cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients.

    Science.gov (United States)

    Pearce, Hayden; Hutton, Paul; Chaudhri, Shalini; Porfiri, Emilio; Patel, Prashant; Viney, Richard; Moss, Paul

    2017-07-01

    Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune-privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T-cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg-specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8 + and CD4 + T-cell responses against MAGE-A family antigens were present in 44% (20/45) of patients' samples assayed by ex vivo IFN-γ ELISPOT. The presence of MAGE-specific CD8 + T cells was further determined following short-term in vitro expansion through the use of pMHC-I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE-specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg-specific T-cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T-cell pool following treatment. Spontaneous T-cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Antigen Expression on Blast Cells and Hematological Parameters at Presentation in Acute Lymphoblastic Leukemia Patients

    International Nuclear Information System (INIS)

    Naeem, S.; Bukhari, M. H.

    2015-01-01

    Objective: To analyze the expression of various antigens on the leukemic blasts and to determine the hematological parameters, in Acute Lymphoblastic Leukemia (ALL) patients at presentation. Study Design: Observational study. Place and Duration of Study: King Edward Medical University, Lahore and Hameed Latif Hospital, Lahore, from February 2013 to March 2014. Methodology: A total of 50 newly diagnosed and untreated patients of ALL were selected from Mayo Hospital and Hameed Latif Hospital. These patients included both genders and all age groups. Hemoglobin, total leukocyte count and platelet count were determined on hematology analyser-Sysmex-Kx-2I. Blast cell percentage was estimated on Giemsa stained blood smears. Immuno phenotyping was done on bone marrow samples by 5 colour flow cytometery on Beckman Counter Navious Flow cytometer. An acute leukemia panel of 23 antibodies was used. The data was entered and analyzed in SPSS version 22. Results: Of the 50 ALL patients, 36 (72 percentage) were B-ALL and 14 (28 percentage) T-ALL. There were 18 (36 percentage) children and 32 (64 percentage) adults. T-ALL included 22 percentage of the childhood and 31 percentage of the adult cases. Immuno phenotypic analysis showed that CD19, CD79a and CD20 were B-lineage specific markers whereas cCD3, CD3 and CD5 were T-lineage specific. CD10 was the most sensitive marker for B-ALL and CD7 was the most sensitive marker of T-ALL. TdT was expressed in 92 percentage B-ALL and 71 percentage T-ALL cases, CD34 in 58 percentage and 43 percentage cases and CD45 in 83 percentage and 100 percentage respectively. High leukocyte count (> 50 x 109/L) was present in 58 percentage cases. Hemoglobin was < 10 g/dl in 74 percentage patients and platelet count was below 20 x 109/Lin 12 percentage patients. Leukocyte count, hemoglobin, platelet count and blast cell percentage did not show a significant difference in the two ALL immuno types. Conclusion: The frequency of T-ALL is higher in childhood

  13. The systems biology of MHC class II antigen presentation

    NARCIS (Netherlands)

    Paul, Petra

    2012-01-01

    Major histocompatibility class II molecules (MHC class II) are one of the key regulators of adaptive immunity because of their specific expression by professional antigen presenting cells (APC). They present peptides derived from endocytosed material to T helper lymphocytes. Consequently, MHC class

  14. Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

    International Nuclear Information System (INIS)

    Kato, H.; Torigoe, T.

    1977-01-01

    A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with other carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All patients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care

  15. Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

    Science.gov (United States)

    Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

    1984-01-01

    HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

  16. Corruption of dendritic cell antigen presentation during acute GVHD leads to regulatory T-cell failure and chronic GVHD.

    Science.gov (United States)

    Leveque-El Mouttie, Lucie; Koyama, Motoko; Le Texier, Laetitia; Markey, Kate A; Cheong, Melody; Kuns, Rachel D; Lineburg, Katie E; Teal, Bianca E; Alexander, Kylie A; Clouston, Andrew D; Blazar, Bruce R; Hill, Geoffrey R; MacDonald, Kelli P A

    2016-08-11

    Chronic graft-versus-host disease (cGVHD) is a major cause of late mortality following allogeneic bone marrow transplantation (BMT) and is characterized by tissue fibrosis manifesting as scleroderma and bronchiolitis obliterans. The development of acute GVHD (aGVHD) is a powerful clinical predictor of subsequent cGVHD, suggesting that aGVHD may invoke the immunologic pathways responsible for cGVHD. In preclinical models in which sclerodermatous cGVHD develops after a preceding period of mild aGVHD, we show that antigen presentation within major histocompatibility complex (MHC) class II of donor dendritic cells (DCs) is markedly impaired early after BMT. This is associated with a failure of regulatory T-cell (Treg) homeostasis and cGVHD. Donor DC-restricted deletion of MHC class II phenocopied this Treg deficiency and cGVHD. Moreover, specific depletion of donor Tregs after BMT also induced cGVHD, whereas adoptive transfer of Tregs ameliorated it. These data demonstrate that the defect in Treg homeostasis seen in cGVHD is a causative lesion and is downstream of defective antigen presentation within MHC class II that is induced by aGVHD. © 2016 by The American Society of Hematology.

  17. Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

    Directory of Open Access Journals (Sweden)

    Khetam Ghannam

    Full Text Available OBJECTIVE: In idiopathic inflammatory myopathies (IIM infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. METHODS: Expression of constitutive (PSMB5, -6, -7 and corresponding immunoproteasomal subunits (PSMB8, -9, -10 was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM and healthy donors (HD. Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. RESULTS: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10 in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. CONCLUSIONS: Immunoproteasomes seem to indicate

  18. Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

    Science.gov (United States)

    Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

    2014-01-01

    Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and

  19. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  20. The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

    Science.gov (United States)

    Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

    2015-05-19

    The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. A novel strategy to improve antigen presentation for active immunotherapy in cancer. Fusion of the human papillomavirus type 16 E7 antigen to a cell penetrating peptide

    International Nuclear Information System (INIS)

    Granadillo, Milaid; Torrens, Isis; Guerra, Maribel

    2012-01-01

    Facilitating the delivery of exogenous antigens to antigen-presenting cells, ensuing processing and presentation via the major histocompatibility complex class I and induction of an effective immune response are fundamental for an effective therapeutic cancer vaccine. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel cell-penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus anti-lipopolysaccharide factor protein (LALF 32-51 ) linked to human papillomavirus 16 E7 antigen (LALF 32-51 -E7). In this work, we demonstrated that the immunization with LALF 32-51 -E7 using the TC-1 mouse model induces a potent and long-lasting anti-tumor response supported on an effective E7-specific CD8 +T -cell response. The finding that therapeutic immunization with LALF 32-51 or E7 alone, or an admixture of LALF32-51 and E7, does not induce significant tumor reduction indicates that covalent linkage between LALF 32-51 and E7 is required for the anti-tumor effect. These results support the use of this novel cell-penetrating peptide as an efficient means for delivering therapeutic targets into cellular compartments with the induction of a cytotoxic CD8 +T lymphocyte immune response. This approach is promissory for the treatment of tumors associated with the human papillomavirus 16, which is responsible for the 50% of cervical cancer cases worldwide and other malignancies. Furthermore, protein-based vaccines can circumvent the major histocompatibility complex specificity limitation associated with peptide vaccines providing a greater extent in their application

  2. Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

    Directory of Open Access Journals (Sweden)

    Coukos George

    2011-08-01

    Full Text Available Abstract Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.

  3. Antigen Presentation Keeps Trending in Immunotherapy Resistance.

    Science.gov (United States)

    Kalbasi, Anusha; Ribas, Antoni

    2018-04-19

    Through a gain-of-function kinome screen, MEX3B was identified as a mediator of resistance to T-cell immunotherapy not previously identified using CRISPR-based screens. MEX3B is a posttranscriptional regulator of HLA-A, validating the critical role of tumor-intrinsic antigen presentation in T-cell immunotherapy and indicating a new putative molecular target. Clin Cancer Res; 24(14); 1-3. ©2018 AACR. See related article by Huang et al., p. xxxx . ©2018 American Association for Cancer Research.

  4. Generation and characterization of antigenic variants induced by exposure of tumor cells to UV radiation in vitro

    International Nuclear Information System (INIS)

    Hostetler, L.L.W.

    1988-01-01

    Antigenic changes present in nonantigenic tumor cells exposed to UV radiation (UV) in vitro were investigated by addressing the following questions: (1) Are antigenic variants (AV) produced that are rejected in normal but not immunosuppressed mice? (2) Does generation of AV depend upon intrinsic properties of the cells exposed or result from the action of UV? (3) Is antigenic modification induced by UV due to increased histocompatibility antigen expression? (4) Do AV crossreact immunologically with parental tumor or with other AV? and (5) Is the UV-associated common antigen expressed on UV-induced tumors present on UV-irradiated tumor cells? AV were generated at different frequencies following in vitro UV irradiation of a spontaneous murine fibrosarcoma, a murine melanoma, and two melanoma clones. Immunological experiments demonstrated that the AV and parental cells shared a determinant that was susceptible to immune recognition, but incapable of inducing immunity. In contrast, the AV were noncrossreactive, suggesting that variant-specific antigens were also expressed. Finally, the AV were recognized by UV-induced suppressor cells, indicating that the UV-associated common antigen expressed by UV-induced tumors was also present

  5. Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen.

    Science.gov (United States)

    Abos Gracia, Beatriz; López Relaño, Juan; Revilla, Ana; Castro, Lourdes; Villalba, Mayte; Martín Adrados, Beatriz; Regueiro, Jose Ramon; Fernández-Malavé, Edgar; Martínez Naves, Eduardo; Gómez Del Moral, Manuel

    2017-01-01

    Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses. © 2017 S. Karger AG, Basel.

  6. Effects of low dose X-ray irradiation on antigen presentation and IL-12 secretion in human dendritic cells in vitro

    International Nuclear Information System (INIS)

    Yan Peng; Jiang Qisheng; Li Fengsheng; He Rui; Wang Cuilan; Li Xiao

    2012-01-01

    Objective: To explore the effects of low dose X-ray irradiation on the ability of antigen presentation and IL-12 secretion in human dendritic cells that had been cultured for different time in vitro. Methods: The human peripheral blood mononuclear cells (PBMC) were collected and differentiated to dendritic cells (DCs) by rhGM-CSF and rhIL-4 treatment in vitro. The DCs were divided into 3 groups, group A: DCs were cultured for 2 d and then irradiated with 0.05, 0.1, 0.2 and 0.5 Gy X-rays; group B: DCs were cultured for 6 d and then irradiated as above; group C:DCs were cultured without irradiation.At 8 d of cell culture, the DCs were applied to activate T cells and CCK-8 was used to detect MLR (mixed lymphocyte reaction), and the antigen presentation ability of DCs was evaluated. MTT assay was also used to test the cell-killing effect of the activated T-cells on A549 cells. IL-12 in the culture medium of DCs was detected by ELISA. Results: After irradiation with 0.2 and 0.5 Gy X-rays, the antigen presentation ability of DCs was decreased in group A (t=2.79 and 3.71, P<0.05), but significantly increased in group B (t=3.60 and 3.11, P<0.05). The ability of the T cell activation was detected and the proliferation of A549 cells was slightly inhibited by the DCs in group A (t=2.89 and 2.91, P<0.05), but was obviously inhibited by the DCs in group B (t=2.91 and 2.82, P<0.05). Meanwhile,the level of IL-12 was dramatically decreased in group A (t=4.44 and 6.93, P<0.05), but was increased in group B (t=3.51 and 4.12, P<0.05). Conclusions: The abilities of antigen presentation and proliferation inhibition of DCs could be down-regulated by low dose (<0.5 Gy) of X-ray irradiation at the early stage of DCs, but was up-regulated at the late stage of DCs culture. (authors)

  7. Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

    International Nuclear Information System (INIS)

    Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

    1986-01-01

    In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

  8. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.

    Science.gov (United States)

    Herce, Henry D; Schumacher, Dominik; Schneider, Anselm F L; Ludwig, Anne K; Mann, Florian A; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M Cristina; Hackenberger, Christian P R

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  9. Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

    Science.gov (United States)

    Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

    2017-08-01

    Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

  10. Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

    Science.gov (United States)

    Zhu, Haiyan; Fang, Xiaoyun; Zhang, Dongmei; Wu, Weicheng; Shao, Miaomiao; Wang, Lan; Gu, Jianxin

    2016-01-01

    Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

  11. Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral-Antigen Pathway.

    Science.gov (United States)

    Hewitt, Rachel E; Robertson, Jack; Haas, Carolin T; Pele, Laetitia C; Powell, Jonathan J

    2017-01-01

    Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer's patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4 + T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4 + T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen-PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer's patch T cell responses.

  12. Minimum information about tolerogenic antigen-presenting cells (MITAP) : a first step towards reproducibility and standardisation of cellular therapies

    NARCIS (Netherlands)

    Lord, Phillip; Aguillon, Juan C; Anderson, Amy E; Appel, Silke; Benitez-Ribas, Daniel; Ten Brinke, Anja; Broere, Femke; Cools, Nathalie; Cuturi, Maria Cristina; Diboll, Julie; Geissler, Edward K; Giannoukakis, Nick; Gregori, Silvia; van Ham, S Marieke; Lattimer, Staci; Marshall, Lindsay; Harry, Rachel A; Hutchinson, James A; Isaacs, John D; Joosten, Irma; van Kooten, Cees; Lopez Diaz de Cerio, Ascension; Nikolic, Tatjana; Oral, Haluk Barbaros; Sofronic-Milosavljevic, Ljiljana; Ritter, Thomas; Riquelme, Paloma; Thomson, Angus W; Trucco, Massimo; Vives-Pi, Marta; Martinez-Caceres, Eva M; Hilkens, Catharien M U

    2016-01-01

    Cellular therapies with tolerogenic antigen-presenting cells (tolAPC) show great promise for the treatment of autoimmune diseases and for the prevention of destructive immune responses after transplantation. The methodologies for generating tolAPC vary greatly between different laboratories, making

  13. Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self-antigens in cancer patients.

    Science.gov (United States)

    Morse, Michael A; Chapman, Robert; Powderly, John; Blackwell, Kimberly; Keler, Tibor; Green, Jennifer; Riggs, Renee; He, Li-Zhen; Ramakrishna, Venky; Vitale, Laura; Zhao, Biwei; Butler, Stephen A; Hobeika, Amy; Osada, Takuya; Davis, Thomas; Clay, Timothy; Lyerly, H Kim

    2011-07-15

    The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self-antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. Two phase I studies were conducted with CDX-1307, a vaccine composed of human chorionic gonadotropin beta-chain (hCG-β) fused to an MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose escalation of single-agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and the Toll-like receptor (TLR) 3 agonist polyinosinic-polycytidylic acid (poly-ICLC) and TLR7/8 agonist resiquimod to activate the APC. CDX-1307 induced consistent humoral and T-cell responses to hCG-β when coadministered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and nonelevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. APC targeting and activation induce adaptive immunity against poorly immunogenic self-antigens which has implications for enhancing the efficacy of cancer immunotherapy.

  14. Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines

    Science.gov (United States)

    Lee Szeto, Gregory; van Egeren, Debra; Worku, Hermoon; Sharei, Armon; Alejandro, Brian; Park, Clara; Frew, Kirubel; Brefo, Mavis; Mao, Shirley; Heimann, Megan; Langer, Robert; Jensen, Klavs; Irvine, Darrell J.

    2015-05-01

    B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

  15. Isolation of a peptide binding protein and its role in antigen presentation

    International Nuclear Information System (INIS)

    Lakey, E.; Pierce, S.K.; Margoliash, E.

    1986-01-01

    A mouse T cell hybrid, TPc9.1, recognizes pigeon cytochrome c (Pc) as processed and presented by histocompatible antigen presenting cells (APC). When paraformaldehyde fixed APC are employed, only a peptide fragment of Pc, Pc 81-104, and not the native Pc, is capable of stimulating TPc9.1 cells. Pc 81-104 appears to associate tightly with the APC surface since paraformaldehyde fixed APC which have been incubated with Pc 81-104 remain stimulatory following extensive washing. When APC are surface labeled with 125 I, solubilized and affinity purified on Pc 81-104-Sepharose 4B columns, two predominant polypeptides of approximately 72 and 74 kd are isolated. Little or no immunoglobulin, Class I or Class II proteins are obtained under these conditions. Antisera from rabbits immunized with the affinity purified material, but not preimmune sera, block the activation of TPc 9.1 cells by Pc as well as Pc 81-104 when presented by live APC. Furthermore, these antisera are even more effective in blocking the activation of TPc9.1 cells by either APC which had been pulsed with Pc and then paraformaldehyde fixed, or by Pc 81-104 when added to paraformaldehyde fixed APC, suggesting that these antisera were not affecting antigen processing. Thus, these peptide binding proteins may play a role in antigen presentation, and they are being further characterized

  16. Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

    International Nuclear Information System (INIS)

    Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

    1982-01-01

    Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants

  17. EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens.

    Science.gov (United States)

    Squadrito, Mario Leonardo; Cianciaruso, Chiara; Hansen, Sarah K; De Palma, Michele

    2018-03-01

    We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8 + T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

  18. Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral–Antigen Pathway

    Science.gov (United States)

    Hewitt, Rachel E.; Robertson, Jack; Haas, Carolin T.; Pele, Laetitia C.; Powell, Jonathan J.

    2017-01-01

    Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer’s patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4+ T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4+ T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen–PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer’s patch T cell responses. PMID:28367148

  19. Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

    Directory of Open Access Journals (Sweden)

    Taguchi Hiroaki

    2011-08-01

    Full Text Available Abstract Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens, carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1 the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2 synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.

  20. Carbohydrates as T-cell antigens with implications in health and disease.

    Science.gov (United States)

    Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

    2016-10-01

    Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation.

    Science.gov (United States)

    Imai, Jun; Otani, Mayu; Sakai, Takahiro; Hatta, Shinichi

    2017-08-21

    Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of naïve CD8 + T cells and memory CD8 + T cells for infectious and tumor immunity but also in the inactivation of self-acting naïve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

  2. Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity.

    Science.gov (United States)

    Almeida, Jorge R; Sauce, Delphine; Price, David A; Papagno, Laura; Shin, So Youn; Moris, Arnaud; Larsen, Martin; Pancino, Gianfranco; Douek, Daniel C; Autran, Brigitte; Sáez-Cirión, Asier; Appay, Victor

    2009-06-18

    CD8(+) T cells are major players in the immune response against HIV. However, recent failures in the development of T cell-based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell-mediated efficacy. CD8(+) T cells from HIV-1-infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8(+) T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8(+) T cells from infected donors. We report that attributes of CD8(+) T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8(+) T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8(+) T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.

  3. Distinct Gut-Derived Bacteria Differentially Affect Three Types of Antigen-Presenting Cells and Impact on NK- and T-Cell Responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Hansen, Anne Marie Valentin; Frøkiær, Hanne

    Objectives Gut bacteria are assumed essential for development and maintenance of a balanced immune system. Specifically, stimulation of antigen-presenting cells (APCs) by gut bacteria is important for polarisation of the immune response. This experiment was designed to reveal similarities...... and differences between the reaction patterns of three types of human APCs when stimulated with intestinal bacteria. Furthermore, the effect of these APCs on NK-cells and T-cells was examined. Methodology The APCs used in this study were blood monocytes, blood dendritic cells, and dendritic cells differentiated...... from monocytes. Monocyte-derived dendritic cells constitute a commonly used model of dendritic cell function. The APCs were cultured for 18 h with four different gut bacteria: Lactobacillus acidophilus X37, Lactobacillus reuteri DSM 12246, E. coli Nissle 1917 or Bifidobacterium longum Q46. Results...

  4. Effect of BSA Antigen Sensitization during the Acute Phase of Influenza A Viral Infection on CD11c+ Pulmonary Antigen Presenting Cells

    Directory of Open Access Journals (Sweden)

    Fumitaka Sato

    2009-01-01

    Conclusions: BSA antigen sensitization during the acute phase of influenza A viral infection enhanced IL-10 production from naive CD4+ T cell interaction with CD11c+ pulmonary APCs. The IL-10 secretion evoked Th2 responses in the lungs with downregulation of Th1 responses and was important for the eosinophil recruitment into the lungs after BSA antigen challenge.

  5. Recipient dendritic cells, but not B cells, are required antigen-presenting cells for peripheral alloreactive CD8+ T-cell tolerance.

    Science.gov (United States)

    Mollov, J L; Lucas, C L; Haspot, F; Gaspar, J Kurtz C; Guzman, A; Sykes, M

    2010-03-01

    Induction of mixed allogeneic chimerism is a promising approach for achieving donor-specific tolerance, thereby obviating the need for life-long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti-CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T-cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient-type B cells from chimeras that were tolerant to donor still promoted CD8 T-cell tolerance, but their role could not be replaced by donor-type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL-10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.

  6. Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

    Science.gov (United States)

    Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

    2015-05-01

    Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

  7. Unusual antigen presentation offers new insight into HIV vaccine design.

    Science.gov (United States)

    McMichael, Andrew J; Picker, Louis J

    2017-06-01

    Recent findings with a rhesus monkey cytomegalovirus based simian immunodeficiency virus vaccine have identified strong CD8+ T cell responses that are restricted by MHC-E. Also mycobacteria specific CD8+ T cells, that are MHC-E restricted, have been identified. MHC-E therefore can present a wide range of epitope peptides to CD8+ T cells, alongside its well defined role in presenting a conserved MHC-class I signal peptide to the NKG2A/C-CD94 receptor on natural killer cells. Here we explore the antigen processing pathways involved in these atypical T cell responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Large, but not small, antigens require time- and temperature-dependent processing in accessory cells before they can be recognized by T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    We have studied if antigens of different size and structure all require processing in antigen-presenting cells of guinea-pigs before they can be recognized by T cells. The method of mild paraformaldehyde fixation was used to stop antigen-processing in the antigen-presenting cells. As a measure...... of antigen presentation we used the proliferative response of appropriately primed T cells during a co-culture with the paraformaldehyde-fixed and antigen-exposed presenting cells. We demonstrate that the large synthetic polypeptide antigen, dinitrophenyl-poly-L-lysine, requires processing. After an initial......-dependent and consequently energy-requiring. Processing is strongly inhibited by the lysosomotrophic drug, chloroquine, suggesting a lysosomal involvement in antigen processing. The existence of a minor, non-lysosomal pathway is suggested, since small amounts of antigen were processed even at 10 degrees C, at which...

  9. B7.1 expression on tumor cells circumvents the need of professional antigen presentation for in vitro propagation of cytotoxic T cell lines.

    Science.gov (United States)

    Iezzi, G; Protti, M P; Rugarli, C; Bellone, M

    1996-01-01

    In vitro propagation of tumor-specific CTLs, to be used for identification of tumor antigens (Ag) and/or adoptive immunotherapy, is hampered by the need of large amounts of professional antigen-presenting cells (APC) used for periodical cycles of restimulation. We evaluated whether RMA T lymphoma cells, stably transfected with the cDNA encoding for the B7.1 costimulatory molecule, provided the activation signals to CD8+ T lymphocytes in the absence of professional APC and CD4+ helper cells. We demonstrate here that long-term CD8+ cell lines can be efficiently propagated in vitro by repeated cycles of stimulation with tumor cells stably expressing B7.1. Professional APC and CD4+ helper cells are not required as far as interleukin 2 is exogenously provided. Furthermore, CD8+ blasts needed both signal 1 (Ag in the contest of the MHC molecule) and signal 2 (interaction of costimulatory molecules) for restimulation. T cell blasts in the presence of signal 1 or 2 only still retained their effector potential but did not undergo clonal expansion. These results are very promising for further applications of specific immunotherapies in humans.

  10. An Antigen-Presenting and Apoptosis-Inducing Polymer Microparticle Prolongs Alloskin Graft Survival by Selectively and Markedly Depleting Alloreactive CD8+ T Cells

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2017-06-01

    Full Text Available Selectively depleting the pathogenic T cells is a fundamental strategy for the treatment of allograft rejection and autoimmune disease since it retains the overall immune function of host. The concept of killer artificial antigen-presenting cells (KaAPCs has been developed by co-coupling peptide–major histocompatibility complex (pMHC multimer and anti-Fas monoclonal antibody (mAb onto the polymeric microparticles (MPs to induce the apoptosis of antigen-specific T cells. But little information is available about its in vivo therapeutic potential and mechanism. In this study, polyethylenimine (PEI-coated poly lactic-co-glycolic acid microparticle (PLGA MP was fabricated as a cell-sized scaffold to covalently co-couple H-2Kb-Ig dimer and anti-Fas mAb for the generation of alloantigen-presenting and apoptosis-inducing MPs. Intravenous infusions of the biodegradable KaAPCs prolonged the alloskin graft survival for 43 days in a single MHC-mismatched murine model, depleted the most of H-2Kb-alloreactive CD8+ T cells in peripheral blood, spleen, and alloskin graft in an antigen-specific manner and anti-Fas-dependent fashion. The cell-sized KaAPCs circulated throughout vasculature into liver, kidney, spleen, lymph nodes, lung, and heart, but few ones into local allograft at early stage, with a retention time up to 36 h in vivo. They colocalized with CD8+ T cells in secondary lymphoid organs while few ones contacted with CD4+ T cells, B cells, macrophage, and dendritic cells, or internalized by phagocytes. Importantly, the KaAPC treatment did not significantly impair the native T cell repertoire or non-pathogenic immune cells, did not obviously suppress the overall immune function of host, and did not lead to visible organ toxicity. Our results strongly document the high potential of PLGA MP-based KaAPCs as a novel antigen-specific immunotherapy for allograft rejection and autoimmune disorder. The in vivo mechanism of alloinhibition, tissue

  11. Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

    2004-01-01

    's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture......B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto...

  12. Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Hegedüs, Laszlo; Leslie, Robert Graham Quinton

    2004-01-01

    B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto......'s thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture...

  13. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

    Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

  14. Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

    Science.gov (United States)

    Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

    2012-04-01

    Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  15. Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P

    2012-10-01

    Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

  16. Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

    Directory of Open Access Journals (Sweden)

    Warren L Denning

    2011-02-01

    Full Text Available Harnessing the ability of cytotoxic T lymphocytes (CTLs to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

  17. Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

    Directory of Open Access Journals (Sweden)

    Shigeo Koido

    2010-01-01

    Full Text Available The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs that can reduce the tumor mass. Dendritic cells (DCs are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

  18. Enteroantigen-presenting B cells efficiently stimulate CD4(+) T cells in vitro

    DEFF Research Database (Denmark)

    Schmidt, Esben Gjerløff Wedebye; Kristensen, Nanna Ny; Claesson, Mogens Helweg

    2011-01-01

    Presentation of enterobacterial antigens by antigen-presenting cells and activation of enteroantigen-specific CD4(+) T cells are considered crucial steps in inflammatory bowel disease (IBD) pathology. The detrimental effects of such CD4(+) T cells have been thoroughly demonstrated in models...... of colitis. Also, we have previously established an in vitro assay where murine enteroantigen-specific colitogenic CD4(+) CD25(-) T cells are activated by splenocytes pulsed with an enterobacterial extract....

  19. Antigen Presenting Cells and Stromal Cells Trigger Human Natural Killer Lymphocytes to Autoreactivity: Evidence for the Involvement of Natural Cytotoxicity Receptors (NCR and NKG2D

    Directory of Open Access Journals (Sweden)

    Alessandro Poggi

    2006-01-01

    Full Text Available Human natural killer (NK lymphocytes should not damage autologous cells due to the engagement of inhibitory receptor superfamily (IRS members by HLA-I. Nevertheless, NK cells kill self cells expressing low levels or lacking HLA-I, as it may occur during viral infections (missing-self hypothesis. Herein, we show that human NK cells can be activated upon binding with self antigen presenting cells or stromal cells despite the expression of HLA-I. Indeed, NK cells can kill and produce pro-inflammatory and regulating cytokines as IFN-γ, TNF-α and IL10 during interaction with autologous dendritic cells or bone marrow stromal cells or skin fibroblasts. The killing of antigen presenting and stromal cells is dependent on LFA1/ICAM1 interaction. Further, the natural cytotoxicity receptors (NCR NKp30 and NKp46 are responsible for the delivery of lethal hit to DC, whereas NKG2D activating receptor, the ligand of the MHC-related molecule MIC-A and the UL16 binding protein, is involved in stromal cell killing. These findings indicate that different activating receptors are involved in cell to self cell interaction. Finally, NK cells can revert the veto effect of stromal cells on mixed lymphocyte reaction further supporting the idea that NK cells may alter the interaction between T lymphocytes and microenvironment leading to autoreactivity.

  20. Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen: Involvement of marginal metallophilic macrophages in the uptake of immune-stimulating complexes

    NARCIS (Netherlands)

    Claassen, I.J.T.M.; Osterhaus, A.D.M.E.; Claassen, E.

    1995-01-01

    Several mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system. However, until

  1. Antigen detection in vivo after immunization with different presentation forms of rabies virus antigen: involvement of marginal metallophilic macrophages in the uptake of immune-stimulating complexes.

    NARCIS (Netherlands)

    I.J.Th.M. Claassen (Ivo); A.D.M.E. Osterhaus (Albert); H.J.H.M. Claassen (Eric)

    1995-01-01

    textabstractSeveral mechanisms have been postulated to explain the relatively high immunogenicity of antigens presented in immune-stimulating complexes (iscom). Their potency can in part be explained by the specific targeting of these structures to cells presenting antigens to the immune system.

  2. Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

    Science.gov (United States)

    Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

    2013-01-01

    In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

  3. Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

    Science.gov (United States)

    Wilson, John T; Postma, Almar; Keller, Salka; Convertine, Anthony J; Moad, Graeme; Rizzardo, Ezio; Meagher, Laurence; Chiefari, John; Stayton, Patrick S

    2015-03-01

    Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell

  4. Cell Wall-Associated Protein Antigens of Streptococcus salivarius: Purification, Properties, and Function in Adherence

    OpenAIRE

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel el...

  5. Interaction of Cowpea Mosaic Virus (CPMV) Nanoparticles with Antigen Presenting Cells In Vitro and In Vivo

    Science.gov (United States)

    Rae, Chris S.; Manchester, Marianne

    2009-01-01

    Background Plant viruses such as Cowpea mosaic virus (CPMV) are increasingly being developed for applications in nanobiotechnology including vaccine development because of their potential for producing large quantities of antigenic material in plant hosts. In order to improve efficacy of viral nanoparticles in these types of roles, an investigation of the individual cell types that interact with the particles is critical. In particular, it is important to understand the interactions of a potential vaccine with antigen presenting cells (APCs) of the immune system. CPMV was previously shown to interact with vimentin displayed on cell surfaces to mediate cell entry, but the expression of surface vimentin on APCs has not been characterized. Methodology The binding and internalization of CPMV by several populations of APCs was investigated both in vitro and in vivo by flow cytometry and fluorescence confocal microscopy. The association of the particles with mouse gastrointestinal epithelium and Peyer's patches was also examined by confocal microscopy. The expression of surface vimentin on APCs was also measured. Conclusions We found that CPMV is bound and internalized by subsets of several populations of APCs both in vitro and in vivo following intravenous, intraperitoneal, and oral administration, and also by cells isolated from the Peyer's patch following gastrointestinal delivery. Surface vimentin was also expressed on APC populations that could internalize CPMV. These experiments demonstrate that APCs capture CPMV particles in vivo, and that further tuning the interaction with surface vimentin may facilitate increased uptake by APCs and priming of antibody responses. These studies also indicate that CPMV particles likely access the systemic circulation following oral delivery via the Peyer's patch. PMID:19956734

  6. Diversity of natural self-derived ligands presented by different HLA class I molecules in transporter antigen processing-deficient cells.

    Directory of Open Access Journals (Sweden)

    Elena Lorente

    Full Text Available The transporter associated with antigen processing (TAP translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P(1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.

  7. Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

  8. Effect of cold nerve allograft preservation on antigen presentation and rejection

    Science.gov (United States)

    Ray, Wilson Z.; Kale, Santosh S.; Kasukurthi, Rahul; Papp, Esther M.; Johnson, Philip J.; Santosa, Katherine B.; Yan, Ying; Hunter, Daniel A.; Mackinnon, Susan E.; Tung, Thomas H.

    2010-01-01

    Object Nerve allotransplantation provides a temporary scaffold for host nerve regeneration and allows for the reconstruction of significant segmental nerve injuries. The need for systemic the current clinical utilization of nerve allografts, although this need is reduced by the practice of cold nerve allograft preservation. Activation of T cells in response to alloantigen presentation occurs in the context of donor antigen presenting cells (direct pathway) or host antigen-presenting cells (indirect pathway). The relative role of each pathway in eliciting an alloimmune response and its potential for rejection of the nerve allograft model has not previously been investigated. The objective of this investigation was to study the effect of progressive periods of cold nerve allograft preservation on antigen presentation and the alloimmune response. Methods The authors used wild type C57Bl/6 (B6), BALB/c, and major histocompatibility Class II–deficient (MHC−/−) C57Bl/6 mice as both nerve allograft recipients and donors. A nonvascularized nerve allograft was used to reconstruct a 1-cm sciatic nerve gap. Progressive cold preservation of donor nerve allografts was used. Quantitative assessment was made after 3 weeks using nerve histomorphometry. Results The donor-recipient combination lacking a functional direct pathway (BALB/c host with MHC−/− graft) rejected nerve allografts as vigorously as wild-type animals. Without an intact indirect pathway (MHC−/− host with BALB/c graft), axonal regeneration was improved (p < 0.052). One week of cold allograft preservation did not improve regeneration to any significant degree in any of the donor-recipient preservation did improve regeneration significantly (p < 0.05) for all combinations compared with wild-type animals without pretreatment. However, only in the presence of an intact indirect pathway (no direct pathway) did 4 weeks of cold preservation improve regeneration significantly compared with 1 week and no

  9. Replication-deficient mutant Herpes Simplex Virus-1 targets professional antigen presenting cells and induces efficient CD4+ T helper responses.

    Science.gov (United States)

    Fiorentini, Simona; Marconi, Peggy; Avolio, Manuela; Marini, Elena; Garrafa, Emirena; Caracciolo, Sonia; Rossi, Daniele; Bozac, Alexandra; Becker, Pablo D; Gentili, Francesca; Facchetti, Fabio; Guzman, Carlos A; Manservigi, Roberto; Caruso, Arnaldo

    2007-07-01

    Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generated a strong p17-specific CD4+ T helper response preceding both p17-specific humoral and effector T cell responses. Moreover, we show that T0-p17 infection did not interfere with the endogenous processing of the transgene encoded antigen, since infected APCs were able to evoke a strong recall response in vitro. Our results demonstrate that replication-deficient HSV vectors can be appealing candidates for the development of vaccines able to trigger T helper responses.

  10. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

    Directory of Open Access Journals (Sweden)

    Kerstin Trautwein-Weidner

    2015-10-01

    Full Text Available Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

  11. CD1d-mediated presentation of endogenous lipid antigens by adipocytes requires microsomal triglyceride transfer protein (MTP)

    DEFF Research Database (Denmark)

    Rakhshandehroo, Maryam; Gijzel, Sanne M W; Siersbæk, Rasmus

    2014-01-01

    -dependent fashion, but little is known about the lipid antigen presentation machinery in adipocytes. Here we show that CD1d, as well as the lipid antigen loading machinery genes pro-saposin (Psap), Niemann Pick type C2 (Npc2), α-galactosidase (Gla), are upregulated in early adipogenesis, and are transcriptionally...... presenting cells (APCs), which may present an important aspect of adipocyte-immune cell communication in the regulation of whole body energy metabolism and immune homeostasis....

  12. An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

    Science.gov (United States)

    Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

    1989-01-01

    A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

  13. Pattern of distribution of blood group antigens on human epidermal cells during maturation

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Buschard, Karsten; Hakomori, Sen-Itiroh

    1984-01-01

    The distribution in human epidermis of A, B, and H blood group antigens and of a precursor carbohydrate chain, N-acetyl-lactosamine, was examined using immunofluorescence staining techniques. The material included tissue from 10 blood group A, 4 blood group B, and 9 blood group O persons. Murine...... on the lower spinous cells whereas H antigen was seen predominantly on upper spinous cells or on the granular cells. Epithelia from blood group A or B persons demonstrated A or B antigens, respectively, but only if the tissue sections were trypsinized before staining. In such cases A or B antigens were found...... monoclonal antibodies were used to identify H antigen (type 2 chain) and N-acetyl-lactosamine. Human antisera were used to identify A and B antigens. In all groups N-acetyl-lactosamine and H antigen were found on the cell membranes of the spinous cell layer. N-acetyl-lactosamine was present mainly...

  14. Role of antigen in migration patterns of T cell subsets arising from gut-associated lymphoid tissue

    International Nuclear Information System (INIS)

    Dunkley, M.L.; Husband, A.J.

    1989-01-01

    Studies of the migration of antigen-specific regulatory T cell subsets responding to gut immunization were undertaken to clarify their migratory potential and the role of antigen in their localization. In initial experiments, lymphocytes collected from the thoracic duct of rats after immunization of Peyer's patches (PP) with keyhole limpet hemocyanin (KLH), were enriched for T helper (Th) cells and labelled with the fluorochrome H33342. In other experiments, a higher frequency of antigen-specific T cells was achieved by short-term culture of the enriched Th cells in the presence of KLH and the blast cells labelled with 3H-thymidine. The distribution of both populations was determined after injection into immunized and unimmunized syngeneic recipients. Whereas the uncultured population (predominantly small Th cells) localized almost exclusively in follicular lymphoid tissues, the cells expanded by secondary culture (predominantly Th blasts) appeared in the gut lamina propria (LP) initially, then in PP and mesenteric lymph nodes. The Th blasts in the LP were almost always seen in close proximity to the gut epithelium. However, the migration of neither population appeared to be influenced significantly by antigen, in contrast to previous findings with regard to IgA-committed B cells. The initial subepithelial location of Th blasts in the gut LP and their subsequent appearance in PP may provide a mechanism by which antigen presented by epithelial cells could influence B cell differentiation in PP through modulation of signals expressed by these T cells

  15. Role of antigen in migration patterns of T cell subsets arising from gut-associated lymphoid tissue

    Energy Technology Data Exchange (ETDEWEB)

    Dunkley, M.L.; Husband, A.J. (Univ. of Newcastle, N.S.W. (Australia))

    1989-07-01

    Studies of the migration of antigen-specific regulatory T cell subsets responding to gut immunization were undertaken to clarify their migratory potential and the role of antigen in their localization. In initial experiments, lymphocytes collected from the thoracic duct of rats after immunization of Peyer's patches (PP) with keyhole limpet hemocyanin (KLH), were enriched for T helper (Th) cells and labelled with the fluorochrome H33342. In other experiments, a higher frequency of antigen-specific T cells was achieved by short-term culture of the enriched Th cells in the presence of KLH and the blast cells labelled with 3H-thymidine. The distribution of both populations was determined after injection into immunized and unimmunized syngeneic recipients. Whereas the uncultured population (predominantly small Th cells) localized almost exclusively in follicular lymphoid tissues, the cells expanded by secondary culture (predominantly Th blasts) appeared in the gut lamina propria (LP) initially, then in PP and mesenteric lymph nodes. The Th blasts in the LP were almost always seen in close proximity to the gut epithelium. However, the migration of neither population appeared to be influenced significantly by antigen, in contrast to previous findings with regard to IgA-committed B cells. The initial subepithelial location of Th blasts in the gut LP and their subsequent appearance in PP may provide a mechanism by which antigen presented by epithelial cells could influence B cell differentiation in PP through modulation of signals expressed by these T cells.

  16. Axotomy induces MHC class I antigen expression on rat nerve cells

    DEFF Research Database (Denmark)

    Maehlen, J; Schröder, H D; Klareskog, L

    1988-01-01

    Immunomorphological staining demonstrates that class I major histocompatibility complex (MHC)-coded antigen expression can be selectively induced on otherwise class I-negative rat nerve cells by peripheral axotomy. Induction of class I as well as class II antigen expression was simultaneously seen...... on non-neural cells in the immediate vicinity of the injured nerve cells. As nerve regeneration after axotomy includes growth of new nerve cell processes and formation of new nerve cell contacts, the present findings raise the question of a role for MHC-coded molecules in cell-cell interactions during...... nerve cell growth....

  17. TNF-Induced Target Cell Killing by CTL Activated through Cross-Presentation

    Directory of Open Access Journals (Sweden)

    Dirk Wohlleber

    2012-09-01

    Full Text Available Viruses can escape cytotoxic T cell (CTL immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF, which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF.

  18. TNF-induced target cell killing by CTL activated through cross-presentation.

    Science.gov (United States)

    Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

    2012-09-27

    Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Co-delivery of PLGA encapsulated invariant NKT cell agonist with antigenic protein induce strong T cell-mediated antitumor immune responses

    NARCIS (Netherlands)

    Dolen, Y.; Kreutz, M.; Gileadi, U.; Tel, J.; Vasaturo, A.; Dinther, E.A.W. van; Hout-Kuijer, M.A. van; Cerundolo, V.; Figdor, C.G.

    2016-01-01

    Antitumor immunity can be enhanced by the coordinated release and delivery of antigens and immune-stimulating agents to antigen-presenting cells via biodegradable vaccine carriers. So far, encapsulation of TLR ligands and tumor-associated antigens augmented cytotoxic T cell (CTLs) responses. Here,

  20. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    International Nuclear Information System (INIS)

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-01-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

  1. A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

    Science.gov (United States)

    Miller, A

    1977-01-01

    The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

  2. Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

    Science.gov (United States)

    Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

    1987-12-01

    We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

  3. Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

    NARCIS (Netherlands)

    Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

    2012-01-01

    Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is

  4. Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells

    DEFF Research Database (Denmark)

    Jensen, A.M.; Llado, Minna Fyhn Lykke; Skov, L.

    1998-01-01

    The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo. Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified. We treated psoriatic patients...... psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen. Epidermal cell suspensions were analysed further by staining...... for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+). Flow cytometric analysis showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin. These results indicate that calcipotriol does not alter either the number of the function of epidermal antigen...

  5. Replication-deficient mutant Herpes Simplex Virus-1 targets professional antigen presenting cells and induces efficient CD4+ T helper responses.

    OpenAIRE

    Fiorentini, Simona; Marconi, Peggy; Avolio, Manuela; Marini, Elena; Garrafa, Emirena; Caracciolo, Sonia; Rossi, Daniele; Bozac, Alexandra; Becker, Pablo D; Gentili, Francesca; Facchetti, Fabio; Guzman, Carlos A; Manservigi, Roberto; Caruso, Arnaldo

    2007-01-01

    Both neutralizing antibodies and cytotoxic T-cells are necessary to control a viral infection. However, vigorous T helper responses are essential for their elicitation and maintenance. Here we show that a recombinant replication-deficient Herpes Simplex Virus (HSV)-1 vector encoding the Human Immunodeficiency Virus (HIV)-1 matrix protein p17 (T0-p17) was capable of infecting professional antigen presenting cells (APCs) in vitro and in vivo. The injection of T0-p17 in the mouse dermis generate...

  6. Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.; Morozov, Giora I.; Mage, Michael G.; Margulies, David H. (NIH); (Hebrew)

    2017-10-12

    Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

  7. Immune responses of dendritic cells after acquiring antigen from apoptotic hepatocholangioma cells caused by γ-ray

    International Nuclear Information System (INIS)

    Wu Gang; Gu Hongguang; Han Benli; Pei Xuetao

    2002-01-01

    Objective: To investigate the induction of cytotoxic T lymphocytes (CTLs) in antitumor responsiveness and therapeutic effects after dendritic cells (DCs) acquired antigen from apoptotic hepatocholangioma cells. Methods: DCs from blood mononuclear cells that maintain the characteristics of immaturity-anti-gen-capturing and-processing capacity were established in vitro by using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Then, apoptosis in hepatocholangioma cells was induced with γ-radiation. The experimental groups included (1) co-culture of DCs, and apoptotic cancer cells and T cells; (2) co-culture of DCs necrotic cancer cells and T cells; (3) co-culture of DCs-cultured cancer cell and T cells. These cells were co-cultured for 7 days. DCs and T cell were enriched separately. Finally, antitumor response test was carried out. Results: These cells had typical dendritic morphology, expressed high levels of CD1a, B7 and acquired antigen from apoptotic cells caused by γ-rays and induced an increased T cell-stimulatory capacity in MLR. Conclusions: DCs obtained from blood mononuclear cells using GM-CSF and IL-4 and DCs can efficiently present antigen driven from apoptotic cells caused by γ-rays and induce T cells increasing obviously. It can probably become an effective approach of DC transduction with antigen

  8. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  9. Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein.

    Science.gov (United States)

    Lorente, Elena; Barriga, Alejandro; García-Arriaza, Juan; Lemonnier, François A; Esteban, Mariano; López, Daniel

    2017-10-01

    The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

  10. Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction

    Science.gov (United States)

    Esterházy, Daria; Loschko, Jakob; London, Mariya; Jove, Veronica; Oliveira, Thiago Y.; Mucida, Daniel

    2016-01-01

    Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development. PMID:27019226

  11. Mature IgM-expressing plasma cells sense antigen and develop competence for cytokine production upon antigenic challenge

    Science.gov (United States)

    Blanc, Pascal; Moro-Sibilot, Ludovic; Barthly, Lucas; Jagot, Ferdinand; This, Sébastien; de Bernard, Simon; Buffat, Laurent; Dussurgey, Sébastien; Colisson, Renaud; Hobeika, Elias; Fest, Thierry; Taillardet, Morgan; Thaunat, Olivier; Sicard, Antoine; Mondière, Paul; Genestier, Laurent; Nutt, Stephen L.; Defrance, Thierry

    2016-01-01

    Dogma holds that plasma cells, as opposed to B cells, cannot bind antigen because they have switched from expression of membrane-bound immunoglobulins (Ig) that constitute the B-cell receptor (BCR) to production of the secreted form of immunoglobulins. Here we compare the phenotypical and functional attributes of plasma cells generated by the T-cell-dependent and T-cell-independent forms of the hapten NP. We show that the nature of the secreted Ig isotype, rather than the chemical structure of the immunizing antigen, defines two functionally distinct populations of plasma cells. Fully mature IgM-expressing plasma cells resident in the bone marrow retain expression of a functional BCR, whereas their IgG+ counterparts do not. Antigen boost modifies the gene expression profile of IgM+ plasma cells and initiates a cytokine production program, characterized by upregulation of CCL5 and IL-10. Our results demonstrate that IgM-expressing plasma cells can sense antigen and acquire competence for cytokine production upon antigenic challenge. PMID:27924814

  12. Evaluation of the ability of N-terminal fragment of lethal factor of Bacillus anthracis for delivery of Mycobacterium T cell antigen ESAT-6 into cytosol of antigen presenting cells to elicit effective cytotoxic T lymphocyte response

    International Nuclear Information System (INIS)

    Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi; Bhatnagar, Rakesh; Banerjee-Bhatnagar, Nirupama

    2006-01-01

    We report the ability of N-terminal fragment of lethal factor of Bacillus anthracis to deliver genetically fused ESAT-6 (early secretory antigen target), a potent T cell antigen of Mycobacterium tuberculosis, into cytosol to elicit Cytotoxic T lymphocyte (CTL) response. In vitro Th1 cytokines data and CTL assay proved that efficient delivery of LFn.ESAT-6 occurs in cytosol, in the presence of protective antigen (PA), and leads to generation of effective CTL response. Since CTL response is essential for protection against intracellular pathogens and, it is well known that only single T cell epitope or single antigenic protein is not sufficient to elicit protective CTL response due to variation or polymorphism in MHC-I alleles among the individuals, we suggest that as a fusion protein LFn can be used to deliver multiepitopes of T cells or multiproteins which can generate effective CTLs against intracellular pathogens like M. tuberculosis. It can be used to enhance the protective efficacy of BCG vaccine

  13. Antigen entrapped in the escheriosomes leads to the generation of CD4(+) helper and CD8(+) cytotoxic T cell response.

    Science.gov (United States)

    Syed, Faisal M; Khan, Masood A; Nasti, Tahseen H; Ahmad, Nadeem; Mohammad, Owais

    2003-06-02

    In previous study, we demonstrated the potential of Escherichia coli (E. coli) lipid liposomes (escheriosomes) to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells including professional antigen presenting cells. Our present study demonstrates that antigen encapsulated in escheriosomes could be successfully delivered simultaneously to the cytosolic as well as endosomal processing pathways of antigen presenting cells, leading to the generation of both CD4(+) T-helper and CD8(+) cytotoxic T cell response. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (egg PC) liposomes, just like antigen-incomplete Freund's adjuvant (IFA) complex, has inefficient access to the cytosolic pathway of MHC I-dependent antigen presentation and failed to generate antigen-specific CD8(+) cytotoxic T cell response. However, both egg PC liposomes as well as escheriosomes-encapsulated antigen elicited strong humoral immune response in immunized animals but antibody titre was significantly higher in the group of animals immunized with escheriosomes-encapsulated antigen. These results imply usage of liposome-based adjuvant as potential candidate vaccine capable of eliciting both cell-mediated as well as humoral immune responses. Furthermore, antigen entrapped in escheriosomes stimulates antigen-specific CD4(+) T cell proliferation and also enhances the level of IL-2, IFN-gamma and IL-4 in the immunized animals.

  14. Lung cancer-associated tumor antigens and the present status of immunotherapy against non-small-cell lung cancer

    International Nuclear Information System (INIS)

    Yasumoto, Kosei; Hanagiri, Takeshi; Takenoyama, Mitsuhiro

    2009-01-01

    Despite recent advances in surgery, irradiation, and chemotherapy, the prognosis of patients with lung cancer is still poor. Therefore, the development and application of new therapeutic strategies are essential for improving the prognosis of this disease. Significant progress in our understanding of tumor immunology and molecular biology has allowed us to identify the tumor-associated antigens recognized by cytotoxic T lymphocytes. Immune responses and tumor-associated antigens against not only malignant melanoma but also lung cancer have been elucidated at the molecular level. In a theoretical sense, tumor eradication is considered possible through antigen-based immunotherapy against such diseases. However, many clinical trials of cancer vaccination with defined tumor antigens have resulted in objective clinical responses in only a small number of patients. Tumor escape mechanisms from host immune surveillance remain a major obstacle for cancer immunotherapy. A better understanding of the immune escape mechanisms employed by tumor cells is necessary before we can develop a more effective immunotherapeutic approach to lung cancer. We review recent studies regarding the identification of tumor antigens in lung cancer, tumor immune escape mechanisms, and clinical vaccine trials in lung cancer. (author)

  15. Inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

    International Nuclear Information System (INIS)

    Vink, A.A.; Roza, L.; Moodycliffe, A.M.; Shreedhar, V.

    1997-01-01

    Exposing skin to UVB (280-320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase, which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosomes treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320-400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function

  16. Rainbow trout CK9, a CCL25-like ancient chemokine that attracts and regulates B cells and macrophages, the main antigen presenting cells in fish.

    Science.gov (United States)

    Aquilino, Carolina; Granja, Aitor G; Castro, Rosario; Wang, Tiehui; Abos, Beatriz; Parra, David; Secombes, Christopher J; Tafalla, Carolina

    2016-04-05

    CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages.

  17. A Genome-wide multidimensional RNAi screen reveals pathways controlling MHC class II antigen presentation

    NARCIS (Netherlands)

    Paul, Petra; van den Hoorn, Tineke; Jongsma, Marlieke L. M.; Bakker, Mark J.; Hengeveld, Rutger; Janssen, Lennert; Cresswell, Peter; Egan, David A.; van Ham, Marieke; ten Brinke, Anja; Ovaa, Huib; Beijersbergen, Roderick L.; Kuijl, Coenraad; Neefjes, Jacques

    2011-01-01

    MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and

  18. Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

    Directory of Open Access Journals (Sweden)

    Kornbluth Richard S

    2009-08-01

    Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

  19. Cell-mediated immune response of synovial fluid lymphocytes to ureaplasma antigen in Reiter's syndrome

    Directory of Open Access Journals (Sweden)

    Pavlica Ljiljana

    2003-01-01

    Full Text Available INTRODUCTION Reiter's syndrome (RS is an seronegative arthritis that occurs after urogenital or enteric infection which in addition with occular and/or mucocutaneous manifestations presents complete form of disease. According to previous understanding arthritis in the RS is the reactive one, which means that it is impossible to isolate its causative agent. However, there are the more and more authors suggesting that arthritis in the urogenital form of disease is caused by the infective agent in the affected joint. This suggestion is based on numerous studies on the presence of Chlmaydia trachomatis and Ureaplasma urealyticum in the inflamed joint by using new diagnostic methods in molecular biology published in the recent literature [1-3]. Besides, numerous studies of the humoral and cell-mediated immune response to "triggering" bacteria in the affected joint have supported previous suggestions [4-7]. Aim of the study was to determine whether synovial fluid T-cells specifically recognize the "triggering" bacteria presumably responsible for the Reiter's syndrome. METHOD The 3H-thymidine uptake procedure for measuring lymphocyte responses was applied to lymphocytes derived concurrently from synovial fluid (SF and from peripheral blood (PB [8]. Ureaplasma antigen and mitogen PHA stimulated lymphocytes in 24 RS patients (24 PB samples, 9 SF samples and the results were compared with those found in 10 patients with rheumatoid arthritis (RA (10 PB samples, 5 SF samples. Preparation of ureaplasma antigen. Ureaplasma was cultured on cell-free liquid medium [9]. Sample of 8 ml was heat-inactivated for 15 minutes at 601C and permanently stirred with magnetic mixer. The sample was centrifuged at 2000 x g for 40 minutes and than deposits carefully carried to other sterile glass tubes (Corex and recentrifuged at 9000 x g for 30 minutes. The deposit was washed 3 times in sterile 0.9% NaCl, and final sediment was resuspended in 1.2 ml sterile 0.9% Na

  20. FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

    Science.gov (United States)

    Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

    2009-12-01

    FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

  1. Protein antigenic structures recognized by T cells: potential applications to vaccine design.

    Science.gov (United States)

    Berzofsky, J A; Cease, K B; Cornette, J L; Spouge, J L; Margalit, H; Berkower, I J; Good, M F; Miller, L H; DeLisi, C

    1987-08-01

    In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic

  2. CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

    Science.gov (United States)

    Chen, Peter; Mesci, Aruz; Carlyle, James R

    2011-01-01

    Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

  3. Modulation of interferon-γ synthesis by the effects of lignin-like enzymatically polymerized polyphenols on antigen-presenting cell activation and the subsequent cell-to-cell interactions.

    Science.gov (United States)

    Yamanaka, Daisuke; Motoi, Masuro; Ishibashi, Ken-ichi; Miura, Noriko N; Adachi, Yoshiyuki; Ohno, Naohito

    2013-12-15

    Lignin-like polymerized polyphenols strongly activate lymphocytes and induce cytokine synthesis. We aimed to characterise the mechanisms of action of polymerized polyphenols on immunomodulating functions. We compared the reactivity of leukocytes from various organs to that of polymerized polyphenols. Splenocytes and resident peritoneal cavity cells (PCCs) responded to polymerized polyphenols and released several cytokines, whereas thymocytes and bone-marrow cells showed no response. Next, we eliminated antigen-presenting cells (APCs) from splenocytes to study their involvement in cytokine synthesis. We found that APC-negative splenocytes showed significantly reduced cytokine production induced by polymerized polyphenols. Additionally, adequate interferon-γ (IFN-γ) induction by polymerized polyphenols was mediated by the coexistence of APCs and T cells because the addition of T cells to PCCs increased IFN-γ production. Furthermore, inhibition of the T cell-APC interaction using neutralising antibodies significantly decreased cytokine production. Thus, cytokine induction by polymerized polyphenols was mediated by the interaction between APCs and T cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Evidence against suppressor cell involvement in naturally acquired tolerance of a minor histocompatibility antigen

    International Nuclear Information System (INIS)

    Johnson, L.L.

    1991-01-01

    The hypothesis was investigated that suppressor cells may be responsible for maintenance of immunologic tolerance of a minor H3 antigen in mice that express the antigen naturally. Lymphoid cell populations from B6.C-H-24c (HW54) mice, a congenic-resistant strain histoincompatible with H-24b-expressing C57BL/6 (B6) mice only with respect to the H-24 locus, were examined in cell-transfer experiments to see if they contained naturally arising H-24c-specific suppressor cells. The H-24 antigen was chosen for these studies because, unlike most other minor and major histocompatibility (H) antigens, it is not detectable on mature lymphoid cells by any of several functional criteria. Thus transfer of HW54 lymphoid cells to B6 hosts could be done without the complication of inducing hyporesponsiveness de novo in the host, as occurs with other minor H antigens that are expressed on lymphocytes. B6 hosts were given HW54 skin grafts along with HW54 lymphoid cells to assess their tolerance of the H-24c-encoded antigen. The hosts were either (1) normal, nonimmune B6 mice; (2) B6 mice rendered immunodeficient by thymectomy and irradiation (TxB) and repopulated with H-24c-immune B6 lymphocytes; or (3) TxB B6 hosts repopulated with nonimmune B6 lymphocytes. In each case it was found that the additionally infused HW54 lymphoid cells did not suppress the ability of these hosts to reject HW54 skin grafts. In other words, HW54 lymphoid cells appear not to possess suppressive activity specific for the H-24c antigen that might maintain antigen-specific natural tolerance. Additional experiments were performed to determine whether HW54 lymphoid cells can inhibit the ability of sublethally irradiated B6 mice to regain the capacity to reject HW54 skin

  5. Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity.

    Science.gov (United States)

    van Montfoort, Nadine; Camps, Marcel G; Khan, Selina; Filippov, Dmitri V; Weterings, Jimmy J; Griffith, Janice M; Geuze, Hans J; van Hall, Thorbald; Verbeek, J Sjef; Melief, Cornelis J; Ossendorp, Ferry

    2009-04-21

    Dendritic cells (DCs) are crucial for priming of naive CD8(+) T lymphocytes to exogenous antigens, so-called "cross-priming." We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte cross-priming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained cross-presentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.

  6. The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

    NARCIS (Netherlands)

    Brakenhoff, R H; Gerretsen, M; Knippels, E M; van Dijk, M.; van Essen, H; Weghuis, D O; Sinke, R J; Snow, G B; van Dongen, G A

    The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose

  7. Do ABO blood group antigens hamper the therapeutic efficacy of mesenchymal stromal cells?

    Science.gov (United States)

    Moll, Guido; Hult, Annika; von Bahr, Lena; Alm, Jessica J; Heldring, Nina; Hamad, Osama A; Stenbeck-Funke, Lillemor; Larsson, Stella; Teramura, Yuji; Roelofs, Helene; Nilsson, Bo; Fibbe, Willem E; Olsson, Martin L; Le Blanc, Katarina

    2014-01-01

    Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

  8. Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Federico Battisti

    2017-09-01

    Full Text Available Dendritic cells (DCs are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

  9. FIBER OPTIC IMMUNOSENSORS BASED ON ENHANCED CHEMILUMINESCENCE AND THEIR APPLICATION TO DETERMINE DIFFERENT ANTIGENS

    NARCIS (Netherlands)

    STARODUB, NF; ARENKOV, PJ; STARODUB, AN; BEREZIN, VA

    The main scheme of the cell for measuring as well as the sensor device itself, the analysis conditions and the results of quantitative determination of several antigens such as estradiol-17, alpha-2-interferon, chorionic honadotropine, antibodies to the influenza virus, total IgG and cells of

  10. Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Mustafa, A S; Amoudy, H A; Wiker, H G

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r......GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

  11. Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. I. Metabolic inactivation impairs both CD and LD antigen signals

    International Nuclear Information System (INIS)

    Kelso, A.; Boyle, W.

    1982-01-01

    The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T/sub c/) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T/sub c/ responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T/sub c/ precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T/sub c/ from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T/sub c/. It is concluded that, in addition to the traditional view that these treatments damage the ''LD'' signal to helper T lymphocytes, metabolic inactivation also impairs recognition of ''CD'' determinants by precursor T/sub c/

  12. CD80 and CD86 Costimulatory Molecules Differentially Regulate OT-II CD4+ T Lymphocyte Proliferation and Cytokine Response in Cocultures with Antigen-Presenting Cells Derived from Pregnant and Pseudopregnant Mice

    Directory of Open Access Journals (Sweden)

    Tomasz Maj

    2014-01-01

    Full Text Available Immune phenomena during the preimplantation period of pregnancy are poorly understood. The aim of our study was to assess the capacity for antigen presentation of splenic antigen-presenting cells (APCs derived from pregnant and pseudopregnant mice in in vitro conditions. Therefore, sorted CD11c+ dendritic cells and macrophages F4/80+ and CD11b+ presenting ovalbumin (OVA were cocultured with CD4+ T cells derived from OT-II mice’s (C57BL6/J-Tg(TcraTcrb1100Mjb/J spleen. After 132 hours of cell culture, proliferation of lymphocytes (ELISA-BrdU, activation of these cells (flow cytometry, cytokine profile (ELISA, and influence of costimulatory molecules blocking on these parameters were measured. We did not detect any differences in regulation of Th1/Th2 cytokine balance. CD86 seems to be the main costimulatory molecule involved in the proliferation response but CD80 is the main costimulatory molecule influencing cytokine secretion in pregnant mice. In conclusion, this study showed that CD80 and CD86 costimulatory molecules regulate OT-II CD4+ T lymphocyte proliferation and cytokine response in cocultures with antigen-presenting cells derived from pregnant and pseudopregnant mice. The implications of these changes still remain unclear.

  13. Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

    Directory of Open Access Journals (Sweden)

    Sergio Cepeda

    2018-01-01

    Full Text Available Although autoimmune disorders are a significant source of morbidity and mortality in older individuals, the mechanisms governing age-associated increases in susceptibility remain incompletely understood. Central T cell tolerance is mediated through presentation of self-antigens by cells constituting the thymic microenvironment, including epithelial cells, dendritic cells, and B cells. Medullary thymic epithelial cells (mTECs and B cells express distinct cohorts of self-antigens, including tissue-restricted self-antigens (TRAs, such that developing T cells are tolerized to antigens from peripheral tissues. We find that expression of the TRA transcriptional regulator Aire, as well as Aire-dependent genes, declines with age in thymic B cells in mice and humans and that cell-intrinsic and cell-extrinsic mechanisms contribute to the diminished capacity of peripheral B cells to express Aire within the thymus. Our findings indicate that aging may diminish the ability of thymic B cells to tolerize T cells, revealing a potential mechanistic link between aging and autoimmunity.

  14. A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

    Science.gov (United States)

    Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

    2012-10-12

    The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

  15. Nuclear localization of Merkel cell polyomavirus large T antigen in Merkel cell carcinoma

    International Nuclear Information System (INIS)

    Nakamura, Tomoyuki; Sato, Yuko; Watanabe, Daisuke; Ito, Hideki; Shimonohara, Nozomi; Tsuji, Takahiro; Nakajima, Noriko; Suzuki, Yoshio; Matsuo, Koma; Nakagawa, Hidemi; Sata, Tetsutaro; Katano, Harutaka

    2010-01-01

    To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

  16. Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

    Science.gov (United States)

    Moguche, Albanus O; Musvosvi, Munyaradzi; Penn-Nicholson, Adam; Plumlee, Courtney R; Mearns, Helen; Geldenhuys, Hennie; Smit, Erica; Abrahams, Deborah; Rozot, Virginie; Dintwe, One; Hoff, Søren T; Kromann, Ingrid; Ruhwald, Morten; Bang, Peter; Larson, Ryan P; Shafiani, Shahin; Ma, Shuyi; Sherman, David R; Sette, Alessandro; Lindestam Arlehamn, Cecilia S; McKinney, Denise M; Maecker, Holden; Hanekom, Willem A; Hatherill, Mark; Andersen, Peter; Scriba, Thomas J; Urdahl, Kevin B

    2017-06-14

    CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

    NARCIS (Netherlands)

    Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

    1985-01-01

    T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

  18. Analysis of antigen-specific B-cell memory directly ex vivo.

    Science.gov (United States)

    McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

    2004-01-01

    Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

  19. Analysis of nuclear accumulation of influenza NP antigen in von Magnus virus-infected cells.

    Science.gov (United States)

    Maeno, K; Aoki, H; Hamaguchi, M; Iinuma, M; Nagai, Y; Matsumoto, T; Takeura, S; Shibata, M

    1981-01-01

    When 1-5C-4 cells were infected with von Magnus virus derived from influenza A/RI/5+ virus by successive undiluted passages in chick embryos, virus-specific proteins were synthesized but production of infectious virus was inhibited. In these cells the synthesis of viral RNA was suppressed and the nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to standard virus-infected cells in which the antigen was distributed throughout the whole cell. The intracellular location and migration of NP were determined by isotope labeling and sucrose gradient centrifugation of subcellular fractions. In standard virus-infected cell NP polypeptide was present predominantly in the cytoplasm in the form of viral ribonucleoprotein (RNP) and intranuclear RNP was detected in reduced amounts. In contrast, in von Magnus virus-infected cells NP polypeptide was present predominantly in the nucleus in a nonassembled, soluble from and the amount of cytoplasmic RNP was considerably reduced. After short-pulse labeling NP was detected exclusively in the cytoplasm in a soluble form and after a chase a large proportion of such soluble NP was seen in the nucleus. It is suggested that a large proportion of the NP synthesized in von Magnus virus-infected cells in not assembled into cytoplasmic RNP because of the lack of available RNA and the NP migrated into the nucleus and remained there.

  20. Dissection of T-cell antigen specificity in human melanoma

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Albæk Thrue, Charlotte; Junker, Niels

    2012-01-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-as...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined....

  1. Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells.

    Science.gov (United States)

    Mascarell, Laurent; Lombardi, Vincent; Louise, Anne; Saint-Lu, Nathalie; Chabre, Henri; Moussu, Hélène; Betbeder, Didier; Balazuc, Anne-Marie; Van Overtvelt, Laurence; Moingeon, Philippe

    2008-09-01

    A detailed characterization of oral antigen-presenting cells is critical to improve second-generation sublingual allergy vaccines. To characterize oral dendritic cells (DCs) within lingual and buccal tissues from BALB/c mice with respect to their surface phenotype, distribution, and capacity to polarize CD4(+) T-cell responses. In situ analysis of oral DCs was performed by immunohistology. Purified DCs were tested in vitro for their capacity to capture, process, and present the ovalbumin antigen to naive CD4(+) T cells. In vivo priming of ovalbumin-specific T cells adoptively transferred to BALB/c mice was analyzed by cytofluorometry in cervical lymph nodes after sublingual administration of mucoadhesive ovalbumin. Three subsets of oral DCs with a distinct tissue distribution were identified: (1) a minor subset of CD207(+) Langerhans cells located in the mucosa itself, (2) a major subpopulation of CD11b(+)CD11c(-) and CD11b(+)CD11c(+) myeloid DCs at the mucosal/submucosal interface, and (3) B220(+)120G8(+) plasmacytoid DCs found in submucosal tissues. Purified myeloid and plasmacytoid oral DCs capture and process the antigen efficiently and are programmed to elicit IFN-gamma and/or IL-10 production together with a suppressive function in naive CD4(+) T cells. Targeting the ovalbumin antigen to oral DCs in vivo by using mucoadhesive particles establishes tolerance in the absence of cell depletion through the stimulation of IFN-gamma and IL-10-producing CD4(+) regulatory T cells in cervical lymph nodes. The oral immune system is composed of various subsets of tolerogenic DCs organized in a compartmentalized manner and programmed to induce T(H)1/regulatory T-cell responses.

  2. Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

    Directory of Open Access Journals (Sweden)

    Benencia Fabian

    2008-04-01

    Full Text Available Abstract Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV B radiation using a convenient tumor model expressing human papilloma virus (HPV E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

  3. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Directory of Open Access Journals (Sweden)

    Serkan Yazıcı

    2015-01-01

    Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

  4. Activation of Antigen-Specific CD8(+) T Cells by Poly-DL-Lactide/Glycolide (PLGA) Nanoparticle-Primed Gr-1(high) Cells.

    Science.gov (United States)

    Luo, Wen-Hui; Yang, Ya-Wun

    2016-04-01

    The aim of this study was to investigate the induction of antigen-specific T cell activation and cell cycle modulation by a poly-DL-lactide/glycolide (PLGA) nanoparticle (NP)-primed CD11b(+)Gr-1(high) subset isolated from mouse bone marrow. PLGA NPs containing the ovalbumin (OVA) antigen were prepared using the double emulsion and solvent evaporation method, and protein release rate and cell viability were determined. The Lin2(¯)CD11b(+)Gr-1(high)Ly6c(low) (Gr-1(high)) subset was sorted from the bone marrow of C57BL/6 J mice by fluorescence-activated cell sorting (FACS) and co-cultured with OT-I CD8(+) splenic T cells. Proliferation of OT-I CD8(+) T cells was monitored, and cell cycles were determined by 5-bromo-2'-deoxyuridine (BrdU) labeling. Treatment of Gr-1(high) cells with PLGA/OVA NPs upregulated expression of the SIINFEKL-H2K(b) complex in the context of MHC I. Co-cultures of OT-I CD8(+) T cells with the PLGA/OVA NP-primed Gr-1(high) cells induced the proliferation of T cells in vitro and modulated cell division and morphology. Treatment of Gr-1(high) cells with PLGA/OVA NPs also induced cell apoptosis and necrosis. This study demonstrated the function of PLGA/OVA NPs in the activation of OT-I CD8(+) T cells and the capability of cross-presentation via the Gr-1(high) polymorphonuclear subset from mouse bone marrow.

  5. Understanding the immunogenicity and antigenicity of nanomaterials: Past, present and future

    International Nuclear Information System (INIS)

    Ilinskaya, Anna N.; Dobrovolskaia, Marina A.

    2016-01-01

    Nanoparticle immunogenicity and antigenicity have been under investigation for many years. During the past decade, significant progress has been made in understanding what makes a nanoparticle immunogenic, how immune cells respond to nanoparticles, what consequences of nanoparticle-specific antibody formation exist and how they challenge the application of nanoparticles for drug delivery. Moreover, it has been recognized that accidental contamination of therapeutic protein formulations with nanosized particulate materials may contribute to the immunogenicity of this type of biotechnology products. While the immunological properties of engineered nanomaterials and their application as vaccine carriers and adjuvants have been given substantial consideration in the current literature, little attention has been paid to nanoparticle immuno- and antigenicity. To fill in this gap, we herein provide an overview of this subject to highlight the current state of the field, review past and present research, and discuss future research directions. - Highlights: • Most engineered nanomaterials are not immunogenic per se. • Generation of nanoparticle-specific antibody can be T-cell dependent or independent. • Antibodies can be generated to particle core, terminal groups or surface coatings. • Engineered and accidental nanomaterials have distinct contribution to immunogenicity. • Tunable physicochemical properties make each nanoparticle unique.

  6. Understanding the immunogenicity and antigenicity of nanomaterials: Past, present and future

    Energy Technology Data Exchange (ETDEWEB)

    Ilinskaya, Anna N.; Dobrovolskaia, Marina A., E-mail: marina@mail.nih.gov

    2016-05-15

    Nanoparticle immunogenicity and antigenicity have been under investigation for many years. During the past decade, significant progress has been made in understanding what makes a nanoparticle immunogenic, how immune cells respond to nanoparticles, what consequences of nanoparticle-specific antibody formation exist and how they challenge the application of nanoparticles for drug delivery. Moreover, it has been recognized that accidental contamination of therapeutic protein formulations with nanosized particulate materials may contribute to the immunogenicity of this type of biotechnology products. While the immunological properties of engineered nanomaterials and their application as vaccine carriers and adjuvants have been given substantial consideration in the current literature, little attention has been paid to nanoparticle immuno- and antigenicity. To fill in this gap, we herein provide an overview of this subject to highlight the current state of the field, review past and present research, and discuss future research directions. - Highlights: • Most engineered nanomaterials are not immunogenic per se. • Generation of nanoparticle-specific antibody can be T-cell dependent or independent. • Antibodies can be generated to particle core, terminal groups or surface coatings. • Engineered and accidental nanomaterials have distinct contribution to immunogenicity. • Tunable physicochemical properties make each nanoparticle unique.

  7. Identification of human tissue cross-presenting dendritic cells

    OpenAIRE

    Haniffa, Muzlifah; Collin, Matthew; Ginhoux, Florent

    2013-01-01

    Dendritic cells (DCs) are a heterogeneous group of functionally specialized antigen-presenting cells. We recently characterized the human tissue cross-presenting DCs and aligned the human and mouse DC subsets. Our findings will facilitate the translation of murine DC studies to the human setting and aid the design of DC-based vaccine strategies for infection and cancer immunotherapy.

  8. Fundamental and clinical evaluation of ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen

    International Nuclear Information System (INIS)

    Koizumi, Mitsuru; Endo, Keigo; Nakajima, Kotoko

    1987-01-01

    A commercial ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen (SCC antigen) was fundamentally and clinically evaluated. Laboratory performance was satisfactory for intra-assay and inter-assay reproducibility, recovery, and dilution, with rapid and simple measurement techniques. Seropositivity for SCC antigen was significantly higher for squamous cell carcinoma of the liver and uterine cervix than the other histology types. In the case of cervical squamous cell carcinoma, it increased with progressing disease. Post-treatment serum levels of SCC antigen returned to negative. SCC antigen is considered to be a useful tumor marker for these diseases. There was a good correlation between the measurement values obtained from the present and conventional (SCC RIAKIT) assays. The present assay remarkably decreased false-positive cases of pulmonary benign diseases. The results showed a ''SCC RIABEAD'' to be a favorable kit for immunoradiometric assay of SSC antigen, as compared with conventional assay kit. (Namekawa, K.)

  9. Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion1

    Science.gov (United States)

    Turner, Michael S.; Kane, Lawrence P.; Morel, Penelope A.

    2009-01-01

    The definitions of tolerogenic vs. immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allo-antigens. However, we have previously reported that mature DC (G4DC) prevented the onset of autoimmune diabetes whereas immature DC (GMDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR Tg mice were stimulated, in the absence of exogenous cytokine, with GMDC or G4DC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both GMDC and G4DC presenting low peptide doses induced weak TCR signaling via the Akt/mTOR pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed G4DC, but not GMDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-Tg T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with antigen dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low antigen doses. These studies indicate that strength of Akt/mTOR signaling, a critical T cell intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production. PMID:19801514

  10. Kefiran suppresses antigen-induced mast cell activation.

    Science.gov (United States)

    Furuno, Tadahide; Nakanishi, Mamoru

    2012-01-01

    Kefir is a traditional fermented milk beverage produced by kefir grains in the Caucasian countries. Kefiran produced by Lactobacillus kefiranofaciens in kefir grains is an exopolysaccharide having a repeating structure with glucose and galactose residues in the chain sequence and has been suggested to exert many health-promoting effects such as immunomodulatory, hypotensive, hypocholesterolemic activities. Here we investigated the effects of kefiran on mast cell activation induced by antigen. Pretreatment with kefiran significantly inhibited antigen-induced Ca(2+) mobilization, degranulation, and tumor necrosis factor-α production in bone marrow-derived mast cells (BMMCs) in a dose-dependent manner. The phosphorylation of Akt, glycogen synthase kinase 3β, and extracellular signal-regulated kinases (ERKs) after antigen stimulation was also suppressed by pretreatment of BMMCs with kefiran. These findings indicate that kefiran suppresses mast cell degranulation and cytokine production by inhibiting the Akt and ERKs pathways, suggesting an anti-inflammatory effect for kefiran.

  11. Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals.

    Science.gov (United States)

    Benson, Micah J; Elgueta, Raul; Schpero, William; Molloy, Michael; Zhang, Weijun; Usherwood, Edward; Noelle, Randolph J

    2009-08-31

    The hypothesis that bystander inflammatory signals promote memory B cell (B(MEM)) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220(+)IgG(+) B(MEM) toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent B(MEM) clonally expand. Surprisingly, proliferating B(MEM) do not acquire germinal center (GC) B cell markers before generating daughter B(MEM) and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of B(MEM) proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of B(MEM) occurred. The absence of a B(MEM) response to nonspecific inflammatory signals clearly shows that B(MEM) proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

  12. Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

    DEFF Research Database (Denmark)

    Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...

  13. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

    Science.gov (United States)

    Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

    2013-04-18

    Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

  14. T cell epitopes on the 36K and 65K Mycobacterium leprae antigens defined by human T cell clones

    NARCIS (Netherlands)

    van Schooten, W. C.; Ottenhoff, T. H.; Klatser, P. R.; Thole, J.; de Vries, R. R.; Kolk, A. H.

    1988-01-01

    To identify the molecular localization and specificity of Mycobacterium leprae antigenic determinants inducing T cell activation, we studied the reactivity of M. leprae-reactive T cell clones from two tuberculoid leprosy patients towards a battery of different mycobacterial strains and purified

  15. The role of CD4 in antigen-independent activation of isolated single T lymphocytes

    DEFF Research Database (Denmark)

    Kelso, A; Owens, T

    1988-01-01

    The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An ass...

  16. Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

    Science.gov (United States)

    Wu, Julie H; Simonette, Rebecca A; Nguyen, Harrison P; Rady, Peter L; Tyring, Stephen K

    2016-06-01

    Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

  17. Critical study and applications of the radioimmunological determination of carcinoembryonic antigen

    International Nuclear Information System (INIS)

    Troupel, Solange.

    1974-01-01

    This paper outlines our research on the development of a radioimmunological method to determine the carcinoembryonic antigen of the digestive system (ACE). The carcinoembryonic antigen is defined and situated in the framework of antigens associated with human tumours. The general principles of the radioimmunological determination are then reviewed. A detailed technical study is devoted to each of the elements involved in the reaction and to the working conditions of each method tried. A labelling procedure and a radioactive protein separation method have been worked out, guaranteeing a high specific radioactivity consistent with a good immunoreactivity. The period of effectiveness of this protein has also been determined, taking account of its deiodination. The antiserum is a very important factor in the sensitivity of the measurement. A ewe antiserum of good antibody content and volume yield was chosen, its disadvantage being the length of the determination imposed by the 48 hour preincubation time. Ammonium sulphate precipitation and double antibody techniques were used for the labelled antigen-antibody separation. In seric solution the ammonium sulphate precipitation carries down non-specifically, in the standards, a large amount of labelled antigen. This disadvantage has been offset by a method of calculation which shows the actual contribution of the labelled complex. The double antibody technique requires a special adjustment to balance quantity of second antiserum and precipitation time. The system sometimes needs an addition of serum from the animal donor of the first antibody in order to obtain an adequate separation. Where techniques are concerned, although the macro-method is suitable for determinations on perchloric extract and is still in common use we prefer to use the one described here under the name of micro-method. Finally the results obtained in experimental and clinical applications are presented [fr

  18. Interleukin-19: a constituent of the regulome that controls antigen presenting cells in the lungs and airway responses to microbial products.

    Directory of Open Access Journals (Sweden)

    Carol Hoffman

    Full Text Available Interleukin (IL-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+ alveolar macrophages and lung dendritic cells.IL-19-deficient (IL-19-/- mice were studied at baseline (naïve and following intranasal challenge with microbial products, or recombinant cytokines. Naïve IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2 expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naïve IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13.Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that

  19. Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

    Directory of Open Access Journals (Sweden)

    Neelkamal Chaudhary

    2010-02-01

    Full Text Available Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H1-T(H17 and destructive allergic (T(H2 immunity. How Aspergillus allergens (Asp f proteins participate in the development of allergic sensitization is unknown.To determine whether Asp f proteins are strictly associated with T(H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17 to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H1-responses to Asp f3 (a putative peroxismal membrane protein, Asp f9/16 (cell wall glucanase, Asp f11 (cyclophilin type peptidyl-prolyl isomerase and Asp f22 (enolase. Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

  20. Changes in antigen-presenting cell function in the spleen and lymph nodes of ultraviolet-irradiated mice

    International Nuclear Information System (INIS)

    Gurish, M.F.; Lynch, D.H.; Daynes, R.A.

    1982-01-01

    It has been previously reported that mice exposed to ultraviolet (UV) radiation exhibit a decrease in splenic antigen-presenting cell (APC) function. The results presented here confirm this observation and further demonstrate that animals exposed daily to UV for extended periods of time (5 weeks instead of 6 days) no longer exhibit this depressed capability. In spite of the depression in splenic APC activity found in 6-day UV-irradiated mice, lymph node APC function from these same animals was elevated compared with that found in the lymph nodes from normal animals. Lymph node APC activity in animals that were splenectomized prior to the UV irradiation, however, was not enhanced over controls. Treatment of animals with a chemical irritant (turpentine) also caused a depression in splenic APC function without modifying lymph node activity. Collectively, our findings suggest that the observed decrease in splenic APC activity, found after the first week of UV exposures, may be attributable to the migration of splenic APC to peripheral lymphoid tissue which drain the site of epidermal inflammation

  1. Antigen Requirements for Efficient Priming of CD8+ T Cells by Leishmania major-Infected Dendritic Cells

    Science.gov (United States)

    Bertholet, Sylvie; Debrabant, Alain; Afrin, Farhat; Caler, Elisabeth; Mendez, Susana; Tabbara, Khaled S.; Belkaid, Yasmine; Sacks, David L.

    2005-01-01

    CD4+ and CD8+ T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4+ or OT-I CD8+ T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3′ nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8+ T cells. The ability of L. major transgenic parasites to activate OT-I CD8+ T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level. PMID:16177338

  2. Antigen dynamics of follicular dendritic cells

    NARCIS (Netherlands)

    Heesters, B.A.

    2015-01-01

    Stromal-derived follicular dendritic cells (FDCs) are a major depot for antigen that are essential for formation of germinal centers, the site where memory and effector B cells differentiate and high-affinity antibody production takes place. Historically, FDCs have been characterized as ‘accessory’

  3. Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

    Science.gov (United States)

    Yap, Jin Yan; Wirasinha, Rushika C; Chan, Anna; Howard, Debbie R; Goodnow, Christopher C; Daley, Stephen R

    2018-02-07

    Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 + regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes. © 2018 John Wiley & Sons Ltd.

  4. Identification of the soluble HVP-associated antigen of the lymphoblastoid cell line established from lymphomatous baboon (Papio hamadryas).

    Science.gov (United States)

    Voevodin, A F; Lapin, B A; Agrba, V Z; Timanovskaya, V V

    1978-01-01

    A new technique (indirect double immunodiffusion) for detection of EBV-associated soluble antigen and corresponding antibodies has been developed. This technique includes three steps: 1) simple double immunodiffusion with extracts of Raji cells (or other EBV-genome positive cells) and human sera containing antibodies against EBV-associated soluble antigen; 2) extensive washing and treatment with anti-human globulin; 3) extensive washing and treatment with tannic acid. Using this test it was shown that the soluble antigen indistinguishable from EBV-associated soluble antigen was present in KMPG-1 cells producing HVP.

  5. Stabilization of Transfected Cells Expressing Low-Incidence Blood Group Antigens: Novel Methods Facilitating Their Use as Reagent-Cells.

    Directory of Open Access Journals (Sweden)

    Cecilia González

    Full Text Available The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs, which coexpress many antigens and are not easily available for low-incidence blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua. High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.

  6. Co-culture with podoplanin+ cells protects leukemic blast cells with leukemia-associated antigens in the tumor microenvironment.

    Science.gov (United States)

    Lee, Ji Yoon; Han, A-Reum; Lee, Sung-Eun; Min, Woo-Sung; Kim, Hee-Je

    2016-05-01

    Podoplanin+ cells are indispensable in the tumor microenvironment. Increasing evidence suggests that podoplanin may support the growth and metastasis of solid tumors; however, to the best of our knowledge no studies have determined whether or not podoplanin serves a supportive role in acute myeloid leukemia (AML). The effects of co‑culture with podoplanin+ cells on the cellular activities of the leukemic cells, such as apoptosis and cell proliferation, in addition to the expression of podoplanin in leukemic cells, were investigated. Due to the fact that genetic abnormalities are the primary cause of leukemogenesis, the overexpression of the fibromyalgia‑like tyrosine kinase‑3 gene in colony forming units was also examined following cell sorting. Podoplanin+ cells were found to play a protective role against apoptosis in leukemic cells and to promote cell proliferation. Tumor‑associated antigens, including Wilms' tumor gene 1 and survivin, were increased when leukemic cells were co‑cultured with podoplanin+ cells. In combination, the present results also suggest that podoplanin+ cells can function as stromal cells for blast cell retention in the AML tumor microenvironment.

  7. Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells

    International Nuclear Information System (INIS)

    Hareyama, Masato

    1988-01-01

    We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-γ. In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G 2 /M phases of the cell cycle than in G 0 /G 1 . The expression of YH206 antigen was enhanced in the S and G 2 /M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens. (author)

  8. Effect of antigen on localization of immunologically specific B cells

    International Nuclear Information System (INIS)

    Ponzio, N.M.; Chapman, J.M.; Thorbecke, G.J.

    1976-01-01

    Studies were conducted to demonstrate homing of memory B cells to sites of antigen localization in lymph nodes, using functional criteria to detect local presence of memory cells at varying intervals after intravenous injection. Cell suspensions were prepared from spleens of donor mice injected with complete Freund's adjuvant. Recipient mice were injected with Escherichia coli endotoxin and immune or normal spleen cells and were gamma-irradiated. Results indicated that passively transferred unilateral B cell memory was established. The development over a period of several days of this difference between left and right lymph nodes suggests that recirculating memory B cells are being progressively selected by antigen in the lymph node, rather than that this difference is due to a specific exit of cells from the circulation towards the antigen

  9. Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen

    Directory of Open Access Journals (Sweden)

    Anita Verma

    2018-02-01

    Full Text Available Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA, the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

  10. Paired Expression Analysis of Tumor Cell Surface Antigens

    Directory of Open Access Journals (Sweden)

    Rimas J. Orentas

    2017-08-01

    Full Text Available Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19 or antibody-based therapy (anti-CD20 in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues. We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK

  11. Shedding of CD9 antigen into cerebrospinal fluid by acute lymphoblastic leukemia cells.

    Science.gov (United States)

    Komada, Y; Ochiai, H; Shimizu, K; Azuma, E; Kamiya, H; Sakurai, M

    1990-07-01

    The accurate identification of small numbers of leukemic cells in the cerebrospinal fluid (CSF) presents a diagnostic problem in the treatment of acute lymphoblastic leukemia (ALL). We demonstrated that soluble CD9 antigen was shed into CSF obtained from children with ALL, using enzyme-linked immunosorbent assay (ELISA), which used the activity of CD9 antigen to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4, simultaneously. Using RCA1/SJ-9A4 ELISA, CD9 antigen was detectable in CSF but not in plasma from 12 cases of CD9+ ALL in central nervous system (CNS) relapse. However, CD9 antigen was not released into CSF from 11 cases of CD9- ALL with CNS involvement, 136 cases of CD9+ ALL in complete remission (CR), 29 cases of CD9- ALL in CR, or 21 cases of aseptic meningitis. Interestingly, the levels of CD9 antigen were elevated in CSF from 7 of 10 CD9+ ALL patients without cytologically proven CNS involvement at diagnosis, with subsequent return to undetectable levels after initial induction chemotherapy was begun. In addition, sequential analysis of CSF from a 5-year-old boy with CD9+ ALL in CNS relapse showed that levels of CD9 antigen correlated well with the number of leukemic cells in CSF. Serial quantitative analysis of CD9 antigen in CSF could be useful to detect the proliferation of residual leukemic cells before the clinical manifestation.

  12. Hepatitis B virus induces IL-23 production in antigen presenting cells and causes liver damage via the IL-23/IL-17 axis.

    Directory of Open Access Journals (Sweden)

    Qinghong Wang

    Full Text Available IL-23 regulates myriad processes in the innate and adaptive immune systems, and is a critical mediator of the proinflammatory effects exerted by Th17 cells in many diseases. In this study, we investigated whether and how hepatitis B virus (HBV causes liver damage directly through the IL-23 signaling pathway. In biopsied liver tissues from HBV-infected patients, expression of both IL-23 and IL-23R was remarkably elevated. In vivo observations also indicated that the main sources of IL-23 were myeloid dendritic cells (mDCs and macrophages. Analysis of in vitro differentiated immature DCs and macrophages isolated from healthy donors revealed that the HBV surface antigen (HBsAg efficiently induces IL-23 secretion in a mannose receptor (MR-dependent manner. Culture with an endosomal acidification inhibitor and the dynamin inhibitor showed that, upon binding to the MR, the HBsAg is taken up by mDCs and macrophages through an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg can also stimulate IL-23 secretion from mDCs, the process was MR- and endocytosis-independent. In addition, IL-23 was shown to be indispensible for HBsAg-stimulated differentiation of naïve CD4(+ T cells into Th17 cells, which were determined to be the primary source of IL-17 in HBV-infected livers. The cognate receptor, IL-17R, was found to exist on the hepatic stellate cells and mDCs, both of which might represent the potential target cells of IL-17 in hepatitis B disease. These data provide novel insights into a yet unrecognized mechanism of HBV-induced hepatitis, by which increases in IL-23 expression, through an MR/endocytosis-dependent or -independent manner, produce liver damage through the IL-23/IL-17 axis.

  13. Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity.

    Directory of Open Access Journals (Sweden)

    Janin Chandra

    Full Text Available Antigen presenting cells (APCs in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.

  14. Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

    Science.gov (United States)

    Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

    2016-01-01

    Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

  15. Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

    Science.gov (United States)

    Hesketh, J; Dobbelaere, D; Griffin, J F; Buchan, G

    1993-01-01

    The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation. PMID:8104884

  16. Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

    Science.gov (United States)

    Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna

    2007-12-01

    Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.

  17. Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment.

    Science.gov (United States)

    Gao, Yingli; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

    2016-08-01

    Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P immersion, which was significantly higher than the levels of uptake measured in the other tissues (P immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish's immune response by the hyperosmotic immersion treatment prior to vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Leukemia-associated antigens in man.

    Science.gov (United States)

    Brown, G; Capellaro, D; Greaves, M

    1975-12-01

    Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

  19. Molecular mimics of the tumour antigen MUC1.

    Directory of Open Access Journals (Sweden)

    Tharappel C James

    Full Text Available A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1 from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

  20. High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

    Science.gov (United States)

    Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

    2017-07-01

    High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  1. Estradiol-induced vaginal mucus inhibits antigen penetration and CD8(+) T cell priming in response to intravaginal immunization.

    Science.gov (United States)

    Seavey, Matthew M; Mosmann, Tim R

    2009-04-14

    Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8(+) T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APCs) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8(+) T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8(+) T cell priming after insemination or vaginal vaccination.

  2. Estradiol-induced vaginal mucus inhibits antigen penetration and CD8+ T cell priming in response to intravaginal immunization

    Science.gov (United States)

    Seavey, Matthew M.; Mosmann, Tim R.

    2010-01-01

    Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8+ T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APC) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8+ T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8+ T cell priming after insemination or vaginal vaccination. PMID:19428849

  3. Chimeric antigen receptor T-cell therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    Kheng Newick

    2016-01-01

    Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

  4. Targeted delivery of antigen to intestinal dendritic cells induces oral tolerance and prevents autoimmune diabetes in NOD mice.

    Science.gov (United States)

    Chen, Yulin; Wu, Jie; Wang, Jiajia; Zhang, Wenjing; Xu, Bohui; Xu, Xiaojun; Zong, Li

    2018-03-15

    The intestinal immune system is an ideal target to induce immune tolerance physiologically. However, the efficiency of oral protein antigen delivery is limited by degradation of the antigen in the gastrointestinal tract and poor uptake by antigen-presenting cells. Gut dendritic cells (DCs) are professional antigen-presenting cells that are prone to inducing antigen-specific immune tolerance. In this study, we delivered the antigen heat shock protein 65-6×P277 (H6P) directly to the gut DCs of NOD mice through oral vaccination with H6P-loaded targeting nanoparticles (NPs), and investigated the ability of this antigen to induce immune tolerance to prevent autoimmune diabetes in NOD mice. A targeting NP delivery system was developed to encapsulate H6P, and the ability of this system to protect and facilitate H6P delivery to gut DCs was assessed. NOD mice were immunised with H6P-loaded targeting NPs orally once a week for 7 weeks and the onset of diabetes was assessed by monitoring blood glucose levels. H6P-loaded targeting NPs protected the encapsulated H6P from degradation in the gastrointestinal tract environment and significantly increased the uptake of H6P by DCs in the gut Peyer's patches (4.1 times higher uptake compared with the control H6P solution group). Oral vaccination with H6P-loaded targeting NPs induced antigen-specific T cell tolerance and prevented diabetes in 100% of NOD mice. Immune deviation (T helper [Th]1 to Th2) and CD4 + CD25 + FOXP3 + regulatory T cells were found to participate in the induction of immune tolerance. In this study, we successfully induced antigen-specific T cell tolerance and prevented the onset of diabetes in NOD mice. To our knowledge, this is the first attempt at delivering antigen to gut DCs using targeting NPs to induce T cell tolerance.

  5. IMMUNOGENICITY OF HUMAN MESENCHYMAL STEM CELLS IN HLA-CLASS I RESTRICTED T CELL RESPONSES AGAINST VIRAL OR TUMOR-ASSOCIATED ANTIGENS

    OpenAIRE

    Morandi, Fabio; Raffaghello, Lizzia; Bianchi, Giovanna; Meloni, Francesca; Salis, Annalisa; Millo, Enrico; Ferrone, Soldano; Barnaba, Vincenzo; Pistoia, Vito

    2008-01-01

    Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic, but may act as antigen-presenting cells (APC) for CD4+ T cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T cell responses.

  6. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  7. Target-specific activation of mast cells by immunoglobulin E reactive with a renal cell carcinoma-associated antigen

    NARCIS (Netherlands)

    Luiten, R. M.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1996-01-01

    Immunoglobulin E (IgE) that specifically binds to antigens present on carcinoma cells may represent a useful tool to combat carcinomas. Induction of an inflammatory response at the tumor site by tumor-specific IgE may result in reduced tumor growth and tumor regression. Local mast cells may be

  8. Redirecting T cells to eradicate B-cell acute lymphoblastic leukemia: bispecific T-cell engagers and chimeric antigen receptors.

    Science.gov (United States)

    Aldoss, I; Bargou, R C; Nagorsen, D; Friberg, G R; Baeuerle, P A; Forman, S J

    2017-04-01

    Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r

  9. Evaluation of accessory cell heterogeneity. III. Role of dendritic cells in the in vitro activation of the antibody response to soluble antigens.

    Science.gov (United States)

    Erb, P; Ramila, G; Sklenar, I; Kennedy, M; Sunshine, G H

    1985-05-01

    Dendritic cells and macrophages obtained from spleen and peritoneal exudate were tested as accessory cells for the activation of lymphokine production by T cells, for supporting T-B cooperation and for the induction of antigen-specific T helper cells. Dendritic cells as well as macrophages were able to activate T cells for interleukin-2 secretion and functioned as accessory cells in T-B cooperation, but only macrophages induced T helper cells, which cooperate with B cells by a linked recognition interaction, to soluble antigens. Dendritic cell- and antigen-activated T cells also did not help B cells in the presence of Con A supernatants which contained various T cell- and B cell-stimulatory factors. The failure of dendritic cells to differentiate memory into functional T helper cells, but their efficient accessory cell function in T-B cooperation, where functional T helper cells are already present, can be best explained by a differential accessory cell requirement for T helper cell activation dependent on the differentiation stage of the T helper cell.

  10. IDENTIFICATION OF ACROSOME AS THE MAIN ANTIGEN OF THE SPERM CELLS PROVOKING AUTOANTIBODIES IN VASECTOMIZED IRANIAN MEN

    Directory of Open Access Journals (Sweden)

    M R Nowroozi

    2008-12-01

    Full Text Available "nVasectomy is one of the extensively used methods of contraception in family planning programs. Antisperm antibodies (ASA develop after vasectomy which can result in auto-immune male infertility. The precise sperm antigens involved in the autoimmune response are still poorly defined, therefore we determined the circulating ASA and identified relevant sperm antigens based on localization of binding sites of ASA to sperm cell antigens, using a rapid, inexpensive and clinically relevant assay in vasectomized men. Results showed that 2.5% of men had ASA at the time of vasectomy, whereas 53.5% of the study population subsequently developed ASA. The numbers of men with circulating ASA increased significantly for the first three months after vasectomy. These antibodies were distinguishable into three groups based on their bindings to different sites of sperm cell antigens including against acrosome and tail in 67.56% and 10.8%, respectively; 21.6% of subjects had antibody to the other parts of the sperm cell antigens. The results of this study are discussed in terms of an autoimmune response against sperm antigens and development of ASA.

  11. Migration of human antigen-presenting cells in a human skin graft onto nude mice model after contact sensitization

    NARCIS (Netherlands)

    Hoefakker, S.; Balk, H.P.; Boersma, W.J.A.; Joost, T. van; Notten, W.R.F.; Claassen, E.

    1995-01-01

    Fluorescent contact chemical allergens provoke sensitization after application on both syngeneic and allogeneic skin grafts in mice. We attempted to determine whether the functional activity in a contact sensitization response of human skin graft was affected at the level of antigen uptake and

  12. Crystallization and X-ray diffraction studies of crustacean proliferating cell nuclear antigen

    International Nuclear Information System (INIS)

    Carrasco-Miranda, Jesus S.; Cardona-Felix, Cesar S.; Lopez-Zavala, Alonso A.; Re Vega, Enrique de la; De la Mora, Eugenio; Rudiño-Piñera, Enrique; Sotelo-Mundo, Rogerio R.; Brieba, Luis G.

    2012-01-01

    Proliferating cell nuclear antigen from Litopenaeus vannamei was recombinantly expressed, purified and crystallized. Diffraction data were obtained and processed to 3 Å. Proliferating cell nuclear antigen (PCNA), a member of the sliding clamp family of proteins, interacts specifically with DNA replication and repair proteins through a small peptide motif called the PCNA-interacting protein or PIP box. PCNA is recognized as one of the key proteins involved in DNA metabolism. In the present study, the recombinant PCNA from Litopenaeus vannamei (LvPCNA) was heterologously overexpressed and purified using metal ion-affinity chromatography. Crystals suitable for diffraction grew overnight using the hanging-drop vapour-diffusion method. LvPCNA crystals belong to space group C2 with unit-cell parameters a = 144.6, b = 83.4, c = 74.3 Å, β = 117.6°. One data set was processed to 3 Å resolution, with an overall R meas of 0.09 and a completeness of 93.3%. Initial phases were obtained by molecular replacement using a homology model of LvPCNA as the search model. Refinement and structural analysis are underway. This report is the first successful crystallographic analysis of a marine crustacean decapod shrimp (L. vannamei) proliferating cell nuclear antigen

  13. Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation

    NARCIS (Netherlands)

    Brok, M.H. den; Bull, C.; Wassink, M.; Graaf, A.M.A. de; Wagenaars, J.A.L.; Minderman, M.; Thakur, M.; Amigorena, S.; Rijke, E.O.; Schrier, C.C.; Adema, G.J.

    2016-01-01

    Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is

  14. A role for NADPH oxidase in antigen presentation

    Directory of Open Access Journals (Sweden)

    Gail J Gardiner

    2013-09-01

    Full Text Available The nicotinamide adenine dinucleotide phosphate (NADPH oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2.-. This radical is an important precursor of hydrogen peroxide (H2O2 and other reactive oxygen species (ROS needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD, a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC crosspresentation by major histocompatibility complex class I molecules (MHC-I through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules (MHC-II.

  15. T lymphocytes from irradiation chimeras repopulated with 13-day fetal liver cells recognize antigens only in association with self-MHC products

    International Nuclear Information System (INIS)

    Nisbet-Brown, E.; Diener, E.

    1986-01-01

    The restriction specificities of maturing thymocytes are determined by the Class II MHC antigens expressed by non-lymphoid thymic tissues. The proliferative response of mature T lymphocytes to antigen-presenting cells (APC) and antigen requires that the APC express the same MHC antigens as the thymus in which the T cells differentiated. Thus, in the two-way bone marrow chimera [A + B----(A x B)F1], T lymphocyte populations of A and B haplotypes have each acquired the potential to recognize antigens associated with either parental haplotype. In spite of the large body of work on MHC restriction, we still do not have a clear understanding of the mechanisms which impose self restriction. The chimeric model systems used previously to study MHC restriction have used adult bone marrow cells as the source of lymphoid precursors. During normal ontogeny, T cells are derived from precursors in the fetal liver and we felt that a direct comparison of T cells from fetal liver and bone marrow-repopulated animals would shed light on the development of MHC restriction specificities during T cell ontogeny in the thymus or prethymically. We found that parental T lymphocyte populations isolated from two-way fetal liver chimeras cooperated only with syngeneic APC, while those from bone marrow chimeras cooperated with APC of either parental haplotype. This suggests that fetal liver and bone marrow may not be equivalent sources of stem cells. Our results may be due to fundamental differences between thymocyte precursors in fetal liver and bone marrow, including the time course of their expression of T cell receptor gene products

  16. A procedure for the radioimmunochemical determination of antigens with different structures

    International Nuclear Information System (INIS)

    Galoci, J.; Machan, V.

    1990-01-01

    To a solution of a specific antibody, its antigen labelled with iodine 125, and of the unknown sample is added animal (e.g., goat) antibody against rabbit immunoglobulin immobilized on modified microcrystalline cellulose; always the same quantities are applied always for the same time. After incubation for 1 hr at 20 degC, the immunochemical complex formed is centrifuged and its radioactivity is measured, and the amount of the antigen in sample is determined by comparison with standards. The procedure is applicable to antigens with any structure. Assets of the separating system include a very good stability in solution, a low value of nonspecific bonding and a low sedimentation velocity of the particles used. The procedure is well suited to the quantitation of antigens present in very low concentrations in blood serum, urine, milk, and extracts from tissues or animal feed. In a modification of the procedure, the animal antibody against rabbit immunoglobulin is immobilized on the surface of modified cellulose particles 5 to 15 μm in size exhibiting a very low sedimentation velocity, activated with 1,1-carbonyldiimidazole. (M.D.)

  17. The brain microvascular endothelium supports T cell proliferation and has potential for alloantigen presentation.

    Directory of Open Access Journals (Sweden)

    Julie Wheway

    Full Text Available Endothelial cells (EC form the inner lining of blood vessels and are positioned between circulating lymphocytes and tissues. Hypotheses have formed that EC may act as antigen presenting cells based on the intimate interactions with T cells, which are seen in diseases like multiple sclerosis, cerebral malaria (CM and viral neuropathologies. Here, we investigated how human brain microvascular EC (HBEC interact with and support the proliferation of T cells. We found HBEC to express MHC II, CD40 and ICOSL, key molecules for antigen presentation and co-stimulation and to take up fluorescently labeled antigens via macropinocytosis. In co-cultures, we showed that HBEC support and promote the proliferation of CD4(+ and CD8(+ T cells, which both are key in CM pathogenesis, particularly following T cell receptor activation and co-stimulation. Our findings provide novel evidence that HBEC can trigger T cell activation, thereby providing a novel mechanism for neuroimmunological complications of infectious diseases.

  18. Cd1b-Mediated T Cell Recognition of a Glycolipid Antigen Generated from Mycobacterial Lipid and Host Carbohydrate during Infection

    Science.gov (United States)

    Moody, D. Branch; Guy, Mark R.; Grant, Ethan; Cheng, Tan-Yun; Brenner, Michael B.; Besra, Gurdyal S.; Porcelli, Steven A.

    2000-01-01

    T cells recognize microbial glycolipids presented by CD1 proteins, but there is no information regarding the generation of natural glycolipid antigens within infected tissues. Therefore, we determined the molecular basis of CD1b-restricted T cell recognition of mycobacterial glycosylated mycolates, including those produced during tissue infection in vivo. Transfection of the T cell receptor (TCR) α and β chains from a glucose monomycolate (GMM)-specific T cell line reconstituted GMM recognition in TCR-deficient T lymphoblastoma cells. This TCR-mediated response was highly specific for natural mycobacterial glucose-6-O-(2R, 3R) monomycolate, including the precise structure of the glucose moiety, the stereochemistry of the mycolate lipid, and the linkage between the carbohydrate and the lipid. Mycobacterial production of antigenic GMM absolutely required a nonmycobacterial source of glucose that could be supplied by adding glucose to media at concentrations found in mammalian tissues or by infecting tissue in vivo. These results indicate that mycobacteria synthesized antigenic GMM by coupling mycobacterial mycolates to host-derived glucose. Specific T cell recognition of an epitope formed by interaction of host and pathogen biosynthetic pathways provides a mechanism for immune response to those pathogenic mycobacteria that have productively infected tissues, as distinguished from ubiquitous, but innocuous, environmental mycobacteria. PMID:11015438

  19. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    International Nuclear Information System (INIS)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-01-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with 111 In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity

  20. Lack of radioimmunodetection and complications associated with monoclonal anticarcinoembryonic antigen antibody cross-reactivity with an antigen on circulating cells

    Energy Technology Data Exchange (ETDEWEB)

    Dillman, R.O.; Beauregard, J.C.; Sobol, R.E.; Royston, I.; Bartholomew, R.M.; Hagan, P.S.; Halpern, S.E.

    1984-05-01

    Characterization of several high-affinity murine monoclonal anticarcinoembryonic antigen (CEA) antibodies suggested good specificity except for cross-reactivity with an antigen on granulocytes and erythrocytes which was different from the previously described normal cross-reacting antigen of granulocytes. In vivo studies in athymic mice using an indium conjugate of an anti-CEA monoclonal antibody (MoAb) revealed excellent specific uptake in colorectal carcinoma xenografts. Studies were conducted in humans to determine the limitations produced by the cross-reactivity with granulocytes and erythrocytes. Patients with metastatic colorectal cancer received 3 to 6 mg of anti-CEA MoAb over 10 min or 2 hr. In five of six trials, the MoAb infusion was associated with a 40 to 90% decrease in circulating granulocytes and systemic toxicity including fever, rigors, and emesis. One patient had no change in cell count and had no toxicity. Radionuclide scans with /sup 111/In-anti-CEA MoAb showed marked uptake in the spleen when cells were eliminated, and in the liver, especially when pretreatment CEA levels were high. Metastatic tumor sites failed to concentrate the isotope. This study emphasizes the potential limitations for radioimmunodetection and/or radioimmunotherapy imposed by reactivity with circulating cells, and suggests that certain toxic reactions associated with MoAb infusions are related to destruction of circulating cells rather than allergic reactions to mouse protein. It also emphasizes how variables such as dose and binding affinity of antibody, radioisotope used, and assessment at different observation points can obscure lack of antibody specificity.

  1. Chimeric Antigen Receptor-Engineered T Cells in Tumor Immunotherapy: From Bench to Beside

    Directory of Open Access Journals (Sweden)

    Peng WANG

    2017-06-01

    Full Text Available Chimeric antigen receptor-engineered T cells (CAR-T cells, a classification of cultured T cells after modification of gene engineering technology, can recognize specific tumor antigens in a major histocompatibility complex (MHC-independent manner, consequently leading to the activation of antitumor function. The recent studies have confirmed that a variety of tumor-associated antigens (TAAs can act as target antigens for CAR-T cells. Nowadays, CAR T-cell therapy, one of the most potential tumor immunotherapies, has made great breakthroughs in hematological malignancies and promising outcomes in solid tumors. In this article, the biological characteristics and antitumor mechanism of CAR-T cells, and their application in tumor treatment were mainly reviewed.

  2. Saponins from soy bean and mung bean inhibit the antigen specific activation of helper T cells by blocking cell cycle progression.

    Science.gov (United States)

    Lee, Suk Jun; Bae, Joonbeom; Kim, Sunhee; Jeong, Seonah; Choi, Chang-Yong; Choi, Sang-Pil; Kim, Hyun-Sook; Jung, Woon-Won; Imm, Jee-Young; Kim, Sae Hun; Chun, Taehoon

    2013-02-01

    Treatment of helper T (Th) cells with saponins from soy bean and mung bean prevented their activation by inhibiting cell proliferation and cytokine secretion. However, the saponins did not affect the expression of major histocompatibility complex class II (A(b)) and co-stimulatory molecule (CD86) on professional antigen-presenting cells. Instead, the saponins directly inhibited Th cell proliferation by blocking the G(1) to S phase cell cycle transition. Moreover, blocking of the cell cycle by the saponins was achieved by decreased expression of cyclin D1 and cyclin E, and constitutive expression of p27(KIP1). Saponins also increased stability of p27(KIP1) in Th cells after antigenic stimulation.

  3. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    2010-07-01

    Full Text Available Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  4. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Science.gov (United States)

    Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

    2010-07-26

    Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  5. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma

    Directory of Open Access Journals (Sweden)

    Long Zheng

    2017-12-01

    Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

  6. A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

    Science.gov (United States)

    Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

    2018-05-07

    Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

  7. Mini-review: Can non-human leucocyte antigen genes determine susceptibility to severe dengue syndromes?

    Science.gov (United States)

    Ng, Dorothy; Ghosh, Aparna; Jit, Mark; Seneviratne, Suranjith L

    2017-09-01

    Dengue viral infections are endemic or epidemic in virtually all tropical countries. Among individuals infected with the dengue virus, severe dengue syndromes (i.e., dengue haemorrhagic fever and dengue shock syndromes) tend to affect only some and this may be due to a combination of host genetic susceptibility and viral factors. In this review article we analyse and discuss the present knowledge of non-human leucocyte antigen host genetic susceptibility to severe dengue syndromes. The relevance of genetic polymorphisms in the pathways of antigen recognition, uptake, processing and presentation, activation of interferon α responses, mast cell and complement activation and T cell activation and dengue disease severity has been reviewed and analysed. © The Author(s) 2018. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. [Radiocompetitive method of H antigen determination].

    Science.gov (United States)

    Semenova, G B; Sokolov, Ia A; Liashenko, V A

    1978-06-01

    The authors describe a radiocompetitive method of H-d-monomere determination with the sensitivity of 2 ng/ml in double antibodies modification; this method was used for comparing the immunological affinity of the affiliated H-antigens. A difference between the immunological affinity to the antibodies in a monomere, polymere and the flagellum was shown.

  9. Invariant NKT cells are required for airway inflammation induced by environmental antigens.

    Science.gov (United States)

    Wingender, Gerhard; Rogers, Paul; Batzer, Glenda; Lee, Myung Steve; Bai, Dong; Pei, Bo; Khurana, Archana; Kronenberg, Mitchell; Horner, Anthony A

    2011-06-06

    Invariant NKT cells (iNKT cells) are a unique subset of T lymphocytes that rapidly carry out effector functions. In this study, we report that a majority of sterile house dust extracts (HDEs) tested contained antigens capable of activating mouse and human iNKT cells. HDEs had adjuvant-like properties in an ovalbumin (OVA)-induced asthma model, which were dependent on Vα14i NKT cells, as vaccinated animals deficient for iNKT cells displayed significantly attenuated immune responses and airway inflammation. Furthermore, the administration of HDEs together with OVA mutually augmented the synthesis of cytokines by Vα14i NKT cells and by conventional CD4(+) T cells in the lung, demonstrating a profound immune response synergy for both Th2 cytokines and IL-17A. These data demonstrate that iNKT cell antigens are far more widely dispersed in the environment than previously anticipated. Furthermore, as the antigenic activity in different houses varied greatly, they further suggest that iNKT cell responses to ambient antigens, particular to certain environments, might promote sensitization to conventional respiratory allergens.

  10. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-07-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

  11. Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

    International Nuclear Information System (INIS)

    Kleinhenz, M.E.; Ellner, J.J.

    1985-01-01

    In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

  12. Chimeric antigen receptor T cells: a novel therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    Shengnan Yu

    2017-03-01

    Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

  13. Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

    Science.gov (United States)

    Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

    2014-09-01

    Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

  14. Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line

    International Nuclear Information System (INIS)

    Shiraishi, Yukimasa; Soma, Hiroaki

    1988-01-01

    The authors have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the panning method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer

  15. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  16. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

    Directory of Open Access Journals (Sweden)

    Iris J Gonzalez-Leal

    2014-09-01

    Full Text Available Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb and L (Ctsl play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC and macrophages (BMM from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12 expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  17. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

  18. Challenges and prospects of chimeric antigen receptor T cell therapy in solid tumors.

    Science.gov (United States)

    Jindal, Vishal; Arora, Ena; Gupta, Sorab

    2018-05-05

    Chimeric antigen receptor (CAR) T cell therapy is a novel and innovative immunotherapy. CAR-T cells are genetically engineered T cells, carrying MHC independent specific antigen receptor and co-stimulatory molecule which can activate an immune response to a cancer specific antigen. This therapy showed great results in hematological malignancies but were unable to prove their worth in solid tumors. Likely reasons for their failure are lack of antigens, poor trafficking, and hostile tumor microenvironment. Excessive amount of research is going on to improve the efficacy of CAR T cell therapy in solid tumors. In this article, we will discuss the challenges faced in improving the outcome of CAR T cell therapy in solid tumors and various strategies adopted to curb them.

  19. Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation.

    Science.gov (United States)

    Kopitar, A N; Ihan Hren, N; Ihan, A

    2006-02-01

    In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T-lymphocyte response. To assess the T-lymphocyte response, the expression of CD25, CD69, intracellular interferon gamma (cIFN-gamma), and intracellular interleukin 4 (cIL-4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL-4 and GM-CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2-12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN-gamma by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T-lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL-4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and T(reg) differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.

  20. T Lymphocyte-Endothelial Interactions: Emerging Understanding of Trafficking and Antigen-Specific Immunity

    Directory of Open Access Journals (Sweden)

    Christopher Vincent Carman

    2015-11-01

    Full Text Available Antigen-specific immunity requires regulated trafficking of T cells in and out of diverse tissues in order to orchestrate lymphocyte development, immune surveillance, responses and memory. The endothelium serves as a unique barrier, as well as a sentinel, between the blood and the tissues and as such it plays an essential locally tuned role in regulating T cell migration and information exchange. While it is well established that chemoattractants and adhesion molecules are major determinants of T cell trafficking, emerging studies have now enumerated a large number of molecular players as well as a range of discrete cellular remodeling activities (e.g. transmigratory cups and invadosome-like protrusions, IPLs that participate in directed migration and pathfinding by T cells. In addition to providing trafficking cues, intimate cell-cell interaction between lymphocytes and endothelial cells provide instruction to T cells that influence their activation and differentiation states. Perhaps the most intriguing and underappreciated of these ‘sentinel’ roles is the ability of the endothelium to act as a non-hematopoietic ‘semi-professional’ antigen-presenting cell. Close contacts between circulating T cells and antigen-presenting endothelium may play unique non-redundant roles in shaping adaptive immune responses within the periphery. A better understanding of the mechanisms directing T cell trafficking and the antigen-presenting role of the endothelium may not only increase our knowledge of the adaptive immune response but also empower the utility of emerging immunomodulatory therapeutics.

  1. Microneedle arrays coated with charge reversal pH-sensitive copolymers improve antigen presenting cells-homing DNA vaccine delivery and immune responses.

    Science.gov (United States)

    Duong, Huu Thuy Trang; Kim, Nak Won; Thambi, Thavasyappan; Giang Phan, V H; Lee, Min Sang; Yin, Yue; Jeong, Ji Hoon; Lee, Doo Sung

    2018-01-10

    Successful delivery of a DNA vaccine to antigen-presenting cells and their subsequent stimulation of CD4 + and CD8 + T cell immunity remains an inefficient process. In general, the delivery of prophylactic vaccines is mainly mired by low transfection efficacy, poor immunogenicity, and safety issues from the materials employed. Currently, several strategies have been exploited to improve immunogenicity, but an effective strategy for safe and pain-free delivery of DNA vaccines is complicated. Herein, we report the rapid delivery of polyplex-based DNA vaccines using microneedle arrays coated with a polyelectrolyte multilayer assembly of charge reversal pH-responsive copolymer and heparin. The charge reversal pH-responsive copolymer, composed of oligo(sulfamethazine)-b-poly(ethylene glycol)-b-poly(amino urethane) (OSM-b-PEG-b-PAEU), was used as a triggering layer in the polyelectrolyte multilayer assembly on microneedles. Charge reversal characteristics of this copolymer, that is, the OSM-b-PEG-b-PAEU copolymer exhibit, positive charge at low pH (pH4.03) and becoming negative charge when exposed to physiological pH conditions (pH7.4), allowing the facile assembly and disassembly of polyelectrolyte multilayers. The electrostatic repulsion between heparin and OSM-b-PEG-b-PAEU charge reversal copolymer triggered the release of DNA vaccines. DNA vaccines laden on microneedles are effectively transfected into RAW 264.7 macrophage cells in vitro. Vaccination of BALB/c mice by DNA vaccine-loaded microneedle arrays coated with a polyelectrolyte multilayer generated antigen-specific robust immune responses. These findings provide potential strategy of charge reversal pH-responsive copolymers coated microneedles for DNA vaccine delivery. Copyright © 2017. Published by Elsevier B.V.

  2. A molecular basis for the presentation of phosphorylated peptides by HLA-B antigens

    NARCIS (Netherlands)

    Alpízar, Adán; Marino, Fabio; Ramos-Fernández, Antonio; Lombardía, Manuel; Jeko, Anita; Pazos, Florencio; Paradela, Alberto; Santiago, César; Heck, Albert J R; Marcilla, Miguel

    2017-01-01

    As aberrant protein phosphorylation is a hallmark of tumor cells, the display of tumor-specific phosphopeptides by Human Leukocyte Antigen (HLA) class I molecules can be exploited in the treatment of cancer by T-cell-based immunotherapy. Yet, the characterization and prediction of HLA-I

  3. The molecular bases of δ/αβ T cell-mediated antigen recognition.

    Science.gov (United States)

    Pellicci, Daniel G; Uldrich, Adam P; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B G; de Boer, Renate; Lim, Ricky T; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H M; Gras, Stephanie; Rossjohn, Jamie; Godfrey, Dale I

    2014-12-15

    αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. © 2014 Pellicci et al.

  4. Synergistic cytotoxic effects of antibodies directed against different cell surface determinants

    Energy Technology Data Exchange (ETDEWEB)

    Elliott, E V; Pindar, A; Stevenson, F K; Stevenson, G T [Southampton General Hospital (UK). Tenovus Research Lab.

    1978-03-01

    Three antibody populations were raised in rabbits against surface antigens on guinea-pig L/sub 2/C leukaemic lymphocytes: against idiotypic determinants on the lambda chain of the surface immunoglobulin, against C region determinants on the lambda chain, and against the surface antigens recognised by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L/sub 2/C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.

  5. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  6. Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

    Directory of Open Access Journals (Sweden)

    Michael C. Gong

    1999-06-01

    Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

  7. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. Copyright © 2016. Published by Elsevier B.V.

  8. Production of prostate-specific antigen by a breast cancer cell line, Sk-Br-3

    International Nuclear Information System (INIS)

    Kamali Sarvestani, E.; Ghaderi, A.

    2002-01-01

    Prostate-specific antigen is a 33-KDa serine protease that is produced predominantly by prostate epithelium. However, it has been shown that about 30-40% of female breast tumors produce prostate-specific antigen and its production is associated with the presence of estrogen and progesterone receptors. We have now developed a new tissue culture system to study prostate-specific antigen production in breast cancer and its association with prognostic factors such as progesterone receptor and c-erbB-2. For this purpose we investigated the ability of prostate-specific antigen production in five different cell lines, including two breast cancer cell lines, Sk-Br-3 and MDA-MB-453. The prostate-specific antigen in tissue culture supernatant and cytoplasm of the Sk-Br-3 cell line was detected by western blotting and immunoperoxidase, respectively. Furthermore, we found lower expression of c-erbB-2 in Sk-Br-3 than non-prostate-specific antigen producer breast cancer cell line, MDA-MB-453. Progesterone receptor was expressed by both prostate-specific antigen-positive and -negative cell lines and only the intensity of staining and the number of positive cells in Sk-Br-3 population was higher than MDA-MB-453. According to our findings prostate-specific antigen can be considered as a good prognostic factor in breast cancer and we suggest that these two cell lines are a good in vitro model to study the relationship of different breast cancer prognostic factors and their regulations

  9. CD8(+)NKT-like cells regulate the immune response by killing antigen-bearing DCs.

    Science.gov (United States)

    Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

    2015-09-15

    CD1d-dependent NKT cells have been extensively studied; however, the function of CD8(+)NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8(+)NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8(+)NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8(+)NKT-like cell development is normal in CD1d(-/-) mice, which suggests that CD8(+)NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8(+)NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

  10. CD8+NKT-like cells regulate the immune response by killing antigen-bearing DCs

    Science.gov (United States)

    Wang, Chao; Liu, Xi; Li, Zhengyuan; Chai, Yijie; Jiang, Yunfeng; Wang, Qian; Ji, Yewei; Zhu, Zhongli; Wan, Ying; Yuan, Zhenglong; Chang, Zhijie; Zhang, Minghui

    2015-01-01

    CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens. PMID:26369936

  11. Longitudinal multiparameter single-cell analysis of macaques immunized with pneumococcal protein-conjugated or unconjugated polysaccharide vaccines reveals distinct antigen specific memory B cell repertoires.

    Directory of Open Access Journals (Sweden)

    Bin Jia

    Full Text Available The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to Streptococcus pneumoniae.We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS using microengraving (a single-cell analysis method and single-cell RT-PCR.Seven days after boosting, the mean frequency of antigen-specific memory B cells was significantly increased in macaques vaccinated with 13vPnC compared to those receiving 23vPS. The 13vPnC-vaccinated macaques also exhibited a more even distribution of antibody specificities to four polysaccharides in the vaccine (PS4, 6B, 14, 23F that were examined. However, single-cell analysis of the antibody variable region sequences from antigen-specific B cells elicited by unconjugated and conjugated vaccines indicated that both the germline gene segments forming the heavy chains and the average lengths of the Complementary Determining Region 3 (CDR3 were similar.Our results confirm that distinctive differences can manifest between antigen-specific memory B cell repertoires in nonhuman primates immunized with conjugated and unconjugated pneumococcal polysaccharide vaccines. The study also supports the notion that the conjugated vaccines have a favorable profile in terms of both the frequency and breadth of the anamnestic response among antigen-specific memory B cells.

  12. Virosomes for antigen and DNA delivery

    NARCIS (Netherlands)

    Daemen, T; de Mare, A; Bungener, L; de Jonge, J; Huckriede, A; Wilschut, J

    2005-01-01

    Specific targeting and delivery as well as the display of antigens on the surface of professional antigen-presenting cells (APCs) are key issues in the design and development of new-generation vaccines aimed at the induction of both humoral and cell-mediated immunity. Prophylactic vaccination

  13. Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

    Science.gov (United States)

    Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

    1982-01-01

    A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.

  14. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

    Science.gov (United States)

    Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

    2008-02-15

    Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

  15. Immunophenotyping of Waldenstroms macroglobulinemia cell lines reveals distinct patterns of surface antigen expression: potential biological and therapeutic implications.

    Directory of Open Access Journals (Sweden)

    Aneel Paulus

    Full Text Available Waldenströms macroglobulinemia (WM is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.1, MWCL-1 and RPCI-WM1. During establishment of RPCI-WM1, we observed loss of the CD19 and CD20 antigens, which are typically present on WM cells. Intrigued by this observation and in an effort to better define the immunophenotypic makeup of this cell line, we conducted a more comprehensive analysis for the presence or absence of other cell surface antigens that are present on the RPCI-WM1 model, as well as those on the two other WM cell lines, BCWM.1 and MWCL-1. We examined expression of 65 extracellular and 4 intracellular antigens, comprising B-cell, plasma cell, T-cell, NK-cell, myeloid and hematopoietic stem cell surface markers by flow cytometry analysis. RPCI-WM1 cells demonstrated decreased expression of CD19, CD20, and CD23 with enhanced expression of CD28, CD38 and CD184, antigens that were differentially expressed on BCWM.1 and MWCL-1 cells. Due to increased expression of CD184/CXCR4 and CD38, RPCI-WM1 represents a valuable model in which to study the effects anti-CXCR4 or anti-CD38 targeted therapies that are actively being developed for treatment of hematologic cancers. Overall, differences in surface antigen expression across the 3 cell lines may reflect the tumor clone population predominant in the index patients, from whom the cell lines were developed. Our analysis defines the utility of the most commonly employed WM cell lines as based on their immunophenotype profiles, highlighting unique differences that can be further studied for

  16. Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

    Directory of Open Access Journals (Sweden)

    Antas Paulo RZ

    2002-01-01

    Full Text Available The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05 on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

  17. Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis.

    Science.gov (United States)

    Knapp, W; Strobl, H; Majdic, O

    1994-12-15

    New technology allows highly sensitive flow cytometric detection and quantitative analysis of intracellular antigens in normal and malignant hemopoietic cells. With this technology, the earliest stages of myeloid and lymphoid differentiation can easily and reliably be identified using antibodies directed against (pro-)myeloperoxidase/MPO, CD22 and CD3 antigens, respectively. Particularly for the analysis of undifferentiated acute myeloblastic leukemia (AML) cells, the immunological demonstration of intracellular MPO or its enzymatically inactive proforms is highly relevant, since other myeloid marker molecules such as CD33, CD13, or CDw65 are either not restricted to the granulomonocytic lineage or appear later in differentiation. By combining MPO staining with staining for lactoferrin (LF), undifferentiated cells can be distinguished from the granulomonocytic maturation compartment in bone marrow, since LF is selectively expressed from the myelocyte stage of differentiation onward. The list of informative intracellular antigens to be used in leukemia cell analysis will certainly expand in the near future. One candidate, intracellular CD68, has already been tested by us, and results are presented. Also dealt within this article are surface marker molecules not (as yet) widely used in leukemia cell analysis but with the potential to provide important additional information. Among them are the surface structures CD15, CD15s, CDw65, CD79a (MB-1), CD79b (B29), CD87 (uPA-R), and CD117 (c-kit).

  18. The extended family of CD1d-restricted T cells: sifting through a mixed bag of TCRs, antigens and functions

    Directory of Open Access Journals (Sweden)

    Elodie eMacho-Fernandez

    2015-07-01

    Full Text Available Natural killer T (NKT cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR usage and antigen-specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer, and type II NKT cells that use a more diverse αβ TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αβ TCRs and CD1d-restricted T cells that use γδ or δ/αβ TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, antitumor immunity, and autoimmunity.

  19. The Extended Family of CD1d-Restricted NKT Cells: Sifting through a Mixed Bag of TCRs, Antigens, and Functions.

    Science.gov (United States)

    Macho-Fernandez, Elodie; Brigl, Manfred

    2015-01-01

    Natural killer T (NKT) cells comprise a family of specialized T cells that recognize lipid antigens presented by CD1d. Based on their T cell receptor (TCR) usage and antigen specificities, CD1d-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR α-chain and recognize α-galactosylceramide (α-GalCer), and type II NKT cells that use a more diverse αβ TCR repertoire and do not recognize α-GalCer. In addition, α-GalCer-reactive NKT cells that use non-canonical αβ TCRs and CD1d-restricted T cells that use γδ or δ/αβ TCRs have recently been identified, revealing further diversity among CD1d-restricted T cells. Importantly, in addition to their distinct antigen specificities, functional differences are beginning to emerge between the different members of the CD1d-restricted T cell family. In this review, while using type I NKT cells as comparison, we will focus on type II NKT cells and the other non-invariant CD1d-restricted T cell subsets, and discuss our current understanding of the antigens they recognize, the formation of stimulatory CD1d/antigen complexes, the modes of TCR-mediated antigen recognition, and the mechanisms and consequences of their activation that underlie their function in antimicrobial responses, anti-tumor immunity, and autoimmunity.

  20. Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

    Directory of Open Access Journals (Sweden)

    Jennifer Gordon

    Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

  1. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  2. Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Rossella Cioncada

    Full Text Available MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs, that are facilitated to engulf antigen and transport it to draining lymph node (dLN where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs. Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of

  3. Studies on antigenic competition. Efforts to identify the cellular basis of competition using a cell transfer system

    International Nuclear Information System (INIS)

    McArthur, W.P.; Siskind, G.W.; Thorbecke, G.J.

    1974-01-01

    Antigenic competition was studied in a cell transfer system. The effect of reconstituting lethally irradiated mice with various numbers of thymus or bone marrow cells on the extent of antigenic competition was evaluated. Brucella or burro erythrocytes both caused inhibition of the immune response (Plaque-forming cell) to sheep erythrocytes when given two days prior to the test antigen. Varying the dose of thymus cells, or of bone marrow cells did not alter the degree of competition. Competition was observed even when the competing antigen was injected with bone marrow cells alone two days before the test antigen and thymus cells. The results suggest that mechanisms other than T-cell activation alone must be considered as operative in some models of antigenic competition

  4. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

    Directory of Open Access Journals (Sweden)

    Damián ePérez-Mazliah

    2012-09-01

    Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

  5. Dynamic visualization of dendritic cell-antigen interactions in the skin following transcutaneous immunization.

    Directory of Open Access Journals (Sweden)

    Teerawan Rattanapak

    Full Text Available Delivery of vaccines into the skin provides many advantages over traditional parenteral vaccination and is a promising approach due to the abundance of antigen presenting cells (APC residing in the skin including Langerhans cells (LC and dermal dendritic cells (DDC. However, the main obstacle for transcutaneous immunization (TCI is the effective delivery of the vaccine through the stratum corneum (SC barrier to the APC in the deeper skin layers. This study therefore utilized microneedles (MN and a lipid-based colloidal delivery system (cubosomes as a synergistic approach for the delivery of vaccines to APC in the skin. The process of vaccine uptake and recruitment by specific types of skin APC was investigated in real-time over 4 hours in B6.Cg-Tg (Itgax-EYFP 1 Mnz/J mice by two-photon microscopy. Incorporation of the vaccine into a particulate delivery system and the use of MN preferentially increased vaccine antigen uptake by a highly motile subpopulation of skin APC known as CD207⁺ DC. No uptake of antigen or any response to immunisation by LC could be detected.

  6. Antigenic relatedness of primate procollagens as determined by a competitive radioimmunoassay

    International Nuclear Information System (INIS)

    Taubman, M.B.; Goldberg, B.

    1978-01-01

    A radioimmunoassay specific for the nonhelical carboxy terminal portion of human type I procollagen was used to study the antigenic relatedness of primate procollagens. The assay identified reactive antigen in primate sera and in the media of primate fibroblast cultures. The displacement curves generated in the assay indicated that human and ape type I procollagens have antigenically identical carboxy terminal determinants which are partially cross-reactive with those from Old and New World monkeys. (author)

  7. Immunofluorescent staining of nuclear antigen in lymphoid cells transformed by Herpesvirus papio (HVP).

    Science.gov (United States)

    Schmitz, H

    1981-01-01

    An improved fixation method for antigen detection in lymphoblastoid cells is described. Herpesvirus papio nuclear antigen (HUPNA) could be stained in several transformed lymphoid cell lines by anti-complement immunofluorescence (ACIF). Antibody to HUPNA was detected in many human sera containing antibodies to Epstein-Barr virus capsid and nuclear antigen (EBNA). Rheumatoid arthritis sera showed a high incidence of both anti-EBNA and anti-HUPNA antibodies.

  8. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...

  9. Cis-acting pathways selectively enforce the non-immunogenicity of shed placental antigen for maternal CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Chin-Siean Tay

    Full Text Available Maternal immune tolerance towards the fetus and placenta is thought to be established in part by pathways that attenuate T cell priming to antigens released from the placenta into maternal blood. These pathways remain largely undefined and their existence, at face value, seems incompatible with a mother's need to maintain a functional immune system during pregnancy. A particular conundrum is evident if we consider that maternal antigen presenting cells, activated in order to prime T cells to pathogen-derived antigens, would also have the capacity to prime T cells to co-ingested placental antigens. Here, we address this paradox using a transgenic system in which placental membranes are tagged with a strong surrogate antigen (ovalbumin. We find that although a remarkably large quantity of acellular ovalbumin-containing placental material is released into maternal blood, splenic CD8 T cells in pregnant mice bearing unmanipulated T cell repertoires are not primed to ovalbumin even if the mice are intravenously injected with adjuvants. This failure was largely independent of regulatory T cells, and instead was linked to the intrinsic characteristics of the released material that rendered it selectively non-immunogenic, potentially by sequestering it from CD8α(+ dendritic cells. The release of ovalbumin-containing placental material into maternal blood thus had no discernable impact on CD8 T cell priming to soluble ovalbumin injected intravenously during pregnancy, nor did it induce long-term tolerance to ovalbumin. Together, these results outline a major pathway governing the maternal immune response to the placenta, and suggest how tolerance to placental antigens can be maintained systemically without being detrimental to host defense.

  10. Cis-Acting Pathways Selectively Enforce the Non-Immunogenicity of Shed Placental Antigen for Maternal CD8 T Cells

    Science.gov (United States)

    Tay, Chin-Siean; Tagliani, Elisa; Collins, Mary K.; Erlebacher, Adrian

    2013-01-01

    Maternal immune tolerance towards the fetus and placenta is thought to be established in part by pathways that attenuate T cell priming to antigens released from the placenta into maternal blood. These pathways remain largely undefined and their existence, at face value, seems incompatible with a mother's need to maintain a functional immune system during pregnancy. A particular conundrum is evident if we consider that maternal antigen presenting cells, activated in order to prime T cells to pathogen-derived antigens, would also have the capacity to prime T cells to co-ingested placental antigens. Here, we address this paradox using a transgenic system in which placental membranes are tagged with a strong surrogate antigen (ovalbumin). We find that although a remarkably large quantity of acellular ovalbumin-containing placental material is released into maternal blood, splenic CD8 T cells in pregnant mice bearing unmanipulated T cell repertoires are not primed to ovalbumin even if the mice are intravenously injected with adjuvants. This failure was largely independent of regulatory T cells, and instead was linked to the intrinsic characteristics of the released material that rendered it selectively non-immunogenic, potentially by sequestering it from CD8α+ dendritic cells. The release of ovalbumin-containing placental material into maternal blood thus had no discernable impact on CD8 T cell priming to soluble ovalbumin injected intravenously during pregnancy, nor did it induce long-term tolerance to ovalbumin. Together, these results outline a major pathway governing the maternal immune response to the placenta, and suggest how tolerance to placental antigens can be maintained systemically without being detrimental to host defense. PMID:24391885

  11. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  12. Accessory signals in T-T cell interactions between antigen- and alloantigen-specific, human memory T cells generated in vitro

    DEFF Research Database (Denmark)

    Odum, N; Ryder, L P; Georgsen, J

    1990-01-01

    The potential of activated HLA class II-positive T cells as antigen-/alloantigen-presenting cells remains controversial. In our model system we use in vitro-primed, HLA class II-specific T cells of the memory T-cell phenotype, CD4+, CD29+ (4B4+), and CD45RO+ (UCHL-1). We have previously shown......), or a calcium ionophore (A23187) enabled Ta to elicit alloantigen-specific memory T-cell responses and to present purified protein derivative (PPD) to PPD-specific T-cell lines. The addition of irradiated, Epstein-Barr virus-transformed B-cell lines (EBV-LCL) (but not their supernatants) had a similar but less...

  13. Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

    Directory of Open Access Journals (Sweden)

    Toshiki Ochi

    2010-01-01

    Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

  14. CD4+ T-cell epitope prediction using antigen processing constraints.

    Science.gov (United States)

    Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

    2016-05-01

    T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

    Science.gov (United States)

    Raff, Adam B.; Gray, Andrew; Kast, W. Martin

    2009-01-01

    The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

  16. Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

    Science.gov (United States)

    Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

    2018-05-01

    Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

  17. Comprehensive Analysis of Cytomegalovirus pp65 Antigen-Specific CD8+ T Cell Responses According to Human Leukocyte Antigen Class I Allotypes and Intraindividual Dominance

    Directory of Open Access Journals (Sweden)

    Seung-Joo Hyun

    2017-11-01

    Full Text Available To define whether individual human leukocyte antigen (HLA class I allotypes are used preferentially in human cytomegalovirus (CMV-specific cytotoxic T lymphocyte responses, CD8+ T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C present in 50 healthy Korean donors. The CD8+ T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8+ T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

  18. Intracellular events regulating cross-presentation

    Directory of Open Access Journals (Sweden)

    Peter eCresswell

    2012-06-01

    Full Text Available Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation (ERAD machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on MHC class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation.

  19. Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates

    Science.gov (United States)

    2011-01-01

    167. [10] E.V. Oaks, T.L. Hale, S.B. Formal, Serum immune response to Shigella protein antigens in rhesus monkeys and humans infected with Shigella ...cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in

  20. Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

    Science.gov (United States)

    Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

    2014-01-01

    Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the

  1. Neutral polymer micelle carriers with pH-responsive, endosome-releasing activity modulate antigen trafficking to enhance CD8(+) T cell responses.

    Science.gov (United States)

    Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S

    2014-10-10

    Synthetic subunit vaccines need to induce CD8(+) cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8(+) cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8(+) T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25-30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8(+) T cell responses (0.4% IFN-γ(+) of CD8(+)) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells

  2. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  3. Expression of the Gastrin-Releasing Peptide Receptor, the Prostate Stem Cell Antigen and the Prostate-Specific Membrane Antigen in Lymph Node and Bone Metastases of Prostate Cancer

    NARCIS (Netherlands)

    Ananias, Hildo J. K.; van den Heuvel, Marius C.; Helfrich, Wijnand; de Jong, Igle J.

    2009-01-01

    OBJECTIVE. Cell membrane antigens like the gastrin-releasing peptide receptor (GRPR), the prostate stem cell antigen (PSCA), and the prostate-specific membrane antigen (PSMA), expressed in prostate cancer, are attractive targets for new therapeutic and diagnostic applications. Therefore, we

  4. Acquired Protective Immunity in Atlantic Salmon Salmo salar against the Myxozoan Kudoa thyrsites Involves Induction of MHIIβ+ CD83+ Antigen-Presenting Cells.

    Science.gov (United States)

    Braden, Laura M; Rasmussen, Karina J; Purcell, Sara L; Ellis, Lauren; Mahony, Amelia; Cho, Steven; Whyte, Shona K; Jones, Simon R M; Fast, Mark D

    2018-01-01

    The histozoic myxozoan parasite Kudoa thyrsites causes postmortem myoliquefaction and is responsible for economic losses to salmon aquaculture in the Pacific Northwest. Despite its importance, little is known about the host-parasite relationship, including the host response to infection. The present work sought to characterize the immune response in Atlantic salmon during infection, recovery, and reexposure to K. thyrsites After exposure to infective seawater, infected and uninfected smolts were sampled three times over 4,275 degree-days. Histological analysis revealed infection severity decreased over time in exposed fish, while in controls there was no evidence of infection. Following a secondary exposure of all fish, severity of infection in the controls was similar to that measured in exposed fish at the first sampling time but was significantly reduced in reexposed fish, suggesting the acquisition of protective immunity. Using immunohistochemistry, we detected a population of MHIIβ + cells in infected muscle that followed a pattern of abundance concordant with parasite prevalence. Infiltration of these cells into infected myocytes preceded destruction of the plasmodium and dissemination of myxospores. Dual labeling indicated a majority of these cells were CD83 + /MHIIβ + Using reverse transcription-quantitative PCR, we detected significant induction of cellular effectors, including macrophage/dendritic cells ( mhii / cd83 / mcsf ), B cells ( igm / igt ), and cytotoxic T cells ( cd8 / nkl ), in the musculature of infected fish. These data support a role for cellular effectors such as antigen-presenting cells (monocyte/macrophage and dendritic cells) along with B and T cells in the acquired protective immune response of Atlantic salmon against K. thyrsites . Copyright © 2017 American Society for Microbiology.

  5. Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity

    Science.gov (United States)

    Lu, Yichen; Friedman, Rachel; Kushner, Nicholas; Doling, Amy; Thomas, Lawrence; Touzjian, Neal; Starnbach, Michael; Lieberman, Judy

    2000-07-01

    Bacillus anthrax lethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8-9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cells in vitro. These results lay the foundation for future clinical vaccine studies.

  6. Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

    International Nuclear Information System (INIS)

    Bidart, J.M.; Troalen, F.; Salesse, R.; Bousfield, G.R.; Bohuon, C.J.; Bellet, D.H.

    1987-01-01

    We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex

  7. Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.

    Science.gov (United States)

    Oka, Tatsuya; Rios, Eon J; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J

    2013-10-01

    Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  8. Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

    Directory of Open Access Journals (Sweden)

    M Jedi-Tehrani

    2005-10-01

    Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

  9. Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptidemajor histocompatibility complex I multimers

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

    2016-01-01

    diversity of T cell recognition in humans. Consequently it has been impossible to comprehensively analyze T cell responsiveness in cancer, infectious and autoimmune diseases. We present and validate a novel technology that enables parallel detection of numerous different peptide-MHC responsive T cells...... with combinatorial encoding of fluorescent-labeled MHC multimers. Finally, we have demonstrated that this technology can be applied for multiplex T cell detection both in limited biological samples, such as uncultured tumor material, and for simultaneous assessment of target recognition and functional capability...... of T cells. This technology enables true high-throughput detection of antigen-responsive T cells and will advance our understanding of immune recognition from model antigens to genomewide immune assessments on a personalized basis....

  10. Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

    Energy Technology Data Exchange (ETDEWEB)

    Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie; (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

    2009-07-10

    Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

  11. The link between CD8⁺ T-cell antigen-sensitivity and HIV-suppressive capacity depends on HLA restriction, target epitope and viral isolate.

    Science.gov (United States)

    Lissina, Anna; Fastenackels, Solène; Inglesias, Maria C; Ladell, Kristin; McLaren, James E; Briceño, Olivia; Gostick, Emma; Papagno, Laura; Autran, Brigitte; Sauce, Delphine; Price, David A; Saez-Cirion, Asier; Appay, Victor

    2014-02-20

    Although it is established that CD8 T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8⁺ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8⁺ T-cell responses against the Gag₂₆₃₋₂₇₂ KK10 epitope restricted by human leukocyte antigen (HLA)-B27. To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8⁺ T-cell clones specific for Nef₇₃₋₈₂ QK10 and Gag₂₀₋₂₉ RY10, both restricted by HLA-A3, alongside CD8⁺ T-cell clones specific for Gag₂₆₃₋₂₇₂ KK10. For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates. Collectively, these data emphasize the central role of the TCR as a determinant of CD8⁺ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.

  12. Antigen-specific and non-specific CD4+ T cell recruitment and proliferation during influenza infection

    International Nuclear Information System (INIS)

    Chapman, Timothy J.; Castrucci, Maria R.; Padrick, Ryan C.; Bradley, Linda M.; Topham, David J.

    2005-01-01

    To track epitope-specific CD4 + T cells at a single-cell level during influenza infection, the MHC class II-restricted OVA 323-339 epitope was engineered into the neuraminidase stalk of influenza/A/WSN, creating a surrogate viral antigen. The recombinant virus, influenza A/WSN/OVA II , replicated well, was cleared normally, and stimulated both wild-type and DO11.10 or OT-II TCR transgenic OVA-specific CD4 + T cells. OVA-specific CD4 T cells proliferated during infection only when the OVA epitope was present. However, previously primed (but not naive) transgenic CD4 + T cells were recruited to the infected lung both in the presence and absence of the OVA 323-339 epitope. These data show that, when primed, CD4 + T cells may traffic to the lung in the absence of antigen, but do not proliferate. These results also document a useful tool for the study of CD4 T cells in influenza infection

  13. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA in the pig ovary.

    Directory of Open Access Journals (Sweden)

    Milan TomĂĄnek

    2007-01-01

    Full Text Available The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.

  14. Cell surface antigens of radiation leukemia virus-induced BALB/c leukemias defined by syngeneic cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Kaneko, Yukio; Oettgen, H.F.; Obata, Yuichi; Nakayama, Eiichi.

    1989-01-01

    Two cell surface antigens of mouse leukemias were defined by BALB/c cytotoxic T lymphocytes (CTL) generated against syngeneic radiation leukemia virus (RadLV)-induced leukemia, BALBRV1 or BALBRVD. Hyperimmunization of BALB/c mice with irradiated leukemias followed by in vitro sensitization of primed spleen cells resulted in the generation of CTL with high killing activity. The specificity of CTL was examined by direct cytotoxicity assays and competitive inhibition assays. A shared cell surface antigen, designated as BALBRV1 antigen, was detected by BALB/c anti-BALBRV1 CTL. BALBRV1 antigen was expressed not only on RadLV-induced BALB/c leukemias except for BALBRVD, but also on spontaneous or X-ray-induced BALB/c leukemias, chemically-induced leukemias with the H-2 d haplotype and some chemically-induced BALB/c sarcomas. In contrast, a unique cell surface antigen, designated as BALBRVD antigen, was detected by BALB/c anti-BALBRVD CTL. BALBRVD antigen was expressed only on BALBRVD, but not on thirty-nine normal lymphoid or tumor cells. These two antigens could be distinguished from those previously defined on Friend, Moloney, Rauscher or Gross murine leukemia virus (MuLV) leukemias, or MuLV-related antigens. Both cytotoxic responses were blocked by antisera against H-2K d , but not H-2D d . The relationship of BALBRV1 antigen and BALBRVD antigen to endogenous MuLV is discussed with regard to the antigenic distribution on tumor cell lines. (author)

  15. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

    Science.gov (United States)

    Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C

    2018-04-16

    The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

  16. A novel and effective cancer immunotherapy mouse model using antigen-specific B cells selected in vitro.

    Directory of Open Access Journals (Sweden)

    Tatsuya Moutai

    Full Text Available Immunotherapies such as adoptive transfer of T cells or natural killer cells, or monoclonal antibody (MoAb treatment have recently been recognized as effective means to treat cancer patients. However, adoptive transfer of B cells or plasma cells producing tumor-specific antibodies has not been applied as a therapy because long-term culture and selective expansion of antigen-specific B cells has been technically very difficult. Here, we describe a novel cancer immunotherapy that uses B-cell adoptive transfer. We demonstrate that germinal-center-like B cells (iGB cells induced in vitro from mouse naïve B cells become plasma cells and produce IgG antibodies for more than a month in the bone marrow of non-irradiated recipient mice. When transferred into mice, iGB cells producing antibody against a surrogate tumor antigen suppressed lung metastasis and growth of mouse melanoma cells expressing the same antigen and prolonged survival of the recipients. In addition, we have developed a novel culture system called FAIS to selectively expand antigen-specific iGB cells utilizing the fact that iGB cells are sensitive to Fas-induced cell death unless their antigen receptors are ligated by membrane-bound antigens. The selected iGB cells efficiently suppressed lung metastasis of melanoma cells in the adoptive immunotherapy model. As human blood B cells can be propagated as iGB cells using culture conditions similar to the mouse iGB cell cultures, our data suggest that it will be possible to treat cancer-bearing patients by the adoptive transfer of cancer-antigen-specific iGB cells selected in vitro. This new adoptive immunotherapy should be an alternative to the laborious development of MoAb drugs against cancers for which no effective treatments currently exist.

  17. Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells. Immunological study using monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Hareyama, Masato

    1988-12-01

    We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-..gamma... In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G/sub 2//M phases of the cell cycle than in G/sub 0//G/sub 1/. The expression of YH206 antigen was enhanced in the S and G/sub 2//M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens.

  18. Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells.

    Directory of Open Access Journals (Sweden)

    JoĂŤl Pestel

    2008-06-01

    Full Text Available Mycobacterium bovis bacillus Calmette-GuĂŠrin (BCG is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

  19. Naive helper T cells from BCG-vaccinated volunteers produce IFN-gamma and IL-5 to mycobacterial antigen-pulsed dendritic cells.

    Science.gov (United States)

    Kowalewicz-Kulbat, Magdalena; Kaźmierczak, Dominik; Donevski, Stefan; Biet, Franck; Pestel, Joël; Rudnicka, Wiesława

    2008-01-01

    Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs) are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

  20. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  1. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    Directory of Open Access Journals (Sweden)

    Marc Cartellieri

    2010-01-01

    Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

  2. Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

    Science.gov (United States)

    Bowers, Edith V.; Horvath, Jeffrey J.; Bond, Jennifer E.; Cianciolo, George J.; Pizzo, Salvatore V.

    2009-01-01

    α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that α2M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α2M*-OVA compared with soluble OVA. The frequency of IFN-γ-producing cells was increased ∼15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*-OVA. These α2M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support α2M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing. PMID:19652028

  3. Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Kurtzhals, J A

    1994-01-01

    of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after...... the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL...... stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from...

  4. A simple assay for the detection of antibodies to endocrine islet cell surface antigens

    International Nuclear Information System (INIS)

    Contreas, G.; Madsen, O.D.; Vissing, H.; Lernmark, Aa.

    1986-01-01

    A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125 I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens. (Auth.)

  5. Organic extract of diesel exhaust particles stimulates expression of Ia and costimulatory molecules associated with antigen presentation in rat peripheral blood monocytes but not in alveolar macrophages

    International Nuclear Information System (INIS)

    Koike, Eiko; Kobayashi, Takahiro

    2005-01-01

    We hypothesized that diesel exhaust particles (DEP) induce the activation of antigen-presenting cells (APC) in lung. The present study was designed to clarify the following about DEP: (1) whether it affects the expression of Ia and B7 molecules in alveolar macrophages (AM) as a mature cell or in peripheral blood monocytes (PBM) as an immature cell (2) if it affects the antigen-presenting (AP) activity of PBM (3) what component of DEP is responsible for the effects, and (4) whether the effect of DEP is related to oxidative stress. DEP was extracted with methylene chloride. Cells were exposed to whole DEP, organic extract, or residual particles for 24 h. Cell-surface molecules were measured by flow cytometry. AP activity was assessed by antigen-specific T cell proliferation. Whole DEP or organic extract significantly increased the expression of Ia and B7 molecules on PBM but not on AM. No significant effect of residual particles was observed. A low concentration of organic extract also increased the AP activity of PBM. When the induction of an antioxidative enzyme was assessed, heme oxygenase-1 protein was found to be significantly increased by exposure to whole DEP, and the organic extract was more effective than the residual particles. Furthermore, the organic extract-induced expression of Ia antigen on PBM was reduced by the addition of an antioxidative agent. These results suggest that DEP may act on immature APC and enhance their AP activity and that the action contributing to oxidative stress may be mediated by organic compounds of DEP

  6. Dissection and manipulation of antigen-specific T cell responses

    NARCIS (Netherlands)

    Schepers, Koen

    2006-01-01

    T cells recognize pathogen-derived antigens and are crucial for fighting pathogens such as viruses and bacteria. In addition, T cells are able to recognize and attack certain types of tumors, in particular virally induced tumors. In this thesis we aimed 1) to obtain more insight into

  7. Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy.

    Science.gov (United States)

    Li Pira, Giuseppina; Di Cecca, Stefano; Biagini, Simone; Girolami, Elia; Cicchetti, Elisabetta; Bertaina, Valentina; Quintarelli, Concetta; Caruana, Ignazio; Lucarelli, Barbarella; Merli, Pietro; Pagliara, Daria; Brescia, Letizia Pomponia; Bertaina, Alice; Montanari, Mauro; Locatelli, Franco

    2017-01-01

    Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

  8. Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy

    Science.gov (United States)

    Li Pira, Giuseppina; Di Cecca, Stefano; Biagini, Simone; Girolami, Elia; Cicchetti, Elisabetta; Bertaina, Valentina; Quintarelli, Concetta; Caruana, Ignazio; Lucarelli, Barbarella; Merli, Pietro; Pagliara, Daria; Brescia, Letizia Pomponia; Bertaina, Alice; Montanari, Mauro; Locatelli, Franco

    2017-01-01

    Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification. PMID:28386262

  9. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  10. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  11. Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

    Directory of Open Access Journals (Sweden)

    Ciprian Tomuleasa

    2018-02-01

    Full Text Available Chimeric antigen receptor (CAR T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

  12. Functional antigen binding by the defective B cells of CBA/N mice.

    Science.gov (United States)

    Snippe, H; Merchant, B; Lizzio, E F; Inman, J K

    1982-01-01

    CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

  13. Novel Antitumor Strategy Utilizing a Plasmid Expressing a Mycobacterium tuberculosis Antigen as a “Danger Signal” to Block Immune Escape of Tumor Cells

    Directory of Open Access Journals (Sweden)

    Yoshiyuki Koyama

    2015-07-01

    Full Text Available Immune escape of tumor cells is one of the main obstacles hindering the effectiveness of cancer immunotherapy. We developed a novel strategy to block immune escape by transfecting tumor cells in vivo with genes of pathogenic antigens from Mycobacterium tuberculosis (TB. This induces presentation of the TB antigen on tumor cell surfaces, which can be recognized by antigen presenting cells (APCs as a “danger signal” to stimulate antitumor immune response. This strategy is also expected to amplify the immune response against tumor-associated antigens, and block immune escape of the tumor. DNA/PEI/chondroitin sulfate ternary complex is a highly effective non-viral gene vector system for in vivo transfection. A therapeutic complex was prepared using a plasmid encoding the TB antigen, early secretory antigenic target-6 (ESAT-6. This was injected intratumorally into syngeneic tumor-bearing mice, and induced significant tumor growth suppression comparable to or higher than similar complexes expressing cytokines such as interleukin-2 (IL-2 and interleukin-12 (IL-12. Co-transfection of the cytokine-genes and the ESAT-6-gene enhanced the antitumor efficacy of either treatment alone. In addition, complete tumor regression was achieved with the combination of ESAT-6 and IL-2 genes.

  14. Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA

    International Nuclear Information System (INIS)

    Pretell, J.O.; Cone, R.E.

    1985-01-01

    Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA

  15. Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray.

    Directory of Open Access Journals (Sweden)

    Daniel S Chen

    2005-10-01

    Full Text Available In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma and tumor necrosis factor-alpha (TNFalpha in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

  16. Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

    Science.gov (United States)

    Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

    2016-11-01

    To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p exosomes significantly suppressed (p exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

  17. Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

    Directory of Open Access Journals (Sweden)

    Kuo-Ching Sheng

    2013-01-01

    Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

  18. Survival and antigenic profile of irradiated malarial sporozoites in infected liver cells

    International Nuclear Information System (INIS)

    Suhrbier, A.; Winger, L.A.; Castellano, E.; Sinden, R.E.

    1990-01-01

    Exoerythrocytic (EE) stages of Plasmodium berghei derived from irradiated sporozoites were cultured in vitro in HepG2 cells. They synthesized several antigens, predominantly but not exclusively those expressed by normal early erythrocytic schizonts. After invasion, over half the intracellular sporozoites, both normal and irradiated, appeared to die. After 24 h, in marked contrast to the normal parasites, EE parasites derived from irradiated sporozoites continued to break open, shedding their antigens into the cytoplasm of the infected host cells. Increasing radiation dosage, which has previously been shown to reduce the ability of irradiated sporozoites to protect animals, correlated with reduced de novo antigen synthesis by EE parasites derived from irradiated sporozoites

  19. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  20. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  1. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Science.gov (United States)

    2012-01-24

    ... Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into Therapeutics for.... 61/473,409 entitled ``Anti-epidermal growth factor receptor variant III chimeric antigen receptors... EGFRvIII chimeric antigen (CARs) and methods of using these engineered T cells to treat and/or prevent...

  2. Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

    Science.gov (United States)

    Lai, Zengzu; Schreiber, John R

    2009-05-21

    Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

  3. Antigen-specific T8+ human clone of cells with a nonspecific augmenting function on the T4 cell-B cell helper interaction

    International Nuclear Information System (INIS)

    Brines, R.D.; Sia, D.Y.; Lehner, T.

    1987-01-01

    The authors isolated a T8 + T3 + Ia + clone of cells from the peripheral blood mononuclear cells of a healthy subject. The clone was expanded and maintained with autologous feed cells, interleukin 2, and a streptococcal antigen. The T8 + clone of cells responded specifically to the streptococcal antigen, in the absence of accessory cells,and released a soluble factor. Both the cloned cells and the corresponding soluble factor expressed augmenting helper but not suppressor activity. The augmenting helper activity for B cell antibody synthesis was demonstrable only in the presence of autologous T 4 cells. Radioimmunoassay was used to measure antibodies. Although stimulation of the T8 + cloned cells was antigen-specific, the resulting soluble factor elicited nonspecific antibody synthesis in the presence of T4 and B cells. The T8 + cloned cell-derived factor was adsorbed by B cells but not by T4 cells. Preliminary studies suggest that the factor has the properties of a B cell growth factor. They suggest that the T8 + population consists of functionally heterogeneous cell subsets, some that have suppressor function and others that augment the T4 + helper-inducer activity in B cell antibody synthesis

  4. Gap junctions at the dendritic cell-T cell interface are key elements for antigen-dependent T cell activation.

    Science.gov (United States)

    Elgueta, Raul; Tobar, Jaime A; Shoji, Kenji F; De Calisto, Jaime; Kalergis, Alexis M; Bono, Maria R; Rosemblatt, Mario; Sáez, Juan C

    2009-07-01

    The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4(+) T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.

  5. Antigen Loss Variants: Catching Hold of Escaping Foes.

    Science.gov (United States)

    Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

    2017-01-01

    Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

  6. Chemoselective ligation and antigen vectorization.

    Science.gov (United States)

    Gras-Masse, H

    2001-01-01

    The interest in cocktail-lipopeptide vaccines has now been confirmed by phase I clinical trials: highly diversified B-, T-helper or cytotoxic T-cell epitopes can be combined with a lipophilic vector for the induction of B- and T-cell responses of predetermined specificity. With the goal of producing an improved vaccine that should ideally induce a multispecific response in non-selected populations, increasing the diversity of the immunizing mixture represents one of the most obvious strategies.The selective delivery of antigens to professional antigen-presenting cells represents another promising approach for the improvement of vaccine efficacy. In this context, the mannose-receptor represents an attractive entry point for the targeting to dendritic cells of antigens linked to clustered glycosides or glycomimetics. In all cases, highly complex but fully characterized molecules must be produced. To develop a modular and flexible strategy which could be generally applicable to a large set of peptide antigens, we elected to explore the potentialities of chemoselective ligation methods. The hydrazone bond was found particularly reliable and fully compatible with sulphide ligation. Hydrazone/thioether orthogonal ligation systems could be developed to account for the nature of the antigens and the solubility of the vector systems. Copyright 2001 The International Association for Biologicals.

  7. Characterization of Yellow Fever Virus Infection of Human and Non-human Primate Antigen Presenting Cells and Their Interaction with CD4+ T Cells.

    Directory of Open Access Journals (Sweden)

    Yu Cong

    2016-05-01

    Full Text Available Humans infected with yellow fever virus (YFV, a mosquito-borne flavivirus, can develop illness ranging from a mild febrile disease to hemorrhagic fever and death. The 17D vaccine strain of YFV was developed in the 1930s, has been used continuously since development and has proven very effective. Genetic differences between vaccine and wild-type viruses are few, yet viral or host mechanisms associated with protection or disease are not fully understood. Over the past 20 years, a number of cases of vaccine-associated disease have been identified following vaccination with 17D; these cases have been correlated with reduced immune status at the time of vaccination. Recently, several studies have evaluated T cell responses to vaccination in both humans and non-human primates, but none have evaluated the response to wild-type virus infection. In the studies described here, monocyte-derived macrophages (MDM and dendritic cells (MoDC from both humans and rhesus macaques were evaluated for their ability to support infection with either wild-type Asibi virus or the 17D vaccine strain and the host cytokine and chemokine response characterized. Human MoDC and MDM were also evaluated for their ability to stimulate CD4+ T cells. It was found that MoDC and MDM supported viral replication and that there were differential cytokine responses to infection with either wild-type or vaccine viruses. Additionally, MoDCs infected with live 17D virus were able to stimulate IFN-γ and IL-2 production in CD4+ T cells, while cells infected with Asibi virus were not. These data demonstrate that wild-type and vaccine YFV stimulate different responses in target antigen presenting cells and that wild-type YFV can inhibit MoDC activation of CD4+ T cells, a critical component in development of protective immunity. These data provide initial, but critical insight into regulatory capabilities of wild-type YFV in development of disease.

  8. Regional Delivery of Chimeric Antigen Receptor (CAR) T-Cells for Cancer Therapy.

    Science.gov (United States)

    Sridhar, Praveen; Petrocca, Fabio

    2017-07-18

    Chimeric Antigen Receptor (CAR) T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

  9. Regional Delivery of Chimeric Antigen Receptor (CAR T-Cells for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Praveen Sridhar

    2017-07-01

    Full Text Available Chimeric Antigen Receptor (CAR T-cells are T-cells with recombinant receptors targeted to tumor antigens. CAR-T cell therapy has emerged as a mode of immunotherapy and is now being extensively explored in hematologic cancer. In contrast, CAR-T cell use in solid tumors has been hampered by multiple obstacles. Several approaches have been taken to circumvent these obstacles, including the regional delivery of CAR-T cells. Regional CAR-T cell delivery can theoretically compensate for poor T-cell trafficking and tumor antigen specificity while avoiding systemic toxicity associated with intravenous delivery. We reviewed completed clinical trials for the treatment of glioblastoma and metastatic colorectal cancer and examined the data in these studies for safety, efficacy, and potential advantages that regional delivery may confer over systemic delivery. Our appraisal of the available literature revealed that regional delivery of CAR-T cells in both glioblastoma and hepatic colorectal metastases was generally well tolerated and efficacious in select instances. We propose that the regional delivery of CAR-T cells is an area of potential growth in the solid tumor immunotherapy, and look towards future clinical trials in head and neck cancer, mesothelioma, and peritoneal carcinomatosis as the use of this technique expands.

  10. Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Tomasz Łęga

    Full Text Available Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human-avian-swine-human M2e (M2eH-A-S-H peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system.

  11. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

    Science.gov (United States)

    Hay, Kevin A; Turtle, Cameron J

    2017-03-01

    Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

  12. Effects of radiation on the expression of antigens on the membranes of human adenocarcinoma cells

    International Nuclear Information System (INIS)

    Hareyama, Masato; Imai, Kozo; Oouchi, Atsushi; Shidou, Mitsuo; Takahashi, Hiroki; Koshiba, Hirohumi; Yachi, Akira; Morita, Kazuo

    1992-01-01

    We have investigated the effects of irradiation on the membranes of tumor cells. X-ray irradiation caused remarkable increases in the expression of tumor-associated antigens (YH206, CEA) and c-erbB-2 protein by flow cytometry, whereas IFN had no obvious effect on the expression of tumor-associated antigens and c-erbB-2 protein. On the other hand, the expression of MHC Class I and ICAM-1 antigen on the membrane by flow cytometry was enhanced by both irradiation and IFN. In addition, the irradiated cells, when analyzed using a CEA specific probe, showed remarkable increases in the CEA mRNA compared to IFN-treated cells. It is possible that enhancement of the expression of tumor-associated antigens and c-erbB-2 protein, together with the enhancement of that of MHC-Class I and ICAM-1, would help cytotoxic killer cells recognize the tumor cells. (author)

  13. Chimeric Antigen Receptor (CAR) T cells: Lessons Learned from Targeting of CD19 in B cell malignancies

    Science.gov (United States)

    Hay, Kevin A; Turtle, Cameron J

    2017-01-01

    Adoptive immunotherapy with chimeric antigen receptor-modified T (CAR-T) cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers. PMID:28110394

  14. IgM and IgD B cell receptors differentially respond to endogenous antigens and control B cell fate

    Science.gov (United States)

    Noviski, Mark; Mueller, James L; Satterthwaite, Anne; Garrett-Sinha, Lee Ann; Brombacher, Frank

    2018-01-01

    Naive B cells co-express two BCR isotypes, IgM and IgD, with identical antigen-binding domains but distinct constant regions. IgM but not IgD is downregulated on autoreactive B cells. Because these isotypes are presumed to be redundant, it is unknown how this could impose tolerance. We introduced the Nur77-eGFP reporter of BCR signaling into mice that express each BCR isotype alone. Despite signaling strongly in vitro, IgD is less sensitive than IgM to endogenous antigen in vivo and developmental fate decisions are skewed accordingly. IgD-only Lyn−/− B cells cannot generate autoantibodies and short-lived plasma cells (SLPCs) in vivo, a fate thought to be driven by intense BCR signaling induced by endogenous antigens. Similarly, IgD-only B cells generate normal germinal center, but impaired IgG1+ SLPC responses to T-dependent immunization. We propose a role for IgD in maintaining the quiescence of autoreactive B cells and restricting their differentiation into autoantibody secreting cells. PMID:29521626

  15. A Critical Analysis of the Role of SNARE Protein SEC22B in Antigen Cross-Presentation

    Directory of Open Access Journals (Sweden)

    S. Julia Wu

    2017-06-01

    Full Text Available Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl mice. Contrary to the paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Although in vitro small hairpin RNA (shRNA-mediated Sec22b silencing in bone-marrow-derived dendritic cells (BMDCs reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNA sequencing of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that contrary to the accepted model, SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

  16. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

    OpenAIRE

    Stroncek, David F.; Lee, Daniel W.; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N.; Kaplan, Rosandra N.; Fry, Terry J.; Mackall, Crystal L.

    2017-01-01

    Background Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Methods Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We ...

  17. Kinetics of human T-cell expression of LFA-1, IL-2 receptor, and ICAM-1 following antigenic stimulation in vitro

    DEFF Research Database (Denmark)

    Hviid, L; Felsing, A; Theander, T G

    1993-01-01

    -specific stimulation is available. In the present study we have examined phenotypic T-cell changes after in vitro stimulation by the antigens purified derivative of tuberculin (PPD) and tetanus toxoid (TT). We show that the well-established differences in kinetics of mitogen- and antigen-induced T-cell proliferation...... of all 3 surface antigens showed similar kinetics, and correlated with the magnitude of the lymphoproliferative response. By day 8 (PHA-stimulation) or day 12 (PPD or TT stimulation), the lymphoproliferative response was essentially completed, the expression of CD11a and CD54 had approached...

  18. Memory control by the B cell antigen receptor.

    Science.gov (United States)

    Engels, Niklas; Wienands, Jürgen

    2018-05-01

    The generation of memory B cells (MBCs) that have undergone immunoglobulin class switching from IgM, which dominates primary antibody responses, to other immunoglobulin isoforms is a hallmark of immune memory. Hence, humoral immunological memory is characterized by the presence of serum immunoglobulins of IgG subtypes known as the γ-globulin fraction of blood plasma proteins. These antibodies reflect the antigen experience of B lymphocytes and their repeated triggering. In fact, efficient protection against a previously encountered pathogen is critically linked to the production of pathogen-specific IgG molecules even in those cases where the primary immune response required cellular immunity, for example, T cell-mediated clearance of intracellular pathogens such as viruses. Besides IgG, also IgA and IgE can provide humoral immunity depending on the microbe's nature and infection route. The molecular mechanisms underlying the preponderance of switched immunoglobulin isotypes during memory antibody responses are a matter of active and controversial debate. Here, we summarize the phenotypic characteristics of distinct MBC subpopulations and discuss the decisive roles of different B cell antigen receptor isotypes for the functional traits of class-switched B cell populations. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

    Directory of Open Access Journals (Sweden)

    Christine Bee

    Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

  20. The 2.5 Å Structure of CD1c in Complex with a Mycobacterial Lipid Reveals an Open Groove Ideally Suited for Diverse Antigen Presentation

    Energy Technology Data Exchange (ETDEWEB)

    Scharf, Louise; Li, Nan-Sheng; Hawk, Andrew J.; Garzón, Diana; Zhang, Tejia; Fox, Lisa M.; Kazen, Allison R.; Shah, Sneha; Haddadian, Esmael J.; Gumperz, Jenny E.; Saghatelian, Alan; Faraldo-Gómez, José D.; Meredith, Stephen C.; Piccirilli, Joseph A.; Adams, Erin J. (Harvard); (UC); (MXPL-G); (UW-MED)

    2011-08-24

    CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 {angstrom} resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-{beta}1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A pocket, aided by a unique exit portal underneath the {alpha}1 helix. Most striking was an open F pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

  1. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  2. Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins.

    Science.gov (United States)

    Sparwasser, T; Vabulas, R M; Villmow, B; Lipford, G B; Wagner, H

    2000-12-01

    Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

  3. Recognition of melanoma-derived antigens by CTL: possible mechanisms involved in down-regulating anti-tumor T-cell reactivity

    DEFF Research Database (Denmark)

    Rivoltini, L; Loftus, D J; Squarcina, P

    1998-01-01

    Several T cell-recognized epitopes presented by melanoma cells have been identified recently. Despite the large array of epitopes potentially available for clinical use, it is still unclear which of these antigens could be effective in mediating anti-tumor responses when used as a vaccine...

  4. Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

    Science.gov (United States)

    Ofran, Yanay; Schlessinger, Avner; Rost, Burkhard

    2008-11-01

    Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

  5. Antigen-Addicted T Cell Reserves Trickle Charge the Frontline Killers.

    Science.gov (United States)

    Kalia, Vandana; Sarkar, Surojit

    2016-07-19

    Highly active killer T cells mediate a stable standoff during controlled persistent infections. In this issue of Immunity, Robey and colleagues describe a unique antigen-addicted T cell population bearing characteristics of both effector and memory CD8(+) T cells that provides a continuous supply of potent killer T cells to curb Toxoplasma gondii growth during latency. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Mass Spectrometry Reveals Changes in MHC I Antigen Presentation After Lentivector Expression of a Gene Regulation System

    Directory of Open Access Journals (Sweden)

    Roland Vogel

    2013-01-01

    Full Text Available The rapamycin-inducible gene regulation system was designed to minimize immune reactions in man and may thus be suited for gene therapy. We assessed whether this system indeed induces no immune responses. The protein components of the regulation system were produced in the human cell lines HEK 293T, D407, and HER 911 following lentiviral transfer of the corresponding genes. Stable cell lines were established, and the peptides presented by major histocompatibility complex class I (MHC I molecules on transduced and wild-type (wt cells were compared by differential mass spectrometry. In all cell lines examined, expression of the transgenes resulted in prominent changes in the repertoire of MHC I-presented self-peptides. No MHC I ligands originating from the transgenic proteins were detected. In vitro analysis of immunogenicity revealed that transduced D407 cells displayed slightly higher capacity than wt controls to promote proliferation of cytotoxic T cells. These results indicate that therapeutic manipulations within the genome of target cells may affect pathways involved in the processing of peptide antigens and their presentation by MHC I. This makes the genomic modifications visible to the immune system which may recognize these events and respond. Ultimately, the findings call attention to a possible immune risk.

  7. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

  8. Suppressor cells in transplantation tolerance. III. The role of antigen in the maintenance of transplantation tolerance

    International Nuclear Information System (INIS)

    Tutschka, P.J.; Hess, A.D.; Beschorner, W.E.; Santos, G.W.

    1982-01-01

    Suppressor cells, which in an alloantigen-specific manner inhibit proliferation of donor cells to host antigens in a mixed lymphocyte culture and adoptively transfer the suppression of graft-versus-host disease (GVHD), undergo a gradual clonal reduction in long-term, allogeneic, histoincompatible rat radiation chimeras until they can no longer be measured in an in vitro suppressor cell assay. When lymphohematopoietic cells from these chimeras are transferred into lethally irradiated secondary recipients of original donor strain, the suppressor cells, now in a target antigen-free environment, undergo a further clonal reduction. After parking for 120 days, the chimeric cells are specifically tolerant to original host antigens, but cannot adoptively transfer suppression of GVHD. When chimeric cells, parked for 120 days in secondary recipients of original donor strain, are stimulated with original host-type antigen repeatedly during or once at the end of the parking period, the suppressor cell clone is expanded, suppressor cells can be identified in vitro, and suppression of GVHD can adoptively be transferred to tertiary recipients

  9. Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Patrycja Anna Kobierecka

    2016-02-01

    Full Text Available Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered

  10. EFFECT OF LIPOSOMAL CLODRONATE-DEPENDENT DEPLETION OF PROFESSIONAL ANTIGEN PRESENTING CELLS ON NUMBERS AND PHENOTYPE OF CANINE CD4+CD25+FOXP3+ REGULATORY T CELLS

    Science.gov (United States)

    Weaver, Kriston F.; Stokes, John V.; Gunnoe, Sagen A.; Follows, Joyce S.; Shafer, Lydia; Ammari, Mais G.; Archer, Todd M.; Thomason, John M.; Mackin, Andrew J.; Pinchuk, Lesya M.

    2015-01-01

    Regulatory T cells (Tregs) are known to control autoreactivity during and subsequent to the development of the peripheral immune system. Professional antigen presenting cells (APCs), dendritic cells (DCs) and monocytes, have an important role in inducing Tregs. For the first time, this study evaluated proportions and phenotypes of Tregs in canine peripheral blood depleted of professional APCs, utilizing liposomal clodronate (LC) and multicolor flow cytometry analysis. Our results demonstrate that LC exposure promoted short term decreases followed by significant increases in the proportions or absolute numbers of CD4+CD25+FOXP3+ Tregs in dogs. In general, the LC-dependent Treg fluctuations were similar to the changes in the levels of CD14+ monocytes in Walker hounds. However, the proportions of monocytes showed more dramatic changes compared to the proportions of Tregs that were visually unchanged after LC treatment over the study period. At the same time, absolute Treg numbers showed, similarly to the levels of CD14+ monocytes, significant compensatory gains as well as the recovery during the normalization period. We confirm the previous data that CD4+ T cells with the highest CD25 expression were highly enriched for FOXP3. Furthermore, for the first time, we report that CD4+CD25lowFOXP3+ is the major regulatory T cell subset affected by LC exposure. The increases within the lowest CD25 expressers of CD4+FOXP3+ cells together with compensatory gains in the proportion of CD14+ monocytes during compensatory and normalization periods suggest the possible direct or indirect roles of monocytes in active recruitment and generation of Tregs from naïve CD4+ T cells. PMID:25950023

  11. Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

    Science.gov (United States)

    Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

    2014-01-01

    Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

  12. How to train your T cell: genetically engineered chimeric antigen receptor T cells versus bispecific T-cell engagers to target CD19 in B acute lymphoblastic leukemia.

    Science.gov (United States)

    Ruella, Marco; Gill, Saar

    2015-06-01

    Antigen-specific T cell-based immunotherapy is getting its day in the sun. The contemporaneous development of two potent CD19-specific immunotherapeutic modalities for the treatment of B-cell malignancies provides exciting opportunities for patients, physicians and scientists alike. Patients with relapsed, refractory or poor-risk B-cell acute lymphoblastic leukemia (ALL) previously had few therapeutic options and now have two potential new lifelines. Physicians will have the choice between two powerful modalities and indeed could potentially enroll some patients on trials exploring both modalities if needed. For scientists interested in tumor immunology, the advent of chimeric antigen receptor T-cell therapy and of bispecific T-cell engagers (BiTEs) provides unprecedented opportunities to explore the promise and limitations of antigen-specific T-cell therapy in the context of human leukemia. In this article, we compare chimeric antigen receptor T cells and BiTEs targeting CD19 in B-cell ALL in the setting of the available clinical literature.

  13. Original antigenic sin responses to influenza viruses.

    Science.gov (United States)

    Kim, Jin Hyang; Skountzou, Ioanna; Compans, Richard; Jacob, Joshy

    2009-09-01

    Most immune responses follow Burnet's rule in that Ag recruits specific lymphocytes from a large repertoire and induces them to proliferate and differentiate into effector cells. However, the phenomenon of "original antigenic sin" stands out as a paradox to Burnet's rule of B cell engagement. Humans, upon infection with a novel influenza strain, produce Abs against older viral strains at the expense of responses to novel, protective antigenic determinants. This exacerbates the severity of the current infection. This blind spot of the immune system and the redirection of responses to the "original Ag" rather than to novel epitopes were described fifty years ago. Recent reports have questioned the existence of this phenomenon. Hence, we revisited this issue to determine the extent to which original antigenic sin is induced by variant influenza viruses. Using two related strains of influenza A virus, we show that original antigenic sin leads to a significant decrease in development of protective immunity and recall responses to the second virus. In addition, we show that sequential infection of mice with two live influenza virus strains leads to almost exclusive Ab responses to the first viral strain, suggesting that original antigenic sin could be a potential strategy by which variant influenza viruses subvert the immune system.

  14. Adsorption of multimeric T cell antigens on carbon nanotubes

    DEFF Research Database (Denmark)

    Fadel, Tarek R; Li, Nan; Shah, Smith

    2013-01-01

    Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated...... to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin....

  15. Distribution of ABO and Rh-Hr blood group antigens, alleles and ...

    African Journals Online (AJOL)

    ABO and Rh-Hr blood group antigens represent a genetically stably determined trait with many-sided biological and clinical significance. The indigenous Ajarian population (105 subjects) was investigated for ABO Rh-Hr red cell blood group antigens. Using immunoserologic methods, seven blood group antigens (A, B, C, c, ...

  16. Improved Activation toward Primary Colorectal Cancer Cells by Antigen-Specific Targeting Autologous Cytokine-Induced Killer Cells

    Directory of Open Access Journals (Sweden)

    Claudia Schlimper

    2012-01-01

    Full Text Available Adoptive therapy of malignant diseases with cytokine-induced killer (CIK cells showed promise in a number of trials; the activation of CIK cells from cancer patients towards their autologous cancer cells still needs to be improved. Here, we generated CIK cells ex vivo from blood lymphocytes of colorectal cancer patients and engineered those cells with a chimeric antigen receptor (CAR with an antibody-defined specificity for carcinoembryonic antigen (CEA. CIK cells thereby gained a new specificity as defined by the CAR and showed increase in activation towards CEA+ colon carcinoma cells, but less in presence of CEA− cells, indicated by increased secretion of proinflammatory cytokines. Redirected CIK activation was superior by CAR-mediated CD28-CD3ζ than CD3ζ signaling only. CAR-engineered CIK cells from colon carcinoma patients showed improved activation against their autologous, primary carcinoma cells from biopsies resulting in more efficient tumour cell lysis. We assume that adoptive therapy with CAR-modified CIK cells shows improved selectivity in targeting autologous tumour lesions.

  17. Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

    DEFF Research Database (Denmark)

    Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole

    2009-01-01

    Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic...

  18. cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

    Science.gov (United States)

    Blachère, Nathalie E; Orange, Dana E; Santomasso, Bianca D; Doerner, Jessica; Foo, Patricia K; Herre, Margaret; Fak, John; Monette, Sébastien; Gantman, Emily C; Frank, Mayu O; Darnell, Robert B

    2014-11-01

    Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Stem Cell Antigen-1 in Skeletal Muscle Function

    OpenAIRE

    Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-01-01

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1...

  20. A clinical study on carcinoembryonic antigens in patients with squamous cell carcinoma of the lung

    International Nuclear Information System (INIS)

    Moriya, Hiroshi; Satoh, Toshihiko; Kimura, Kazuei; Togawa, Takafumi; Higuchi, Yoshisuke

    1986-01-01

    The serum levels of carcinoembryonic antigens (CEA) were determined in 57 patients with squamous cell carcinoma of the lung. The overall positive ratio was 45.6 %. The patients were classified into 2 groups, a peripheral type and a central type, according to bronchoscopic findings. The positive ratio in patients with peripheral type was 66.7 %. And the ratio with central type was 26.7 %. There was a significant difference (p < 0.005) between peripheral type and central type of squamous cell carcinoma of the lung. (author)

  1. Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria

    Directory of Open Access Journals (Sweden)

    Laurence Rolland

    1992-06-01

    Full Text Available In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17 XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weigth ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection,in parallel to the parasitemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.

  2. A compound chimeric antigen receptor strategy for targeting multiple myeloma.

    Science.gov (United States)

    Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

    2018-02-01

    Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

  3. The Langerhans cell

    International Nuclear Information System (INIS)

    Wolff, K.; Stingl, G.

    1983-01-01

    Langerhans cells are the bone-marrow-derived immune cells of the epidermis; they express Ia antigens and receptors for the Fc portion of IgG and complement components and are required for epidermal-cell-induced antigen-specific, syngeneic and allogeneic T-cell activitation and the generation of epidermal-cell-induced cytotoxic T cells. Their presence within the epidermis and functional integrity determine whether topical application of haptens leads to specific sensitization or unresponsiveness, and in skin grafts of only I region disparate donors, they represent the cells responsible for the critical allosensitizing signal. UV radiation abrogates most of Langerhans cell functions in vitro; under certain conditions in vivo, it prevents contact sensitization favoring the development of specific unresponsiveness. UV radiation abrogates antigen-presenting capacities of epidermal cells by interfering both with the processing of antigen by Langerhans cells and the production of the epidermal-cell-derived thymocyte activating factor required for optimal T-cell responses

  4. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

  5. Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

    Directory of Open Access Journals (Sweden)

    Karasuyama Hajime

    2011-04-01

    Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

  6. Efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy in patients with haematological and solid malignancies: protocol for a systematic review and meta-analysis.

    Science.gov (United States)

    Grigor, Emma J M; Fergusson, Dean A; Haggar, Fatima; Kekre, Natasha; Atkins, Harold; Shorr, Risa; Holt, Robert A; Hutton, Brian; Ramsay, Tim; Seftel, Matthew; Jonker, Derek; Daugaard, Mads; Thavorn, Kednapa; Presseau, Justin; Lalu, Manoj M

    2017-12-29

    Patients with relapsed or refractory malignancies have a poor prognosis. Immunotherapy with chimeric antigen receptor T (CAR-T) cells redirects a patient's immune cells against the tumour antigen. CAR-T cell therapy has demonstrated promise in treating patients with several haematological malignancies, including acute B-cell lymphoblastic leukaemia and B-cell lymphomas. CAR-T cell therapy for patients with other solid tumours is also being tested. Safety is an important consideration in CAR-T cell therapy given the potential for serious adverse events, including death. Previous reviews on CAR-T cell therapy have been limited in scope and methodology. Herein, we present a protocol for a systematic review to identify CAR-T cell interventional studies and examine the safety and efficacy of this therapy in patients with haematology malignancies and solid tumours. We will search MEDLINE, including In-Process and Epub Ahead of Print, EMBASE and the Cochrane Central Register of Controlled Trials from 1946 to 22 February 2017. Studies will be screened by title, abstract and full text independently and in duplicate. Studies that report administering CAR-T cells of any chimeric antigen receptor construct targeting antigens in patients with haematological malignancies and solid tumours will be eligible for inclusion. Outcomes to be extracted will include complete response rate (primary outcome), overall response rate, overall survival, relapse and adverse events. A meta-analysis will be performed to synthesise the prevalence of outcomes reported as proportions with 95% CIs. The potential for bias within included studies will be assessed using a modified Institute of Health Economics tool. Heterogeneity of effect sizes will be determined using the Cochrane I 2 statistic. The review findings will be submitted for peer-reviewed journal publication and presented at relevant conferences and scientific meetings to promote knowledge transfer. CRD42017075331. © Article author(s) (or

  7. Merkel Cell Polyomavirus Small T Antigen Initiates Merkel Cell Carcinoma-like Tumor Development in Mice.

    Science.gov (United States)

    Verhaegen, Monique E; Mangelberger, Doris; Harms, Paul W; Eberl, Markus; Wilbert, Dawn M; Meireles, Julia; Bichakjian, Christopher K; Saunders, Thomas L; Wong, Sunny Y; Dlugosz, Andrzej A

    2017-06-15

    Merkel cell carcinoma (MCC) tumor cells express several markers detected in normal Merkel cells, a nonproliferative population of neuroendocrine cells that arise from epidermis. MCCs frequently contain Merkel cell polyomavirus (MCPyV) DNA and express viral transforming antigens, sT and tLT, but the role of these putative oncogenes in MCC development, and this tumor's cell of origin, are unknown. Using a panel of preterm transgenic mice, we show that epidermis-targeted coexpression of sT and the cell fate-determinant atonal bHLH transcription factor 1 (ATOH1) leads to development of widespread cellular aggregates, with histology and marker expression mimicking that of human intraepidermal MCC. The MCC-like tumor phenotype was dependent on the FBXW7-binding domain of sT, but not the sT-PP2A binding domain. Coexpression of MCPyV tLT did not appreciably alter the phenotype driven by either sT or sT combined with ATOH1. MCPyV sT, when coexpressed with ATOH1, is thus sufficient to initiate development of epidermis-derived MCC-like tumors in mice. Cancer Res; 77(12); 3151-7. ©2017 AACR . ©2017 American Association for Cancer Research.

  8. Prognostic value of pretreatment serum carcinoembryonic antigen and squamous cell carcinoma antigen levels for patients with stage I-III non-small cell lung cancer treated with radiation therapy alone

    International Nuclear Information System (INIS)

    Saito, Yoshihiro; Mitsuhashi, Norio; Hayakawa, Kazushige

    1998-01-01

    Serum carcinoembryonic antigen (CEA) and serum squamous cell carcinoma antigen (SCC Ag) levels have been reported to be useful as prognostic factors, indicators of clinical response, and predictors for recurrence in patients with lung cancer treated by surgery or chemotherapy. We investigated whether pretreatment serum CEA and SCC Ag levels were useful as independent prognostic factors in patients with stage I to III non-small cell lung cancer who were treated with radiation therapy alone. The serum CEA and SCC Ag levels were measured in 158 and 47 patients, respectively, before radiation therapy. Serum CEA and SCC Ag levels were measured by sandwich radioimmunoassay using the CEA-RIA (radioimmunoassay) kit and the SCC-RIA kit. Serum CEA and SCC Ag levels were above reference values in 19% and 30% of the patients, respectively. The 5-year survival rates were significantly better for patients with a negative SCC Ag result than for those with positive SCC Ag levels (p=0.0001), though no significant difference in survival rates was seen by CEA positivity (p=0.25). SCC Ag positivity (p=0.0006) and stage (p=0.04) were the important prognostic factors, as determined by multivariate analyses. Pretreatment serum SCC Ag level may be useful as an independent prognostic factor in patients with stage I to III non-small cell lung cancer who are treated with radiation therapy alone. (author)

  9. Equine peripheral blood mononuclear cells proliferate in response to tetanus toxoid antigen.

    Science.gov (United States)

    McKelvie, J; Little, S; Foster, A P; Cunningham, F M; Hamblin, A

    1998-01-01

    It has been reported that equine peripheral blood mononuclear cells (PBMNs) do not proliferate in response to tetanus toxoid (TT) (Frayne and Stokes 1995, Research in Veterinary Science 59, 79-81). Here we demonstrate that lymphocyte proliferation responses to TT, which are characteristic of a recall antigen, may be achieved under certain culture conditions. Given that TT vaccination is routinely applied to many horses, TT is a suitable antigen for the investigation of cellular immune responses by peripheral blood mononuclear cells in the horse.

  10. Epstein-Barr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23

    International Nuclear Information System (INIS)

    Wang, F.; Gregory, C.D.; Rowe, M.; Rickinson, A.B.; Wang, D.; Birkenbach, M.; Kikutani, H.; Kishimoto, T.; Kieff, E.

    1987-01-01

    Epstein-Barr virus (EBV) infection of EBV-negative Burkitt lymphoma (BL) cells includes some changes similar to those seen in normal B lymphocytes that have been growth transformed by EBV. The role of individual EBV genes in this process was evaluated by introducing each of the viral genes that are normally expressed in EBV growth-transformed and latently infected lymphoblasts into an EBV-negative BL cell line, using recombinant retrovirus-mediated transfer. Clones of cells were derived that stably express the EBV nuclear antigen 1 (EBNA-1), EBNA-2, EBNA-3, EBNA-leader protein, or EBV latent membrane protein (LMP). These were compared with control clones infected with the retrovirus vector. All 10 clones converted to EBNA-2 expression differed from control clones or clones expressing other EBV proteins by growth in tight clumps and by markedly increased expression of one particular surface marker of B-cell activation, CD23. Other activation antigens were unaffected by EBNA-2 expression, as were markers already expressed on the parent BL cell line. The results indicate that EBNA-2 is a specific direct or indirect trans-activator of CD23. This establishes a link between an EBV gene and cell gene expression. Since CD23 has been implicated in the transduction of B-cell growth signals, its specific induction by EBNA-2 could be important in EBV induction of B-lymphocyte transformation

  11. Determinants of wheat antigen and fungal alpha-amylase exposure in bakeries.

    Science.gov (United States)

    Burstyn, I; Teschke, K; Bartlett, K; Kennedy, S M

    1998-05-01

    The study's objectives were to measure flour antigen exposure in bakeries and define the determinants of exposure. Ninety-six bakery workers, employed in seven different bakeries, participated in the study. Two side-by-side full-shift inhalable dust samples were obtained from each study participant on a single occasion. The flour antigen exposure was measured as wheat antigen and fungal alpha-amylase content of the water-soluble fraction of inhalable dust, assayed via enzyme-linked immunosorbent assays. During the entire sampling period bakers were observed and information on 14 different tasks was recorded at 15-minute intervals. Other production characteristics were also recorded for each sampling day and used in statistical modeling to identify significant predictors of exposure. The mean alpha-amylase antigen exposure was 22.0 ng/m3 (ranging from below the limit of detection of 0.1 ng/m3 to 307.1 ng/m3) and the mean wheat antigen exposure was 109 micrograms/m3 (ranging from below the limit of detection of 1 microgram/m3 to 1018 micrograms/m3). Regression models that explained 74% of variability in wheat antigen and alpha-amylase antigen exposures were constructed. The models indicated that tasks such as weighing, pouring, and operating dough-brakers increased flour antigen exposure, while packing and decorating resulted in lower exposures. Croissant, puff-pastry, and bread/bun production lines were associated with increased exposure, while cake production and substitution of dusting with the use of divider oil were associated with decreased exposure. Exposure levels can be reduced by the automation of forming tasks, alteration of tasks requiring pouring of flour, and changes to the types of products manufactured.

  12. Determination of carcinoembryonic antigen: experiences with a new radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Lamerz, R [Univ., Munich; Ruider, H

    1976-04-01

    The determination of the carcinoembryonic antigen (CEA) as RIA-test was examined and tested. Labelling was carried out with /sup 125/I according to the chloramin T-method, as RIA in the form of a competitive double antibody examination. The method was tested on patients with colonic and pancreatic carcinomas.

  13. T cell recognition of breast cancer antigens

    DEFF Research Database (Denmark)

    Petersen, Nadia Viborg; Andersen, Sofie Ramskov; Andersen, Rikke Sick

    Recent studies are encouraging research of breast cancer immunogenicity to evaluate the applicability ofimmunotherapy as a treatment strategy. The epitope landscape in breast cancer is minimally described, thus it is necessary to identify T cell targets to develop immune mediated therapies.......This project investigates four proteins commonly upregulated in breast cancer and thus probable tumor associated antigens (TAAs). Aromatase, prolactin, NEK3, and PIAS3 contribute to increase growth, survival, and motility of malignant cells. Aspiring to uncover novel epitopes for cytotoxic T cells, a reverse...... recognition utilizing DNA barcode labeled MHC multimers to screen peripheral blood lymphocytes from breast cancer patients and healthy donor samples. Signif-icantly more TAA specific T cell responses were detected in breast cancer patients than healthy donors for both HLA-A*0201 (P

  14. Screening Immunomodulators To Skew the Antigen-Specific Autoimmune Response.

    Science.gov (United States)

    Northrup, Laura; Sullivan, Bradley P; Hartwell, Brittany L; Garza, Aaron; Berkland, Cory

    2017-01-03

    Current therapies to treat autoimmune diseases often result in side effects such as nonspecific immunosuppression. Therapies that can induce antigen-specific immune tolerance provide an opportunity to reverse autoimmunity and mitigate the risks associated with global immunosuppression. In an effort to induce antigen-specific immune tolerance, co-administration of immunomodulators with autoantigens has been investigated in an effort to reprogram autoimmunity. To date, identifying immunomodulators that may skew the antigen-specific immune response has been ad hoc at best. To address this need, we utilized splenocytes obtained from mice with experimental autoimmune encephalomyelitis (EAE) in order to determine if certain immunomodulators may induce markers of immune tolerance following antigen rechallenge. Of the immunomodulatory compounds investigated, only dexamethasone modified the antigen-specific immune response by skewing the cytokine response and decreasing T-cell populations at a concentration corresponding to a relevant in vivo dose. Thus, antigen-educated EAE splenocytes provide an ex vivo screen for investigating compounds capable of skewing the antigen-specific immune response, and this approach could be extrapolated to antigen-educated cells from other diseases or human tissues.

  15. Mucosal-Associated Invariant T Cells: New Insights into Antigen Recognition and Activation

    Directory of Open Access Journals (Sweden)

    Xingxing Xiao

    2017-11-01

    Full Text Available Mucosal-associated invariant T (MAIT cells, a novel subpopulation of innate-like T cells that express an invariant T cell receptor (TCRα chain and a diverse TCRβ chain, can recognize a distinct set of small molecules, vitamin B metabolites, derived from some bacteria, fungi but not viruses, in the context of an evolutionarily conserved major histocompatibility complex-related molecule 1 (MR1. This implies that MAIT cells may play unique and important roles in host immunity. Although viral antigens are not recognized by this limited TCR repertoire, MAIT cells are known to be activated in a TCR-independent mechanism during some viral infections, such as hepatitis C virus and influenza virus. In this article, we will review recent works in MAIT cell antigen recognition, activation and the role MAIT cells may play in the process of bacterial and viral infections and pathogenesis of non-infectious diseases.

  16. IRF8-dependent DCs play a key role in the regulation of CD8 T cell responses to epithelial-derived antigen in the steady state but not in inflammation

    DEFF Research Database (Denmark)

    Joeris, Thorsten

    Along the process of epithelial self-renewal, antigens derived from apoptotic intestinal epithelial cells (IECs) are taken up by antigen presenting cells (APCs), transported to gut-draining lymph nodes and crosspresented to CD8 T cells. In steady state, rapid tolerization of CD8 T cells reactive...... towards epithelialderived antigens is crucial to maintain tissue homeostasis. Since IRF8-dependent dendritic dells (IRF8-DCs) have superior cross-presenting capabilities, we aimed to investigate their role in this process. IFABP-tOva mice, expressing the model-antigen Ovalbumin (Ova) in IECs, were used...... as recipients to set up chimeras using either CD11c-cre.Irf8fl/fl bone marrow, which cannot generate IRF8-DCs, or cre-negative Irf8fl/fl control bone marrow. Whereas transfer of Ova-specific CD8 T cells (OT-I cells) to control chimeras resulted in their rapid tolerization, OT-I cells transferred to CD11c...

  17. Targeting Antigens to Dec-205 on Dendritic Cells Induces Immune Protection in Experimental Colitis in Mice

    Science.gov (United States)

    Wadwa, Munisch; Klopfleisch, Robert; Buer, Jan; Westendorf, Astrid M.

    2016-01-01

    The endocytotic c-type lectin receptor DEC-205 is highly expressed on immature dendritic cells. In previous studies, it was shown that antigen-targeting to DEC-205 is a useful tool for the induction of antigen-specific Foxp3+ regulatory T cells and thereby can prevent inflammatory processes. However, whether this approach is sufficient to mediate tolerance in mucosal tissues like the gut is unknown. In this study, we established a new mouse model in which the adoptive transfer of naive hemagglutinin (HA)-specific CD4+Foxp3– T cells into VILLIN-HA transgenic mice leads to severe colitis. To analyze if antigen-targeting to DEC-205 could protect against inflammation of the gut, VILLIN-HA transgenic mice were injected with an antibody–antigen complex consisting of the immunogenic HA110–120 peptide coupled to an α-DEC-205 antibody (DEC-HA) before adoptive T cell transfer. DEC-HA-treated mice showed significantly less signs of intestinal inflammation as was demonstrated by reduced loss of body weight and histopathology in the gut. Strikingly, abrogated intestinal inflammation was mediated via the conversion of naive HA-specific CD4+Foxp3– T cells into HA-specific CD4+Foxp3+ regulatory T cells. In this study, we provide evidence that antigen-targeting to DEC-205 can be utilized for the induction of tolerance in mucosal organs that are confronted with large numbers of exogenous antigens. PMID:27141310

  18. Lea blood group antigen on human platelets

    International Nuclear Information System (INIS)

    Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

    1985-01-01

    One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125 I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125 I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125 I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined

  19. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-12-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

  20. Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

    International Nuclear Information System (INIS)

    Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

    1985-01-01

    Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

  1. Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

    Science.gov (United States)

    Wu, Lisa Y; Johnson, Jacqueline M; Simmons, Jessica K; Mendes, Desiree E; Geruntho, Jonathan J; Liu, Tiancheng; Dirksen, Wessel P; Rosol, Thomas J; Davis, William C; Berkman, Clifford E

    2014-05-01

    Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1)  mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50  = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly

  2. Phenotype of Antigen Unexperienced TH Cells in the Inflamed Central Nervous System in Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Franck, Sophia; Paterka, Magdalena; Birkenstock, Jerome; Zipp, Frauke; Siffrin, Volker; Witsch, Esther

    2017-06-01

    Multiple sclerosis is a chronic, disseminated inflammation of the central nervous system which is thought to be driven by autoimmune T cells. Genetic association studies in multiple sclerosis and a large number of studies in the animal model of the disease support a role for effector/memory T helper cells. However, the mechanisms underlying relapses, remission and chronic progression in multiple sclerosis or the animal model experimental autoimmune encephalomyelitis, are not clear. In particular, there is only scarce information on the role of central nervous system-invading naive T helper cells in these processes. By applying two-photon laser scanning microscopy we could show in vivo that antigen unexperienced T helper cells migrated into the deep parenchyma of the inflamed central nervous system in experimental autoimmune encephalomyelitis, independent of their antigen specificity. Using flow cytometric analyses of central nervous system-derived lymphocytes we found that only antigen-specific, formerly naive T helper cells became activated during inflammation of the central nervous system encountering their corresponding antigen.

  3. B cell antigen receptor signaling and internalization are mutually exclusive events.

    Directory of Open Access Journals (Sweden)

    Ping Hou

    2006-07-01

    Full Text Available Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

  4. In vitro stimulation of rabbit T lymphocytes by cells expressing herpes simplex antigens.

    Science.gov (United States)

    Kapoor, A K; Ling, N R; Nash, A A; Bachan, A; Wildy, P

    1982-04-01

    Lymphocyte stimulation responses to herpes antigens were studied using virus-infected X-irradiated cells. Rabbits were immunized with herpes simplex virus type 1 (strain HFEM) grown in RK 13 cells. For in vitro stimulation assay BHK21 cells were X-irradiated (15 000 rad) and infected with a high m.o.i. of a temperature-sensitive (ts) mutant (N102) of HFEM strain at the non-permissive temperature (38.5 degrees C) of virus. Virus antigens were expressed on the infected cells and there was no leakage of infectious virus into the medium at 38.5 degrees C. T lymphocytes from rabbits immunized with herpes simplex virus were specifically activated by herpesvirus-infected X-irradiated cells; lymph node cells from rabbits immunized with RK13 cells and from non-immune rabbits showed no proliferative response.

  5. T cells from fully H-2 allogeneic (A replaced by B) radiation bone marrow chimeras are functionally competent and host restricted but are alloreactive against hybrid Ia determinants expressed on (A x B)F1 cells

    International Nuclear Information System (INIS)

    Kruisbeek, A.M.; Hathcock, K.S.; Hodes, R.J.; Singer, A.

    1982-01-01

    In this communication it is demonstrated that T cells from fully allogeneic A replaced by B radiation bone marrow chimeras are alloreactive against the hybrid Ia molecules expressed on the surface of heterozygous A X B cells. These results suggested that previous failures to generate cytotoxic T lymphocyte (CTL) responses from fully allogeneic chimeras by sensitizing the chimeric T cells to antigen in an (A X B)F1-priming environment might have been confounded by an ongoing alloreaction against determinants created by hybrid Ia molecules expressed on F1 cells. Consequently, the ability to generate CTL responses from fully allogeneic chimeras was re-examined by sensitizing the chimeric T cells to antigen presented by homozygous rather that F1 stimulator cells. It was found that T cells of donor bone marrow origin that mediate cytotoxic responses to trinitrophenyl-modified self determinants do differentiate into functional competence in an H-2-incompatible host environment and are restricted to the host H-2 haplotype

  6. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S

    1987-01-01

    A detailed analysis of the residues within an immunogenic peptide that endow it with the capacity to interact with Ia and to be recognized by T cells is presented. Ia interacts with only a few of the peptide residues and overall exhibits a very broad specificity. Some residues appear to interact...... both with Ia and with T cells, leading to a model in which a peptide antigen is 'sandwiched' between Ia and the T-cell receptor....

  7. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujiwara

    2014-12-01

    Full Text Available Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs, transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

  8. MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation

    DEFF Research Database (Denmark)

    Rosloniec, E F; Vitez, L J; Buus, S

    1990-01-01

    the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further...... found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323......-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3...

  9. TANTIGEN: a comprehensive database of tumor T cell antigens

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Tongchusak, Songsak; Lin, Honghuang

    2017-01-01

    Tumor T cell antigens are both diagnostically and therapeutically valuable molecules. A large number of new peptides are examined as potential tumor epitopes each year, yet there is no infrastructure for storing and accessing the results of these experiments. We have retroactively cataloged more ...

  10. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    Science.gov (United States)

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

  11. Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

    Science.gov (United States)

    Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J.; Rivière, Isabelle

    2009-01-01

    Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor. PMID:19238016

  12. Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

    International Nuclear Information System (INIS)

    Sadeghi, H.; Da Silva, A.M.; Klein, C.

    1988-01-01

    The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

  13. Effect of radiation on the expression of carcinoembryonic antigen of human gastric adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hareyama, M.; Imai, K.; Kubo, K.; Takahashi, H.; Koshiba, H.; Hinoda, Y.; Shidou, M.; Oouchi, A.; Yachi, A.; Morita, K. (Sapporo Medical College (Japan))

    1991-05-01

    The changes of antigenic expression of cultured human gastric adenocarcinoma MKN45 cells caused by irradiation were investigated to elucidate the immune responses to localized irradiation. The expression of carcinoembryonic antigen (CEA) showed remarkable increases in the culture supernatant and on the surface of the membrane of irradiated cells. The expression of major histocompatibility complex Class I antigen on the membrane also was enhanced by irradiation. In addition, the irradiated cell groups, when analyzed using a CEA-specific probe, showed remarkable increases in the CEA mRNA. These enhancements increased in the 10-Gy and 15-Gy irradiated populations compared with the 5-Gy irradiated population. These results suggest that the enhancement of expression of CEA by radiation takes place at the CEA gene expression (mRNA) level but not at the protein level.

  14. Technical and clinical performance of a new assay to detect squamous cell carcinoma antigen levels for the differential diagnosis of cervical, lung, and head and neck cancer.

    Science.gov (United States)

    Holdenrieder, Stefan; Molina, Rafael; Qiu, Ling; Zhi, Xiuyi; Rutz, Sandra; Engel, Christine; Kasper-Sauer, Pia; Dayyani, Farshid; Korse, Catharina M

    2018-04-01

    In squamous cell carcinoma, squamous cell carcinoma antigen levels are often elevated. This multi-center study evaluated the technical performance of a new Elecsys ® squamous cell carcinoma assay, which measures serum squamous cell carcinoma antigen 1 and 2 levels in an equimolar manner, and investigated the potential of squamous cell carcinoma antigen for differential diagnosis of cervical, lung, and head and neck squamous cell carcinoma.Assay precision and method comparison experiments were performed across three European sites. Reference ranges for reportedly healthy individuals were determined using samples from banked European and Chinese populations. Differential diagnosis experiments determined whether cervical, lung, or head and neck cancer could be differentiated from apparently healthy, benign, or other malignant cohorts using squamous cell carcinoma antigen levels alone. Squamous cell carcinoma antigen cut-off levels were calculated based on squamous cell carcinoma antigen levels at 95% specificity. Repeatability coefficients of variation across nine analyte concentrations were ≤5.3%, and intermediate precision coefficients of variation were ≤10.3%. Method comparisons showed good correlations with Architect and Kryptor systems (slopes of 1.1 and 1.5, respectively). Reference ranges for 95th percentiles for apparently healthy individuals were 2.3 ng/mL (95% confidence interval: 1.9-3.8; European cohort, n = 153) and 2.7 ng/mL (95% confidence interval: 2.2-3.3; Chinese cohort, n = 146). Strongest differential diagnosis results were observed for cervical squamous cell carcinoma: receiver operating characteristic analysis showed that squamous cell carcinoma antigen levels (2.9 ng/mL cut-off) differentiate cervical squamous cell carcinoma (n = 127) from apparently healthy females (n = 286; area under the curve: 86.2%; 95% confidence interval: 81.8-90.6; sensitivity: 61.4%; specificity: 95.6%), benign diseases (n = 187; area

  15. Functional, Antigen-Specific Stem Cell Memory (TSCM CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

    Directory of Open Access Journals (Sweden)

    Cheleka A. M. Mpande

    2018-03-01

    Full Text Available BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM, which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM or effector (TEFF T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb, the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+ were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also

  16. Enhanced vaccine-induced CD8+ T cell responses to malaria antigen ME-TRAP by fusion to MHC class ii invariant chain.

    Directory of Open Access Journals (Sweden)

    Alexandra J Spencer

    Full Text Available The orthodox role of the invariant chain (CD74; Ii is in antigen presentation to CD4+ T cells, but enhanced CD8+ T cells responses have been reported after vaccination with vectored viral vaccines encoding a fusion of Ii to the antigen of interest. In this study we assessed whether fusion of the malarial antigen, ME-TRAP, to Ii could increase the vaccine-induced CD8+ T cell response. Following single or heterologous prime-boost vaccination of mice with a recombinant chimpanzee adenovirus vector, ChAd63, or recombinant modified vaccinia virus Ankara (MVA, higher frequencies of antigen-specific CD4+ and CD8+ T cells were observed, with the largest increases observed following a ChAd63-MVA heterologous prime-boost regimen. Studies in non-human primates confirmed the ability of Ii-fusion to augment the T cell response, where a 4-fold increase was maintained up to 11 weeks after the MVA boost. Of the numerous different approaches explored to increase vectored vaccine induced immunogenicity over the years, fusion to the invariant chain showed a consistent enhancement in CD8+ T cell responses across different animal species and may therefore find application in the development of vaccines against human malaria and other diseases where high levels of cell-mediated immunity are required.

  17. Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8+ T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells

    OpenAIRE

    Coelho-dos-Reis, Jordana G.; Huang, Jing; Tsao, Tiffany; Pereira, Felipe V.; Funakoshi, Ryota; Nakajima, Hiroko; Sugiyama, Haruo; Tsuji, Moriya

    2016-01-01

    In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8+ T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8+ T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immuniza...

  18. Reduction-sensitive dextran nanogels aimed for intracellular delivery of antigens

    NARCIS (Netherlands)

    Li, Dandan; Kordalivand, Neda; Fransen, Marieke F.; Ossendorp, Ferry; Raemdonck, Koen; Vermonden, Tina; Hennink, Wim E.; Van Nostrum, Cornelus F.

    2015-01-01

    Targeting of antigens to dendritic cells (DCs) to induce strong cellular immune response can be established by loading in a nano-sized carrier and keeping the antigen associated with the particles until they are internalized by DCs. In the present study, a model antigen (ovalbumin, OVA) is

  19. Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

    Science.gov (United States)

    Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

    2009-05-15

    The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, pautomated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

  20. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  1. HIV p24 as scaffold for presenting conformational HIV Env antigens.

    Directory of Open Access Journals (Sweden)

    Maria Tagliamonte

    Full Text Available Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4(+ and CD8(+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.

  2. Committed T lymphocyte stem cells of rats. Characterization by surface W3/13 antigen and radiosensitivity

    International Nuclear Information System (INIS)

    Dyer, M.J.; Hunt, S.V.

    1981-01-01

    The existence of stem cells committed to the T lymphoid lineage was deduced from studying how rat T and B stem cells differ in their expression of membrane W3/13 antigen and in their susceptibility in vivo to gamma irradiation. Stem cell activity of rat bone marrow and fetal liver was measured in long-term radiation chimeras using B and T cell alloantigenic surface markers to identify the progeny of donor cells. Monoclonal mouse anti-rat thymocyte antibody W3/13 labeled approximately 40% of fetal liver cells and 60-70% of young rat bone marrow cells (40% brightly, 25% dimly). Bright, dim, and negative cells were separated on a fluorescence-activated cell sorter. All B and T lymphoid stem cells in fetal liver were W3/13 bright, as were B lymphoid stem cells in bone marrow. W3/13 dim bone marrow had over half the T cell repopulating activity of unseparated marrow but gave virtually no B cell repopulation. In further experiments, the radiosensitivity of endogenous B and T lymphoid stem cells was determined by exposing host rats to between 4.5 and 10 Gy of gamma irradiation before repopulation with genetically marked marrow. The results depended on whether chimerism was assayed before day 50 or after day 100. At early times, a radioresistant T stem cell was indicated, whose activity waned later. Thus committed T stem cells of rats carry moderate amounts of W3/13 antigen and are more radioresistant but less permanently chimeragenic than the stem cells that regenerate B lymphocytes

  3. Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cells

    Directory of Open Access Journals (Sweden)

    Diana Magalhães de Oliveira

    1998-01-01

    Full Text Available Nitric oxide (NO is an important effector molecule involved in immune regulation and defense. NO produced by cytokine-activated macrophages was reported to be cytotoxic against the helminth Schistosoma mansoni. Identification and characterization of S. mansoni antigens that can provide protective immunity is crucial for understanding the complex immunoregulatory events that modulate the immune response in schistosomiasis. It is, then, essential to have available defined, purified parasite antigens. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP, named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SEA (soluble egg antigen or to SWAP. In the present work we report the effect of different in vivo trials with mice on their spleen cells ability to produce NO. We demonstrate that PIII-immunization is able to significantly increase NO production by spleen cells after in vitro stimulation with LPS. These data suggest a possible role for NO on the protective immunity induced by PIII.

  4. Present and future of allogeneic natural killer cell therapy

    Directory of Open Access Journals (Sweden)

    Okjae eLim

    2015-06-01

    Full Text Available Natural killer (NK cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA-haploidentical hematopoietic stem cell transplantation revealed the anti-tumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor (KIR ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

  5. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    International Nuclear Information System (INIS)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

    1990-01-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

  6. Presentation of human minor histocompatibility antigens by HLA-B35 and HLA-B38 molecules

    International Nuclear Information System (INIS)

    Yamamoto, Junji; Kariyone, Ai; Kano, Kyoichi; Takiguchi, Masafumi; Akiyama, Nobuo

    1990-01-01

    Cytotoxic T lymphocyte (CTL) clones specific for human minor histocompatibility antigens (hmHAs) were produced from a patient who had been grafted with the kidneys from his mother and two HLA-identical sisters. Of eight CTL clones generated, four recognized an hmHA (hmHA-1) expressed on cells from the mother and sister 3 (second donor); two recognized another antigen (hmHA-2) on cells from the father, sister (third donor), and sister 3; and the remaining two clones recognized still another antigen (hmHA-3) on cells from the father and sister 3. Panel studies revealed that CTL recognition of hmHA-1 was restricted by HLA-B35 and that of hmHA-2 and hmHA-3 was restricted by HLA-B38. The HLA-B35 restriction of the hmHA-1 -specific CTL clones was substantiated by the fact that they killed HLA-A null/HLA-B null Hmy2CIR targets transfected with HLA-B35 but not HLA-B51, -Bw52, or -Bw53 transfected Hmy2CIR targets. These data demonstrated that the five amino acids substitutions on the α 1 domain between HLA-B35 and -Bw53, which are associated with Bw4/Bw6 epitopes, play a critical role in the relationship of hmHA-1 to HLA-B35 molecules. The fact that the hmHA-1-specific CTLs failed to kill Hmy2CIR cells expressing HLA-B35/51 chimeric molecules composed of the α 1 domain of HLA-B35 and other domains of HLA-B51 indicated that eight residues on the α 2 domain also affect the interaction of hmHA-1 and the HLA-B35 molecules

  7. Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

    Directory of Open Access Journals (Sweden)

    Paulo RZ Antas

    2004-02-01

    Full Text Available The production of interferon gamma (IFNgamma guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r² = 0.9266; p = 0.0102. Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.

  8. Determining T-cell specificity to understand and treat disease

    DEFF Research Database (Denmark)

    Hadrup, Sine Reker; Newell, Evan W.

    2017-01-01

    Adaptive immune responses and immunopathogeneses are based on the ability of T cells to respond to specific antigens. Consequently, understanding T-cell recognition patterns in health and disease involves studying the complexity and genetic heterogeneity of the antigen recognition pathway, which...

  9. CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature.

    Directory of Open Access Journals (Sweden)

    Phillip A Swanson

    2016-12-01

    Full Text Available Cerebral malaria (CM is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM, we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs, where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4 therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen

  10. Improved Prediction of Bovine Leucocyte Antigens (BoLA) Presented Ligands by Use of Mass-Spectrometry-Determined Ligand and in Vitro Binding Data

    DEFF Research Database (Denmark)

    Nielsen, Morten; Connelley, Tim; Ternette, Nicola

    2018-01-01

    Peptide binding to MHC class I molecules is the single most selective step in antigen presentation and the strongest single correlate to peptide cellular immunogenicity. The cost of experimentally characterizing the rules of peptide presentation for a given MHC-I molecule is extensive, and predic...... available at http://www.cbs.dtu.dk/services/NetMHCpan/NetBoLApan . The approach has here been applied to the BoLA-I system, but the pipeline is readily applicable to MHC systems in other species....

  11. Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form.

    Science.gov (United States)

    Smith, Mark L; Mason, Hugh S; Shuler, Michael L

    2002-12-30

    The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed. Copyright 2002 Wiley Periodicals, Inc.

  12. [Role of helminth antigens in the abnormal mitosis of bone marrow cells in laboratory animals].

    Science.gov (United States)

    Sivkova, T N; Tatarnikova, N A; Berezhko, V K; Benediktov, I I

    2013-01-01

    The intraabdominal administration of somatic extracts of the cestodes Hydatigera taeniaformis Batsch 1786, Lamarck, 1816 and Diphyllobothrium latum Linnaeus, 1758 and the nematodes Anisakis simplex larva Rudolphi 1809, Toxocara canis Railliet et Henry, 1912 in albino mice proved that these helminths had a karyopathic effect on the bone marrow cells of the animals. The antigenic composition of these extracts was investigated using the agar gel immunodiffusion test. The antigenic composition of the parasites was ascertained to affect their karyopathic properties. The amount of antigens and their foreignness caused a marked karyopathic effect on the bone marrow cells of laboratory animals during intraabdominal administration.

  13. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    International Nuclear Information System (INIS)

    Holers, V.M.; Kotzin, B.L.

    1985-01-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

  14. Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

    Science.gov (United States)

    Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

    1986-06-01

    Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

  15. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    Directory of Open Access Journals (Sweden)

    Yukiko Kiniwa

    Full Text Available Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+ T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+ T helper (Th cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+ T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1 as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+ Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+ T cell-mediated immunotherapy in melanoma.

  16. Identification of DRG-1 As a Melanoma-Associated Antigen Recognized by CD4+ Th1 Cells.

    Science.gov (United States)

    Kiniwa, Yukiko; Li, Jiang; Wang, Mingjun; Sun, Chuang; Lee, Jeffrey E; Wang, Rong-Fu; Wang, Helen Y

    2015-01-01

    Immunotherapy has emerged as a promising strategy for the treatment of metastatic melanoma. Clinical studies have demonstrated the feasibility of cancer immunotherapy using tumor antigens recognized by CD8(+) T cells. However, the overall immune responses induced by these antigens are too weak and transient to induce tumor regression in the majority of patients who received immunization. A growing body of evidence suggests that CD4(+) T helper (Th) cells play an important role in antitumor immunity. Therefore, the identification of MHC class II-restricted tumor antigens capable of stimulating CD4(+) T cells may provide opportunities for developing effective cancer vaccines. To this end, we describe the identification of developmentally regulated GTP-binding protein 1 (DRG-1) as a melanoma-associated antigen recognized by HLA-DR11-restricted CD4(+) Th1 cells. Epitope mapping analysis showed that the DRG1248-268 epitope of DRG-1 was required for T cell recognition. Reverse transcription-polymerase chain reaction revealed that DRG-1 was highly expressed in melanoma cell lines but not in normal tissues. DRG-1 knockdown by lentiviral-based shRNA suppressed melanoma cell proliferation and soft agar colony formation. Taken together, these data suggest that DRG-1 plays an important role in melanoma cell growth and transformation, indicating that DRG1 may represent a novel target for CD4(+) T cell-mediated immunotherapy in melanoma.

  17. Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia

    Science.gov (United States)

    Ghiotto, Fabio; Fais, Franco; Valetto, Angelo; Albesiano, Emilia; Hashimoto, Shiori; Dono, Mariella; Ikematsu, Hideyuki; Allen, Steven L.; Kolitz, Jonathan; Rai, Kanti R.; Nardini, Marco; Tramontano, Anna; Ferrarini, Manlio; Chiorazzi, Nicholas

    2004-01-01

    Studies of B cell antigen receptors (BCRs) expressed by leukemic lymphocytes from patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that B lymphocytes with some level of BCR structural restriction become transformed. While analyzing rearranged VHDJH and VLJL genes of 25 non–IgM-producing B-CLL cases, we found five IgG+ cases that display strikingly similar BCRs (use of the same H- and L-chain V gene segments with unique, shared heavy chain third complementarity-determining region [HCDR3] and light chain third complementarity-determining region [LCDR3] motifs). These H- and L-chain characteristics were not identified in other B-CLL cases or in normal B lymphocytes whose sequences are available in the public databases. Three-dimensional modeling studies suggest that these BCRs could bind the same antigenic epitope. The structural features of the B-CLL BCRs resemble those of mAb’s reactive with carbohydrate determinants of bacterial capsules or viral coats and with certain autoantigens. These findings suggest that the B lymphocytes that gave rise to these IgG+ B-CLL cells were selected for this unique BCR structure. This selection could have occurred because the precursors of the B-CLL cells were chosen for their antigen-binding capabilities by antigen(s) of restricted nature and structure, or because the precursors derived from a B cell subpopulation with limited BCR heterogeneity, or both. PMID:15057307

  18. Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss skin epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kristoffer Lindell

    Full Text Available Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.

  19. Lipopolysaccharide O-Antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss) Skin Epithelial Cells

    Science.gov (United States)

    Lindell, Kristoffer; Fahlgren, Anna; Hjerde, Erik; Willassen, Nils-Peder; Fällman, Maria; Milton, Debra L.

    2012-01-01

    Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues. PMID:22662189

  20. Magnitude of Alloresponses to MHC Class I/II Expressing Human Cardiac Myocytes is Limited by their Intrinsic Ability to Process and Present Antigenic Peptides

    Directory of Open Access Journals (Sweden)

    Aftab A. Ansari

    2003-01-01

    Full Text Available In this investigation we have explored the relationship between the weak allogenicity of cardiac myocytes and their capacity to present allo-antigens by examining the ability of a human cardiac myocyte cell line (W-1 to process and present nominal antigens. W-1 cells (HLA-A*0201 and HLA-DR β1*0301 pulsed with the influenza A matrix 1 (58-66 peptide (M1 were able to serve as targets for the HLA-A*0201 restricted CTL line PG, specific for M1-peptide. However, PG-CTLs were unable to lyse W-1 target cells infected with a recombinant vaccinia virus expressing the M1 protein (M1-VAC. Pretreatment of these M1-VAC targets with IFN-γ partially restored their ability to process and present the M1 peptide. However, parallel studies demonstrated that IFN-γ pretreated W-1's could not process tetanus toxin (TT or present the TT(830-843 peptide to HLA-DR3 restricted TT-primed T cells. Semi-quantitative RT-PCR measurements revealed significantly lower constitutive levels of expression for MHC class I, TAP-1/2, and LMP-2/7 genes in W-1s that could be elevated by pretreatment with IFN-γ to values equal to or greater than those expressed in EBV-PBLs. However, mRNA levels for the genes encoding MHC class II, Ii, CIITA, and DMA/B were markedly lower in both untreated and IFN-γ pretreated W-1s relative to EBV-PBLs. Furthermore, pulse-chase analysis of the corresponding genes revealed significantly lower protein levels and longer half-life expression in W-1s relative to EBV-PBLs. These results suggest that weak allogenicity of cardiac myocytes may be governed by their limited expression of MHC genes and gene products critical for antigen processing and presentation.

  1. Chimeric antigen receptor T cell therapy in pancreatic cancer: from research to practice.

    Science.gov (United States)

    Jindal, Vishal; Arora, Ena; Masab, Muhammad; Gupta, Sorab

    2018-05-04

    Chimeric antigen receptor (CAR) T cell therapy is genetically engineered tumor antigen-specific anticancer immunotherapy, which after showing great success in hematological malignancies is currently being tried in advanced solid tumors like pancreatic cancer. Immunosuppressive tumor microenvironment and dense fibrous stroma are some of the limitation in the success of this novel therapy. However, genetic modifications and combination therapy is the topic of the research to improve its efficacy. In this article, we summarize the current state of knowledge, limitations, and future prospects for CAR T cell therapy in pancreatic cancer.

  2. In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

    Science.gov (United States)

    Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

    2017-07-14

    Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Systems Analysis Reveals High Genetic and Antigen-Driven Predetermination of Antibody Repertoires throughout B Cell Development

    Directory of Open Access Journals (Sweden)

    Victor Greiff

    2017-05-01

    Full Text Available Antibody repertoire diversity and plasticity is crucial for broad protective immunity. Repertoires change in size and diversity across multiple B cell developmental stages and in response to antigen exposure. However, we still lack fundamental quantitative understanding of the extent to which repertoire diversity is predetermined. Therefore, we implemented a systems immunology framework for quantifying repertoire predetermination on three distinct levels: (1 B cell development (pre-B cell, naive B cell, plasma cell, (2 antigen exposure (three structurally different proteins, and (3 four antibody repertoire components (V-gene usage, clonal expansion, clonal diversity, repertoire size extracted from antibody repertoire sequencing data (400 million reads. Across all three levels, we detected a dynamic balance of high genetic (e.g., >90% for V-gene usage and clonal expansion in naive B cells and antigen-driven (e.g., 40% for clonal diversity in plasma cells predetermination and stochastic variation. Our study has implications for the prediction and manipulation of humoral immunity.

  4. Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.

    Directory of Open Access Journals (Sweden)

    Emerence Crompot

    Full Text Available Microparticles (MPs, also called microvesicles (MVs are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets. Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM and Scanning Electron microscopy (SEM.Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins.We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

  5. Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

    Science.gov (United States)

    Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

    2017-01-01

    Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

  6. Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

    Science.gov (United States)

    O'Neill, F J; Hu, Y; Chen, T; Carney, H

    1997-02-27

    In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

  7. Chimeric Antigen Receptor-Modified T Cells Redirected to EphA2 for the Immunotherapy of Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ning Li

    2018-02-01

    Full Text Available Erythropoietin-producing hepatocellular carcinoma A2 (EphA2 is overexpressed in more than 90% of non-small cell lung cancer (NSCLC but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC.

  8. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

    Science.gov (United States)

    Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin; Wang, Lei

    2017-11-01

    Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

  9. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

    Directory of Open Access Journals (Sweden)

    Yushi Yao

    2017-11-01

    Full Text Available Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM, which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

  10. Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

    International Nuclear Information System (INIS)

    Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

    2011-01-01

    Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

  11. Cell-free soluble-phase radioimmunoassay for Thy-1 antigen

    Energy Technology Data Exchange (ETDEWEB)

    Shalev, A.; Zuckerman, F. (Ben-Gurion Univ. of the Negev, Beersheba (Israel))

    1983-12-01

    A cell-free, soluble-phase, radioimmunoassay has been developed for Thy-1 antigen. The method is based on immunoprecipitation of radiolabelled Thy-1 molecules with specific antibodies, antiimmunoglobulin serum and polyethyleneglycol (PEG). The method can be used with convenience to screen for the presence of Thy-1 in various fluids as well as on cell surfaces for qualitative or quantitative purposes. Presence of antibodies or autoantibodies against Thy-1 can also be detected specifically. Evidence that the dog, carp, hamster and goldfish carry Thy-1-like molecules on neuronal (brain) cells is demonstrated by this method.

  12. A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy

    Directory of Open Access Journals (Sweden)

    Erhao Zhang

    2017-01-01

    Full Text Available Abstract Adoptive cell therapy using chimeric antigen receptor (CAR-engineered T cells has emerged as a very promising approach to combating cancer. Despite its ability to eliminate tumors shown in some clinical trials, CAR-T cell therapy involves some significant safety challenges, such as cytokine release syndrome (CRS and “on-target, off-tumor” toxicity, which is related to poor control of the dose, location, and timing of T cell activity. In the past few years, some strategies to avoid the side effects of CAR-T cell therapy have been reported, including suicide gene, inhibitory CAR, dual-antigen receptor, and the use of exogenous molecules as switches to control the CAR-T cell functions. Because of the advances of the CAR paradigm and other forms of cancer immunotherapy, the most effective means of defeating the cancer has become the integration therapy with the combinatorial control system of switchable dual-receptor CAR-T cell and immune checkpoint blockade.

  13. MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

    Energy Technology Data Exchange (ETDEWEB)

    Menachery, Vineet D.; Schafer, Alexandra; Burnum-Johnson, Kristin E.; Mitchell, Hugh D.; Eisfeld-Fenney, Amie J.; Walters, Kevin B.; Nicora, Carrie D.; Purvine, Samuel O.; Casey, Cameron P.; Monroe, Matthew E.; Weitz, Karl K.; Stratton, Kelly G.; Webb-Robertson, Bobbie-Jo M.; Gralinski, Lisa; Metz, Thomas O.; Smith, Richard D.; Waters, Katrina M.; Sims, Amy C.; Kawaoka, Yoshihiro; Baric, Ralph

    2018-01-16

    Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigen presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.

  14. Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

    Science.gov (United States)

    Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

    1987-09-01

    A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

  15. The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

    Science.gov (United States)

    Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

    2016-01-01

    Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

  16. Isolation and characterization of antigen-Ia complexes involved in T cell recognition

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1986-01-01

    Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

  17. Vaccination and the TAP-independent antigen processing pathways.

    Science.gov (United States)

    López, Daniel; Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen

    2013-09-01

    The cytotoxic CD8(+) T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8(+) T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.

  18. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

    DEFF Research Database (Denmark)

    Drent, Esther; Groen, Richard W. J.; Noort, Willy A. Noort

    2016-01-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody...... sequences to generate second-generation retroviral CD38- chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence......, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite...

  19. A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway.

    Directory of Open Access Journals (Sweden)

    Smanla Tundup

    Full Text Available The milk pentasaccharide LNFPIII has therapeutic action for metabolic and autoimmune diseases and prolongs transplant survival in mice when presented as a neoglycoconjugate. Within LNFPIII is the Lewisx trisaccharide, expressed by many helminth parasites. In humans, LNFPIII is found in human milk and also known as stage-specific embryonic antigen-1. LNFPIII-NGC drives alternative activation of macrophages and dendritic cells via NFκB activation in a TLR4 dependent mechanism. However, the connection between LNFPIII-NGC activation of APCs, TLR4 signaling and subsequent MAP kinase signaling leading to anti-inflammatory activation of APCs remains unknown. In this study we determined that the innate receptor CD14 was essential for LNFPIII-NGC induction of both ERK and NFkB activation in APCs. Induction of ERK activation by LNFPIII-NGC was completely dependent on CD14/TLR4-Ras-Raf1/TPL2-MEK axis in bone marrow derived dendritic cells (BMDCs. In addition, LNFPIII-NGC preferentially induced the production of Th2 "favoring" chemokines CCL22 and matrix metalloprotease protein-9 in a CD14 dependent manner in BMDCs. In contrast, LNFPIII-NGC induces significantly lower levels of Th1 "favoring" chemokines, MIP1α, MIP1β and MIP-2 compared to levels in LPS stimulated cells. Interestingly, NGC of the identical human milk sugar LNnT, minus the alpha 1-3 linked fucose, failed to activate APCs via TLR4/MD2/CD14 receptor complex, suggesting that the alpha 1-3 linked fucose in LNFPIII and not on LNnT, is required for this process. Using specific chemical inhibitors of the MAPK pathway, we found that LNFPIII-NGC induction of CCL22, MMP9 and IL-10 production was dependent on ERK activation. Over all, this study suggests that LNFPIII-NGC utilizes CD14/TLR4-MAPK (ERK axis in modulating APC activation to produce anti-inflammatory chemokines and cytokines in a manner distinct from that seen for the pro-inflammatory PAMP LPS. These pathways may explain the in vivo

  20. Tissue distribution of histo-blood group antigens

    DEFF Research Database (Denmark)

    Ravn, V; Dabelsteen, Erik

    2000-01-01

    carrier carbohydrate chains. Histo-blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histoblood group antigens, i.e. the ABO, Lewis, and saliva-secretor type of the individual, and the cell- and tissue......The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo-blood group antigens and related carbohydrate structures. The present paper summarizes the available data...... concerning the histological distribution of histo-blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo-blood group antigens carried by type 1, 2, and 3 chain...