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Sample records for activation antigen cd23

  1. 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

  2. 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...

  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. Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

    Science.gov (United States)

    Lecoanet-Henchoz, S; Plater-Zyberk, C; Graber, P; Gretener, D; Aubry, J P; Conrad, D H; Bonnefoy, J Y

    1997-09-01

    CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

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

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    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.

  6. 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

  7. 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.

  8. 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

  9. Natural CD4+ T-cell responses against indoleamine 2,3-dioxygenase

    DEFF Research Database (Denmark)

    Munir, Shamaila; Larsen, Stine Kiaer; Iversen, Trine Zeeberg

    2012-01-01

    The enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tum...... antigens. Recently, we described cytotoxic CD8(+) T-cell reactivity towards IDO-derived peptides.......The enzyme indoleamine 2,3-dioxygenase (IDO) contributes to immune tolerance in a variety of settings. In cancer IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it endorses the establishment of peripheral immune tolerance to tumor...

  10. 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.

  11. 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.

  12. Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Daniela F Angelini

    Full Text Available It has long been known that multiple sclerosis (MS is associated with an increased Epstein-Barr virus (EBV seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A and lytic (BZLF-1, BMLF-1 antigens in relapsing-remitting MS patients (n = 113 and healthy donors (HD (n = 43 and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.

  13. Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan); Chiba, Joe, E-mail: chibaj@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278-8510 (Japan)

    2009-09-11

    We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

  14. Long-term Maintenance of CD4 T Cell Memory Responses to Malaria Antigens in Malian Children Coinfected with Schistosoma haematobium

    Directory of Open Access Journals (Sweden)

    Kirsten E. Lyke

    2018-02-01

    Full Text Available Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP Malian children, aged 4–8 years, are protected from malaria compared to matched schistosomiasis-negative (SN children. The effect of concomitant schistosomiasis upon acquisition of T cell memory is unknown. We examined antigen-specific T cell frequencies in 48 Malian children aged 4–14 to a pool of malaria blood stage antigens, and a pool of schistosomal antigens, at a time point during a malaria episode and at a convalescent time point ~6 months later, following cessation of malaria transmission. CD4+ T cell-derived memory responses, defined as one or more significant cytokine (IFN-γ, TNF-α, IL-2, and/or IL-17A responses, was measured to schistoma antigens in 18/23 SP children at one or both time points, compared to 4/23 SN children (P < 0.0001. At the time of malaria infection, 12/24 SN children and 15/23 SP children (P = 0.29 stimulated with malaria antigens demonstrated memory recall as defined by CD4-derived cytokine production. This compares to 7/23 SN children and 16/23 SP children (P = 0.009 at the convalescent timepoint. 46.2% of cytokine-producing CD4+ T cells expressed a single cytokine after stimulation with malaria antigen during the malaria episode. This fell to 40.9% at follow-up with a compensatory rise of multifunctional cytokine secretion over time, a phenomenon consistent with memory maturation. The majority (53.2–59.5% of responses derived from CD45RA−CD62L− effector memory T cells with little variation in the phenotype depending upon the time point or the study cohort. We conclude that detectable T cell memory responses can be measured against both malaria and schistosoma antigens and that the presence of Schistosoma haematobium may be associated with long-term maintenance of T memory to malaria.

  15. Involvement of CD244 in regulating CD4+ T cell immunity in patients with active tuberculosis.

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    Bingfen Yang

    Full Text Available CD244 (2B4 is a member of the signaling lymphocyte activation molecule (SLAM family of immune cell receptors and it plays an important role in modulating NK cell and CD8(+ T cell immunity. In this study, we investigated the expression and function of CD244/2B4 on CD4(+ T cells from active TB patients and latent infection individuals. Active TB patients had significantly elevated CD244/2B4 expression on M. tuberculosis antigen-specific CD4(+ T cells compared with latent infection individuals. The frequencies of CD244/2B4-expressing antigen-specific CD4(+ T cells were significantly higher in retreatment active TB patients than in new active TB patients. Compared with CD244/2B4-dull and -middle CD4(+ T cells, CD244/2B4-bright CD4(+ T cell subset had significantly reduced expression of IFN-γ, suggesting that CD244/2B4 expression may modulate IFN-γ production in M. tuberculosis antigen-responsive CD4(+ T cells. Activation of CD244/2B4 signaling by cross-linking led to significantly decreased production of IFN-γ. Blockage of CD244/2B4 signaling pathway of T cells from patients with active TB resulted in significantly increased production of IFN-γ, compared with isotype antibody control. In conclusion, CD244/2B4 signaling pathway has an inhibitory role on M. tuberculosis antigen-specific CD4(+ T cell function.

  16. Neutral Polymer Micelle Carriers with pH-Responsive, Endosome-Releasing Activity Modulate Antigen Trafficking to Enhance CD8 T-Cell Responses

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    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

  17. 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

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

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    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.

  19. CD21+ (B2 antigen+) cell decrement and CD4+CD29+ (helper-inducer) cell increment suggest an activation of cell immune reactivity in multiple sclerosis.

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    Gambi, D; Porrini, A M; Giampietro, A; Macor, S

    1991-08-01

    Two-color flow cytometric analysis on peripheral blood lymphocytes of 35 untreated multiple sclerosis (MS) patients, 17 other medical disease (OMD) patients and 14 healthy control (HC) subjects was performed to evaluate the levels of different T and B cell subpopulations. In MS patients we observed an increase in CD4+CD29+ helper-inducer cells but this increase was not related to the different phases of the disease. We hypothesize that this change is related to the reduction of CD21+ cells expressing B2 antigen, a 140 kDa molecule disappearing after B cell activation. An increased level of CD4+CD45RA- (helper-inducer-like cells) and a reduction of CD4+CD29- (suppressor-inducer-like cells) were also present in our patients. These findings demonstrate an immune 'disequilibrium' in MS, which is linked with an increased level of CD25+ cells expressing the interleukin-2 (IL-2) receptor. IL-2, besides being a T cell growth factor, is also a B cell growth factor. These data let us hypothesize that an activation of the immune response is present in MS.

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

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    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.

  1. Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

    Science.gov (United States)

    Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

    2010-02-01

    The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

  2. Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

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    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.

  3. Oral delivery of the Sj23LHD-GST antigen by Salmonella typhimurium type III secretion system protects against Schistosoma japonicum infection in mice.

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    Guo Chen

    2011-09-01

    Full Text Available BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease and oral vaccine delivery system would be benefit for prevention of this disease. Although attenuated salmonella has been used as an antigen expression vector for oral vaccine development, the membrane-bound vacuoles in which bacteria reside hinders the presentation of expressed heterologous antigens to the major histocompatibility complex (MHC molecules. The present work used an attenuated Salmonella typhimurium strain VNP20009 to secretory expression of Sj23LHDGST bivalent antigen from Schistosoma japonicum and tested the protective efficacy against S. japonicum infection in orally immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: Promoters (nirB or pagC were used to express the antigen (Sj23LHDGST and the Salmonella type III or α-hemolysin secretion system was employed to secrete it. The immunoblotting analysis and fluorescent microscopy revealed that the antigen was effectively expressed and delivered to the cytosol of macrophages in vitro. Among recombinant vaccine strains, an engineered VNP20009 which expressed the antigen by nirB promoter and secreted it through type III secretion system (nirB-sopE(1-104-Sj23LHD-GST efficiently protected against S. japonicum infection in a mouse model. This strain elicited a predominantly IgG(2a antibody response and a markedly increase in the production of IL-12 and IFN-γ. The flow cytometric analysis demonstrated that this strain caused T cell activation as evidenced by significantly increased expression of CD44 and CD69. CONCLUSION/SIGNIFICANCE: Oral delivery of antigen by nirB-driven Salmonella typhimurium type III secretion system is a novel, safe, inexpensive, efficient and convenient approach for schistosome vaccine development.

  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. Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

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    Williams, L A; McLellan, A D; Summers, K L; Sorg, R V; Fearnley, D B; Hart, D N

    1996-01-01

    Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro. PMID:8911149

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

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    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

  7. CD4(+)and CD8(+)T-cell reactions against leukemia-associated- or minor-histocompatibility-antigens in AML-patients after allogeneic SCT.

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    Steger, Brigitte; Milosevic, Slavoljub; Doessinger, Georg; Reuther, Susanne; Liepert, Anja; Braeu, Marion; Schick, Julia; Vogt, Valentin; Schuster, Friedhelm; Kroell, Tanja; Busch, Dirk H; Borkhardt, Arndt; Kolb, Hans-Jochem; Tischer, Johanna; Buhmann, Raymund; Schmetzer, Helga

    2014-04-01

    T-cells play an important role in the remission-maintenance in AML-patients (pts) after SCT, however the role of LAA- (WT1, PR1, PRAME) or minor-histocompatibility (mHag, HA1) antigen-specific CD4(+) and CD8(+)T-cells is not defined. A LAA/HA1-peptide/protein stimulation, cloning and monitoring strategy for specific CD8(+)/CD4(+)T-cells in AML-pts after SCT is given. Our results show that (1) LAA-peptide-specific CD8+T-cells are detectable in every AML-pt after SCT. CD8(+)T-cells, recognizing two different antigens detectable in 5 of 7 cases correlate with long-lasting remissions. Clonal TCR-Vβ-restriction exemplarily proven by spectratyping in PRAME-specific CD8(+)T-cells; high PRAME-peptide-reactivity was CD4(+)-associated, as shown by IFN-γ-release. (2) Two types of antigen-presenting cells (APCs) were tested for presentation of LAA/HA1-proteins to CD4(+)T-cells: miniEBV-transduced lymphoblastoid cells (B-cell-source) and CD4-depleted MNC (source for B-cell/monocyte/DC). We provide a refined cloning-system for proliferating, CD40L(+)CD4(+)T-cells after LAA/HA1-stimulation. CD4(+)T-cells produced cytokines (GM-CSF, IFN-γ) upon exposure to LAA/HA1-stimulation until after at least 7 restimulations and demonstrated cytotoxic activity against naive blasts, but not fibroblasts. Antileukemic activity of unstimulated, stimulated or cloned CD4(+)T-cells correlated with defined T-cell-subtypes and the clinical course of the disease. In conclusion we provide immunological tools to enrich and monitor LAA/HA1-CD4(+)- and CD8(+)T-cells in AML-pts after SCT and generate data with relevant prognostic value. We were able to demonstrate the presence of LAA-peptide-specific CD8(+)T-cell clones in AML-pts after SCT. In addition, we were also able to enrich specific antileukemic reactive CD4(+)T-cells without GvH-reactivity upon repeated LAA/HA1-protein stimulation and limiting dilution cloning. Copyright © 2013 Elsevier GmbH. All rights reserved.

  8. 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.

  9. 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

  10. Antigen-Experienced CD4lo T Cells Are Linked to Deficient Contraction of the Immune Response in Autoimmune Diabetes

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    Sean Linkes

    2010-01-01

    Full Text Available Following proper activation, naïve “CD4lo” T cells differentiate into effector T cells with enhanced expression of CD4 -“CD4hi” effectors. Autoimmune diabetes-prone NOD mice display a unique set of antigen-experienced “CD4lo” T cells that persist after primary stimulation. Here, we report that a population of such cells remained after secondary and tertiary TCR stimulation and produced cytokines upon antigenic challenge. However, when NOD blasts were induced in the presence of rIL-15, the number of antigen-experienced “CD4lo” T cells was significantly reduced. Clonal contraction, mediated in part by CD95-dependent activation-induced cell death (AICD, normally regulates the accumulation of “CD4hi” effectors. Interestingly, CD95 expression was dramatically reduced on the AICD-resistant NOD “CD4lo” T cells. Thus, while autoimmune disease has often been attributed to the engagement of robust autoimmunity, we suggest that the inability to effectively contract the immune response distinguishes benign autoimmunity from progressive autoimmune diseases that are characterized by chronic T cell-mediated inflammation.

  11. 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.

  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

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    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. Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue

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    Marcus Nascimento Borges

    Full Text Available CONTEXT AND OBJECTIVE: Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. DESIGN AND SETTING: This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. METHODS: The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. RESULTS: Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13 in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75 was statistically larger (P < 0.001. Several clinical features were analyzed, but only parity was shown to influence CD83 antigen expression in the adjacent breast tissue, such that positive expression was more evident in nulliparous women (P = 0.042. CONCLUSIONS: The expression of the CD83 antigen in the fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

  14. Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

    Science.gov (United States)

    Liu, Shuangshuang; Li, Shasha; Zhang, Yang; Wang, Ye; Zhu, Yumeng; Wang, Bin; Chen, Zhi-Nan

    2017-01-01

    The immunoglobulin superfamily member CD147 is a widely expressed glycoprotein that occurs in both a membrane-spanning and soluble form. Sandwich ELISA is a powerful tool for analyzing soluble antigens. The aim of the present study was to obtain a highly specific polyclonal antibody against human CD147 that can be used for sandwich ELISA analysis. Expression of recombinant CD147 by a eukaryotic expression system was used to immunize rabbits to obtain antiserum. A highly specific polyclonal antibody that was able to detect soluble CD147 in sandwich ELISA was obtained by antigen-immunoaffinity chromatography purification. The purity of rabbit anti-CD147 polyclonal antibodies was ~99%, and ELISA analysis was able to determine the titer of the rabbit anti-CD147 polyclonal antibodies at 1:512,000. The lowest concentration of the standard CD147 antigen that the sandwich ELISA was able to detect was 31.25 pg/ml. The sandwich ELISA system was composed of anti-hepatoma HAb18 monoclonal antibodies and purified rabbit anti-CD147 polyclonal antibodies. The present study demonstrated that antigen-immunoaffinity chromatography may be a good technique for the purification of polyclonal antibodies, which may be used to detect antigen in sandwich ELISAs. PMID:28487989

  15. The simultaneous ex vivo detection of low-frequency antigen-specific CD4+ and CD8+ T-cell responses using overlapping peptide pools.

    Science.gov (United States)

    Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H

    2012-11-01

    The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.

  16. Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

    Science.gov (United States)

    Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

    2017-01-01

    CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

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

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    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.

  18. Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki; Yoichiro; Tanabe, Kazumi; Umeki, Shigeko; Nakamura, Nori; Yamakido, Michio; Hamamoto, Kazuko.

    1990-04-01

    The T-cell receptor CD3 (TCR/CD3) complex plays a central role in antigen recognition and activation of mature T cells, and therefore abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of surface TCR/CD3 expression among human mature CD4 + T cells. The presence of variant CD4 + T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T-cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T-cell dysfunction. (author)

  19. The extended family of CD1d-restricted T cells: sifting through a mixed bag of TCRs, antigens and functions

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    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.

  20. 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.

  1. Role of 4-1BB receptor in the control played by CD8(+ T cells on IFN-gamma production by Mycobacterium tuberculosis antigen-specific CD4(+ T Cells.

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    Carla Palma

    Full Text Available BACKGROUND: Antigen-specific IFN-gamma producing CD4(+ T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+ T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+ T cells which suppressed IFN-gamma-secreting CD4(+ T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+ T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+ T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+ T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+ T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+ T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+ T cells. The selective

  2. Cellular size as a means of tracking mTOR activity and cell fate of CD4+ T cells upon antigen recognition.

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    Kristen N Pollizzi

    Full Text Available mTOR is a central integrator of metabolic and immunological stimuli, dictating immune cell activation, proliferation and differentiation. In this study, we demonstrate that within a clonal population of activated T cells, there exist both mTORhi and mTORlo cells exhibiting highly divergent metabolic and immunologic functions. By taking advantage of the role of mTOR activation in controlling cellular size, we demonstrate that upon antigen recognition, mTORhi CD4+ T cells are destined to become highly glycolytic effector cells. Conversely, mTORlo T cells preferentially develop into long-lived cells that express high levels of Bcl-2, CD25, and CD62L. Furthermore, mTORlo T cells have a greater propensity to differentiate into suppressive Foxp3+ T regulatory cells, and this paradigm was also observed in human CD4+ T cells. Overall, these studies provide the opportunity to track the development of effector and memory T cells from naïve precursors, as well as facilitate the interrogation of immunologic and metabolic programs that inform these fates.

  3. The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases.

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    Janossy, G; Coustan-Smith, E; Campana, D

    1989-03-01

    Current views about the origin of acute lymphoid leukemia (ALL) emphasize the importance of maturation arrest at a precursor cell level. Recently, the CD22 antigen has been identified in the cytoplasm of normal bone marrow-borne immature B lineage cells, while the CD3 antigen (epsilon chain) has been detected within normal immature thymic blasts. In the first part our study performed on 100 cases of known acute leukemias, the expression of such cytoplasmic molecules, referred to as cCD22 and cCD3, was analyzed together with their appearance in the leukemic cells' membrane (mCD22 and mCD3). The presence of cCD22 in B-lineage ALL and that of cCD3 in T-ALL has indeed fully confirmed the diagnosis reached by other markers, and mCD22 and mCD3 were expressed on only a few cases of B- and T-lineage ALL, also revealing a degree of developmental asynchrony within leukemic blasts. In the subsequent analysis both cCD22 and cCD3 have been included in a standard panel of diagnostic reagents applied on 500 consecutive cases of acute leukemia. Here the aim was to analyze both the diagnostic precision of individual markers and the heterogeneity of various leukemic types in terms of the expression of membrane and intracellular antigens and their cytochemical features (Sudan Black B and esterases). It has been found that cCD22 and cCD3 are exquisitely specific for B-precursor ALL (TdT+, CD19+) and T-ALL (TdT+, CD7+), respectively, while both markers are absent in acute myeloblastic leukemia (AML) and acute myelomonocytic and monocytic leukemia (AMML/AMoL). These observations contrast the findings which demonstrate that 31% of cases among nonlymphoid acute leukemia (including AML and AMML) express CD7 and/or TdT. The study of myeloid antigens detected by CD13, CD33, and CD14 is also informative and complementary, both in diagnosing and subdividing the AML and AMML/AMoL groups. The peculiar main observation of this study is that only with the combined use of these markers in a

  4. Serum levels of total IgE and soluble CD23 in bronchial asthma

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    G. Di Lorenzo

    1996-01-01

    Full Text Available The aim of the present study was to compare, during the pollen season, serum levels of total IgE and soluble CD23 (sCD23 from patients with allergic bronchial asthma, with those from healthy subjects. Significantly higher levels of total IgE and sCD23 were found in patients with asthma compared to the control group. Both in normal controls and in asthmatic patients, a significant correlation was shown between the levels of these two molecules. In asthmatic patients, significant correlations were found for both total IgE and sCD23, with lung function measured as bronchial responsiveness to inhaled methacholine. These results suggest that in asthmatic patients, in addition to the study of total serum IgE levels, the assessment of sCD23 serum levels may be helpful in the evaluation of disease activity.

  5. Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

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    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.

  6. Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue.

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    Borges, Marcus Nascimento; Facina, Gil; Silva, Ismael Dale Cotrin Guerreiro; Waitzberg, Angela Flávia Logullo; Nazario, Afonso Celso Pinto

    2011-12-01

    Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13) in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75) was statistically larger (P fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

  7. Allopurinol reduces antigen-specific and polyclonal activation of human T cells

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    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.

  8. 77 FR 62520 - Prospective Grant of Exclusive License: The Development of Anti-CD22 Chimeric Antigen Receptors...

    Science.gov (United States)

    2012-10-15

    ... Exclusive License: The Development of Anti- CD22 Chimeric Antigen Receptors (CARs) for the Treatment of B... ``Anti-CD22 Chimeric Antigen Receptors'' [HHS Ref. E-265-2011/0-US-01], and (b) U.S. Patent Application... CD22 on their cell surface using chimeric antigen receptors which contain the HA22 or BL22 antibody...

  9. Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells.

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    Tang, Jinle; Li, Jialu; Zhu, Xuejun; Yu, Yuan; Chen, Dan; Yuan, Lei; Gu, Zhenyang; Zhang, Xingding; Qi, Lin; Gong, Zhishu; Jiang, Pengjun; Yu, Juhua; Meng, Huimin; An, Gangli; Zheng, Huyong; Yang, Lin

    2016-06-07

    Various CD7-targeting immunotoxins have been tested for its potential in treating CD7+ malignant patients but none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Here we successfully constructed the monovalent and bivalent CD7 nanobody-based immunotoxins PG001 and PG002, both conjugated with a truncated derivative of Pseudomonas exotoxin A respectively. The prokaryotic system expressed immunotoxins not only maintained their binding specificity for CD7-positive cells with a Kd of 16.74 nM and 3.6 nM for PG001 and PG002 respectively, but also efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, and primary T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with an in vitro cytotoxic activity (EC50) in the range of 23-30 pM for PG002. In NOD/SCID mice transplanted with CEM cells, PG001 and PG002 prevented engraftment of the cells and markedly prolonged mouse survival. Owing to the efficient antigen-restricted anti-leukemic activity of PG002, this CD7 nanobody-based immunotoxin exhibited a superior anti-CD7 positive malignancies activity than previously reported immunotoxins, and may represent a promising therapeutic strategy in treating CD7-positive leukemia and lymphoma, which still remain a significant clinical challenge.

  10. IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

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    da Silva, Thiago Aparecido; Mariano, Vania Sammartino; Sardinha-Silva, Aline; de Souza, Maria Aparecida; Mineo, Tiago Wilson Patriarca; Roque-Barreira, Maria Cristina

    2016-01-01

    ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production

  11. IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

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    Thiago Aparecido da Silva

    Full Text Available ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs, as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, Artin

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

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    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.

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

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    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.

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

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    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.

  15. The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

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    Gunter Rappl

    Full Text Available Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+ CD57(+ CD7(- phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

  16. CD4 Depletion or CD40L Blockade Results in Antigen-Specific Tolerance in a Red Blood Cell Alloimmunization Model

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    Prabitha Natarajan

    2017-08-01

    Full Text Available Approximately 3–10% of human red blood cell (RBC transfusion recipients form alloantibodies to non-self, non-ABO blood group antigens expressed on donor RBCs, with these alloantibodies having the potential to be clinically significant in transfusion and pregnancy settings. However, the majority of transfused individuals never form detectable alloantibodies. Expanding upon observations that children initially transfused with RBCs at a young age are less likely to form alloantibodies throughout their lives, we hypothesized that “non-responders” may not only be ignorant of antigens on RBCs but instead tolerized. We investigated this question in a reductionist murine model, in which transgenic donors express the human glycophorin A (hGPA antigen in an RBC-specific manner. Although wild-type mice treated with poly IC and transfused with hGPA RBCs generated robust anti-hGPA IgG alloantibodies that led to rapid clearance of incompatible RBCs, those transfused in the absence of an adjuvant failed to become alloimmunized. Animals depleted of CD4+ cells or treated with CD40L blockade prior to initial hGPA RBC exposure, in the presence of poly IC, failed to generate detectable anti-hGPA IgG alloantibodies. These non-responders to a primary transfusion remained unable to generate anti-hGPA IgG alloantibodies upon secondary hGPA exposure and did not prematurely clear transfused hGPA RBCs even after their CD4 cells had returned or their CD40L blockade had resolved. This observed tolerance was antigen (hGPA specific, as robust IgG responses to transfused RBCs expressing a third-party antigen occurred in all studied groups. Experiments completed in an RBC alloimmunization model that allowed evaluation of antigen-specific CD4+ T-cells (HOD (hen egg lysozyme, ovalbumin, and human duffyb demonstrated that CD40L blockade prevented the expansion of ovalbumin 323-339 specific T-cells after HOD RBC transfusion and also prevented germinal center formation. Taken

  17. T helper-independent activation of human CD8+ cells: the role of CD28 costimulation.

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    Van Gool, S W; Zhang, Y; Kasran, A; de Boer, M; Ceuppens, J L

    1996-07-01

    The concept that activation of MHC class I-restricted CD8+ cells entirely depends on help from MHC class II-restricted CD4+ T cells has recently been supplemented with an alternative model in which CD8+ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8+ T cells in two different in vitro models. Freshly isolated CD8+ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting Fc gamma R+ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8+ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8+ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8+ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8+ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.

  18. Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

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    Koguchi, Yoshinobu; Gardell, Jennifer L.; Thauland, Timothy J.; Parker, David C.

    2011-01-01

    CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery. PMID:21677130

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

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    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.

  20. Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection

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    Janice K. P. Lam

    2018-03-01

    Full Text Available Long term carriers were shown to generate robust polyfunctional T cell (PFC responses against lytic and latent antigens of Epstein-Barr virus (EBV. However, the time of emergence of PFC responses against EBV antigens, pattern of immunodominance and difference between CD4+ and CD8+ T cell responses during various stages of EBV infection are not clearly understood. A longitudinal study was performed to assess the development of antigen-specific PFC responses in children diagnosed to have primary symptomatic (infectious mononucleosis [IM] and asymptomatic (AS EBV infection. Evaluation of IFN-γ secreting CD8+ T cell responses upon stimulation by HLA class I-specific peptides of EBV lytic and latent proteins by ELISPOT assay followed by assessment of CD4+ and CD8+ PFC responses upon stimulation by a panel of overlapping EBV peptides for co-expression of IFN-γ, TNF-α, IL-2, perforin and CD107a by flow cytometry were performed. Cytotoxicity of T cells against autologous lymphoblastoid cell lines (LCLs as well as EBV loads in PBMC and plasma were also determined. Both IM and AS patients had elevated PBMC and plasma viral loads which declined steadily during a 12-month period from the time of diagnosis whilst decrease in the magnitude of CD8+ T cell responses toward EBV lytic peptides in contrast to increase toward latent peptides was shown with no significant difference between those of IM and AS patients. Both lytic and latent antigen-specific CD4+ and CD8+ T cells demonstrated polyfunctionality (defined as greater or equal to three functions concurrent with enhanced cytotoxicity against autologous LCLs and steady decrease in plasma and PBMC viral loads over time. Immunodominant peptides derived from BZLF1, BRLF1, BMLF1 and EBNA3A-C proteins induced the highest proportion of CD8+ as well as CD4+ PFC responses. Diverse functional subtypes of both CD4+ and CD8+ PFCs were shown to emerge at 6–12 months. In conclusion, EBV antigen-specific CD4+ and CD

  1. Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

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    Maud Condomines

    Full Text Available Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1 costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

  2. 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

  3. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

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    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.

  4. 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.

  5. Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

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    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.

  6. 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.

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

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    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

  8. Cognate antigen stimulation generates potent CD8+ inflammatory effector T cells.

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    Hsueh-Cheng eSung

    2013-12-01

    Full Text Available Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88 deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8+ inflammatory effector cells in a lymph node can mobilize 107 cells in 24h, including lymphocytes, natural killer cells and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

  9. 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.

  10. In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

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    Kentaro Minagawa

    Full Text Available Approximately fifty percent of patients with acute myeloid leukemia can be cured with current therapeutic strategies which include, standard dose chemotherapy for patients at standard risk of relapse as assessed by cytogenetic and molecular analysis, or high-dose chemotherapy with allogeneic hematopoietic stem cell transplant for high-risk patients. Despite allogeneic hematopoietic stem cell transplant about 25% of patients still succumb to disease relapse, therefore, novel strategies are needed to improve the outcome of patients with acute myeloid leukemia.We developed an immunotherapeutic strategy targeting the CD33 myeloid antigen, expressed in ~ 85-90% of patients with acute myeloid leukemia, using chimeric antigen receptor redirected T-cells. Considering that administration of CAR T-cells has been associated with cytokine release syndrome and other potential off-tumor effects in patients, safety measures were here investigated and reported. We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the inducible Caspase9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen receptor recognizing human CD33. ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells had a 75±3.8% (average ± standard error of the mean chimeric antigen receptor expression, were able to specifically lyse CD33+ targets in vitro, including freshly isolated leukemic blasts from patients, produce significant amount of tumor-necrosis-factor-alpha and interferon-gamma, express the CD107a degranulation marker, and proliferate upon antigen specific stimulation. Challenging ΔCD19 selected inducible Caspase9-CAR.CD33 T-cells with programmed-death-ligand-1 enriched leukemia blasts resulted in significant killing like observed for the programmed-death-ligand-1 negative leukemic blasts fraction. Since the administration of 10 nanomolar of a non

  11. 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

    Science.gov (United States)

    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 immunization regimens were able to induce higher levels of CD8+ T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44+CD62L−NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation. PMID:27132023

  12. Dietary Fructo-Oligosaccharides Attenuate Early Activation of CD4+ T Cells Which Produce both Th1 and Th2 Cytokines in the Intestinal Lymphoid Tissues of a Murine Food Allergy Model.

    Science.gov (United States)

    Tsuda, Masato; Arakawa, Haruka; Ishii, Narumi; Ubukata, Chihiro; Michimori, Mana; Noda, Masanari; Takahashi, Kyoko; Kaminogawa, Shuichi; Hosono, Akira

    2017-01-01

    Fructo-oligosaccharides (FOS) are prebiotic agents with immunomodulatory effects involving improvement of the intestinal microbiota and metabolome. In this study, we investigated the cellular mechanisms through which FOS modulate intestinal antigen-specific CD4+ T cell responses in food allergy, using OVA23-3 mice. OVA23-3 mice were fed an experimental diet containing either ovalbumin (OVA) or OVA and FOS for 1 week. Body weight and mucosal mast cell protease 1 in the serum were measured as the indicator of intestinal inflammation. Single-cell suspensions were prepared from intestinal and systemic lymphoid tissues for cellular analysis. Cytokine production was measured by ELISA. Activation markers and intracellular cytokines in CD4+ T cells were analyzed by flow cytometry. Activated CD4+ T cells were purified to examine cytokine production. Dietary intake of FOS provided moderate protection from the intestinal inflammation induced by the OVA-containing diet. FOS significantly reduced food allergy-induced Th2 cytokine responses in intestinal tissues but not in systemic tissues. FOS decreased OVA diet-induced IFN-γ+IL-4+ double-positive CD4+ T cells and early-activated CD45RBhighCD69+CD4+ T cells in the mesenteric lymph nodes. Furthermore, we confirmed that these CD45RBhighCD69+CD4+ T cells are able to produce high levels of IFN-γ and moderate level of IL-4, IL-10, and IL-13. Dietary intake of FOS during the development of food allergy attenuates the induction of intestinal Th2 cytokine responses by regulating early activation of naïve CD4+ T cells, which produce both Th1 and Th2 cytokines. Our results suggest FOS might be a potential food agent for the prevention of food allergy by modulating oral sensitization to food antigens. © 2017 S. Karger AG, Basel.

  13. Cis-acting pathways selectively enforce the non-immunogenicity of shed placental antigen for maternal CD8 T cells.

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    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.

  14. 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

  15. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23

    Science.gov (United States)

    Weinhold, Mario; Eisenblätter, Martin; Jasny, Edith; Fehlings, Michael; Finke, Antje; Gayum, Hermine; Rüschendorf, Ursula; Renner Viveros, Pablo; Moos, Verena; Allers, Kristina; Schneider, Thomas; Schaible, Ulrich E.; Schumann, Ralf R.; Mielke, Martin E.; Ignatius, Ralf

    2013-01-01

    Background Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4+ and CD8+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs), which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S) 19, which has previously been employed successfully to vaccinate cattle. Methodology/Principal findings We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR)2. Conclusions/Significance Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2) human DCs to produce Th1-promoting cytokines. PMID:23805193

  16. The Attenuated Brucella abortus Strain 19 Invades, Persists in, and Activates Human Dendritic Cells, and Induces the Secretion of IL-12p70 but Not IL-23.

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    Mario Weinhold

    Full Text Available Bacterial vectors have been proposed as novel vaccine strategies to induce strong cellular immunity. Attenuated strains of Brucella abortus comprise promising vector candidates since they have the potential to induce strong CD4(+ and CD8(+ T-cell mediated immune responses in the absence of excessive inflammation as observed with other Gram-negative bacteria. However, some Brucella strains interfere with the maturation of dendritic cells (DCs, which is essential for antigen-specific T-cell priming. In the present study, we investigated the interaction of human monocyte-derived DCs with the smooth attenuated B. abortus strain (S 19, which has previously been employed successfully to vaccinate cattle.We first looked into the potential of S19 to hamper the cytokine-induced maturation of DCs; however, infected cells expressed CD25, CD40, CD80, and CD86 to a comparable extent as uninfected, cytokine-matured DCs. Furthermore, S19 activated DCs in the absence of exogeneous stimuli, enhanced the expression of HLA-ABC and HLA-DR, and was able to persist intracellularly without causing cytotoxicity. Thus, DCs provide a cellular niche for persisting brucellae in vivo as a permanent source of antigen. S19-infected DCs produced IL-12/23p40, IL-12p70, and IL-10, but not IL-23. While heat-killed bacteria also activated DCs, soluble mediators were not involved in S19-induced activation of human DCs. HEK 293 transfectants revealed cellular activation by S19 primarily through engagement of Toll-like receptor (TLR2.Thus, as an immunological prerequisite for vaccine efficacy, B. abortus S19 potently infects and potently activates (most likely via TLR2 human DCs to produce Th1-promoting cytokines.

  17. Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

    Science.gov (United States)

    Sitt, Tatjana; Pelle, Roger; Chepkwony, Maurine; Morrison, W Ivan; Toye, Philip

    2018-05-06

    The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

  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. Myeloid antigens in childhood lymphoblastic leukemia:clinical data point to regulation of CD66c distinct from other myeloid antigens

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    Madzo Jozef

    2005-04-01

    Full Text Available Abstract Background Aberrant expression of myeloid antigens (MyAgs on acute lymphoblastic leukemia (ALL cells is a well-documented phenomenon, although its regulating mechanisms are unclear. MyAgs in ALL are interpreted e.g. as hallmarks of early differentiation stage and/or lineage indecisiveness. Granulocytic marker CD66c – Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6 is aberrantly expressed on ALL with strong correlation to genotype (negative in TEL/AML1 and MLL/AF4, positive in BCR/ABL and hyperdiploid cases. Methods In a cohort of 365 consecutively diagnosed Czech B-precursor ALL patients, we analyze distribution of MyAg+ cases and mutual relationship among CD13, CD15, CD33, CD65 and CD66c. The most frequent MyAg (CD66c is studied further regarding its stability from diagnosis to relapse, prognostic significance and regulation of surface expression. For the latter, flow cytometry, Western blot and quantitative RT-PCR on sorted cells is used. Results We show CD66c is expressed in 43% patients, which is more frequent than other MyAgs studied. In addition, CD66c expression negatively correlates with CD13 (p Conclusion In contrast to general notion we show that different MyAgs in lymphoblastic leukemia represent different biological circumstances. We chose the most frequent and tightly genotype-associated MyAg CD66c to show its stabile expression in patients from diagnosis to relapse, which differs from what is known on the other MyAgs. Surface expression of CD66c is regulated at the gene transcription level, in contrast to previous reports.

  20. 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.

    Science.gov (United States)

    Coelho-Dos-Reis, Jordana G; Huang, Jing; Tsao, Tiffany; Pereira, Felipe V; Funakoshi, Ryota; Nakajima, Hiroko; Sugiyama, Haruo; Tsuji, Moriya

    2016-07-01

    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 immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  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. Malignant T cells exhibit CD45 resistant Stat 3 activation and proliferation in cutaneous T cell lymphoma

    DEFF Research Database (Denmark)

    Krejsgaard, T; Helvad, Rikke; Ralfkiær, Elisabeth

    2010-01-01

    CD45 is a protein tyrosine phosphatase, which is well-known for regulating antigen receptor signalling in T and B cells via its effect on Src kinases. It has recently been shown that CD45 can also dephosphorylate Janus kinases (Jaks) and thereby regulate Signal transducer and activator of transcr......CD45 is a protein tyrosine phosphatase, which is well-known for regulating antigen receptor signalling in T and B cells via its effect on Src kinases. It has recently been shown that CD45 can also dephosphorylate Janus kinases (Jaks) and thereby regulate Signal transducer and activator...... of transcription (Stat) activation and cytokine-induced proliferation in lymphocytes. Consequently, CD45 dysregulation could be implicated in aberrant Jak/Stat activation and proliferation in lymphoproliferative diseases. Despite high expression of the CD45 ligand, Galectin-1, in skin lesions from cutaneous T......-cell lymphoma (CTCL), the malignant T cells exhibit constitutive activation of the Jak3/Stat3 signalling pathway and uncontrolled proliferation. We show that CD45 expression is down-regulated on malignant T cells when compared to non-malignant T cells established from CTCL skin lesions. Moreover, CD45 cross...

  11. Increased prevalence of late stage T cell activation antigen (VLA-1) in active juvenile chronic arthritis

    DEFF Research Database (Denmark)

    Ødum, Niels; Morling, Niels; Platz, P

    1987-01-01

    The presence of activated T cells as judged from the reaction with monoclonal antibodies (MoAb) against (a) a late stage T cell activation antigen (VLA-1), (b) the interleukin 2 (IL2) receptor (CD25), and (c) four different HLA class II molecules (HLA-DR, DRw52, DQ, and DP) was studied in 15 pati...

  12. Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

    Science.gov (United States)

    Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

    2018-05-09

    When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

  13. Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyBac Transposon-Engineered T Cells

    Directory of Open Access Journals (Sweden)

    Daisuke Morita

    2018-03-01

    Full Text Available Adoptive T cell therapy using chimeric antigen receptor (CAR-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs as feeder cells and virus-specific T cell receptor (TCR stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.

  14. Multiple dendritic cell populations activate CD4+ T cells after viral stimulation.

    Directory of Open Access Journals (Sweden)

    Adele M Mount

    2008-02-01

    Full Text Available Dendritic cells (DC are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+ T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+ T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+ and CD8(+ T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+ and CD4(+ T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+ T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+ T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+ T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

  15. 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.

  16. 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

  17. CD1 and mycobacterial lipids activate human T cells.

    Science.gov (United States)

    Van Rhijn, Ildiko; Moody, D Branch

    2015-03-01

    For decades, proteins were thought to be the sole or at least the dominant source of antigens for T cells. Studies in the 1990s demonstrated that CD1 proteins and mycobacterial lipids form specific targets of human αβ T cells. The molecular basis by which T-cell receptors (TCRs) recognize CD1-lipid complexes is now well understood. Many types of mycobacterial lipids function as antigens in the CD1 system, and new studies done with CD1 tetramers identify T-cell populations in the blood of tuberculosis patients. In human populations, a fundamental difference between the CD1 and major histocompatibility complex systems is that all humans express nearly identical CD1 proteins. Correspondingly, human CD1 responsive T cells show evidence of conserved TCRs. In addition to natural killer T cells and mucosal-associated invariant T (MAIT cells), conserved TCRs define other subsets of human T cells, including germline-encoded mycolyl-reactive (GEM) T cells. The simple immunogenetics of the CD1 system and new investigative tools to measure T-cell responses in humans now creates a situation in which known lipid antigens can be developed as immunodiagnostic and immunotherapeutic reagents for tuberculosis disease. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice

    DEFF Research Database (Denmark)

    Liu, Yawei; Teige, Anna; Mondoc, Emma

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition...... in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated...... with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells...

  19. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  20. CD117 expression on blast cells in acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Goryainova N.V.

    2015-09-01

    Full Text Available The aim of the present work was to analyze the frequency of CD117 (c-KIT antigen expression on the blast cells in acute myeloid leukemia (AML, evaluation of the presence of the relationship between the expression of the c-KIT and leukemia according to the FAB classification and definition of co-expression of the antigen CD117, antigens CD33 and CD34. The data of 47 patients with AML were diagnosed. M0 AML variant was established in 3 (6% patients, M1 – in 2 (4%, M2 – in 9 (20%, M4 – in 22 (47% and M5 – in 11 (23%. For immunophenotypic stu¬dies monoclonal antibodies (mAb that detect antigens of anti-CD34, anti-CD33 and anti-CD117 (Becton Dickinson, USA were used. The presence of the antigen CD117 was detected in 39 people, accounting for 83% of all surveyed. Antigen c-KIT was present in 48.117.0% cells on average: in all 3 cases – AML M0, in2 cases of AML M1, in 6 cases – AML M2, 20 of 22 cases – AML M4 and in 8 of 11 AML M5 cases. Average levels of CD117 in investigated leukemia cases statistically differed significantly (p=0.0067. Among 39 CD117- positive patients in 25 (53% co-expression of CD117+/CD34+ was revealed. Expression of CD117+/CD34- was observed in 14 cases (30%, CD117-/CD34+ – in 4 cases (8,5%, CD117-/CD34- – in 4 cases (8.5%. CD34 had of 64% of cells of myeloid origin. A high positive cor¬relation between expression of CD117 and CD34 (r=+0,5169 was determined, being statistically significant (p0,0067.

  1. Endolysosomal‐escape nanovaccines through adjuvant‐induced tumor antigen assembly for enhanced effector CD8+ T cell activation

    Czech Academy of Sciences Publication Activity Database

    Qiu, L.; Valente, M.; Dolen, Y.; Jäger, Eliezer; ter Beest, M.; Zheng, L.; Figdor, C. G.; Verdoes, M.

    2018-01-01

    Roč. 14, č. 15 (2018), s. 1-11, č. článku 1703539. ISSN 1613-6810 Grant - others:AV ČR(CZ) MSM200501602 Program:Program na podporu mezinárodní spolupráce začínajících výzkumných pracovníků Institutional support: RVO:61389013 Keywords : antigen/adjuvant codelivery * cancer nanovaccines * cross-presentation Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 8.643, year: 2016

  2. GM-CSF/IL-3/IL-5 receptor common β chain (CD131 expression as a biomarker of antigen-stimulated CD8+ T cells

    Directory of Open Access Journals (Sweden)

    Maric Dragan

    2008-04-01

    Full Text Available Abstract Background Upon Ag-activation cytotoxic T cells (CTLs produce IFN-γ GM-CSF and TNF-α, which deliver simultaneously pro-apoptotic and pro-inflammatory signals to the surrounding microenvironment. Whether this secretion affects in an autocrine loop the CTLs themselves is unknown. Methods Here, we compared the transcriptional profile of Ag-activated, Flu-specific CTL stimulated with the FLU M1:58-66 peptide to that of convivial CTLs expanded in vitro in the same culture. PBMCs from 6 HLA-A*0201 expressing donors were expanded for 7 days in culture following Flu M1:58-66 stimulation in the presence of 300 IU/ml of interleukin-2 and than sorted by high speed sorting to high purity CD8+ expressing T cells gated according to FluM1:58-66 tetrameric human leukocyte antigen complexes expression. Results Ag-activated CTLs displayed higher levels of IFN-γ, GM-CSF (CSF2 and GM-CSF/IL-3/IL-5 receptor common β- chain (CD131 but lacked completely expression of IFN-γ receptor-II and IFN-stimulated genes (ISGs. This observation suggested that Ag-activated CTLs in preparation for the release of IFN-γ and GM-CSF shield themselves from the potentially apoptotic effects of the former entrusting their survival to GM-SCF. In vitro phenotyping confirmed the selective surface expression of CD131 by Ag-activated CTLs and their increased proliferation upon exogenous administration of GM-CSF. Conclusion The selective responsiveness of Ag-activated CTLs to GM-CSF may provide an alternative explanation to the usefulness of this chemokine as an adjuvant for T cell aimed vaccines. Moreover, the selective expression of CD131 by Ag-activated CTLs proposes CD131 as a novel biomarker of Ag-dependent CTL activation.

  3. 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.

  4. Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

    Science.gov (United States)

    Heijnen, Ingmar A F M; Barnett, David; Arroz, Maria J; Barry, Simon M; Bonneville, Marc; Brando, Bruno; D'hautcourt, Jean-Luc; Kern, Florian; Tötterman, Thomas H; Marijt, Erik W A; Bossy, David; Preijers, Frank W M B; Rothe, Gregor; Gratama, Jan W

    2004-11-01

    HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells. (c) 2004 Wiley-Liss, Inc.

  5. 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.

  6. Metabolic Adaptation of Human CD4+ and CD8+ T-Cells to T-Cell Receptor-Mediated Stimulation

    Directory of Open Access Journals (Sweden)

    Nicholas Jones

    2017-11-01

    Full Text Available Linking immunometabolic adaptation to T-cell function provides insight for the development of new therapeutic approaches in multiple disease settings. T-cell activation and downstream effector functions of CD4+ and CD8+ T-cells are controlled by the strength of interaction between the T-cell receptor (TCR and peptides presented by human leukocyte antigens (pHLA. The role of TCR–pHLA interactions in modulating T-cell metabolism is unknown. Here, for the first time, we explore the relative contributions of the main metabolic pathways to functional responses in human CD4+ and CD8+ T-cells. Increased expression of hexokinase II accompanied by higher basal glycolysis is demonstrated in CD4+ T-cells; cytokine production in CD8+ T-cells is more reliant on oxidative phosphorylation. Using antigen-specific CD4+ and CD8+ T-cell clones and altered peptide ligands, we demonstrate that binding affinity tunes the underlying metabolic shift. Overall, this study provides important new insight into how metabolic pathways are controlled during antigen-specific activation of human T-cells.

  7. CD4 lymphocyte counts and serum p24 antigen of no diagnostic value in monitoring HIV-infected patients with pulmonary symptoms

    DEFF Research Database (Denmark)

    Orholm, M; Nielsen, T L; Nielsen, Jens Ole

    1990-01-01

    The diagnostic value of the CD4 cell counts and the HIV p24 antigen were evaluated in a consecutive series of 105 HIV-infected patients experiencing 128 episodes of pulmonary symptoms which required bronchoscopy. One-third of patients with opportunistic infection (OI) had CD4 counts greater than ....... In conclusion, the CD4 cell counts and the presence of p24 antigen in serum had a very limited predictive value for the presence of OI in HIV-infected patients with pulmonary symptoms.......The diagnostic value of the CD4 cell counts and the HIV p24 antigen were evaluated in a consecutive series of 105 HIV-infected patients experiencing 128 episodes of pulmonary symptoms which required bronchoscopy. One-third of patients with opportunistic infection (OI) had CD4 counts greater than 0.......200 x 10(9)/l, and 60% of patients without OI had CD4 counts less than 0.200 x 10(9)/l; 47 and 42% of patients with and without OI, respectively, had detectable p24 antigen in serum. Only 36% of the patients with OI presented the combination of CD4 cells less than 0.200 x 10(9)/l and p24 in serum...

  8. Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice.

    Science.gov (United States)

    Liu, Yawei; Teige, Anna; Mondoc, Emma; Ibrahim, Saleh; Holmdahl, Rikard; Issazadeh-Navikas, Shohreh

    2011-01-01

    NKT cells in the mouse recognize antigen in the context of the MHC class I-like molecule CD1d and play an important role in peripheral tolerance and protection against autoimmune and other diseases. NKT cells are usually activated by CD1d-presented lipid antigens. However, peptide recognition in the context of CD1 has also been documented, although no self-peptide ligands have been reported to date. Here, we have identified an endogenous peptide that is presented by CD1d to activate mouse NKT cells. This peptide, the immunodominant epitope from mouse collagen type II (mCII707-721), was not associated with either MHC class I or II. Activation of CD1d-restricted mCII707-721-specific NKT cells was induced via TCR signaling and classical costimulation. In addition, mCII707-721-specific NKT cells induced T cell death through Fas/FasL, in an IL-17A-independent fashion. Moreover, mCII707-721-specific NKT cells suppressed a range of in vivo inflammatory conditions, including delayed-type hypersensitivity, antigen-induced airway inflammation, collagen-induced arthritis, and EAE, which were all ameliorated by mCII707-721 vaccination. The findings presented here offer new insight into the intrinsic roles of NKT cells in health and disease. Given the results, endogenous collagen peptide activators of NKT cells may offer promise as novel therapeutics in tissue-specific autoimmune and inflammatory diseases.

  9. EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

    Science.gov (United States)

    Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

    2017-09-01

    Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as

  10. HIV-1 transgenic rat CD4+ T cells develop decreased CD28 responsiveness and suboptimal Lck tyrosine dephosphorylation following activation

    International Nuclear Information System (INIS)

    Yadav, Anjana; Pati, Shibani; Nyugen, Anhthu; Barabitskaja, Oxana; Mondal, Prosanta; Anderson, Michael; Gallo, Robert C.; Huso, David L.; Reid, William

    2006-01-01

    Impaired CD4+ T cell responses, resulting in dysregulated T-helper 1 (Th1) effector and memory responses, are a common result of HIV-1 infection. These defects are often preceded by decreased expression and function of the α/β T cell receptor (TCR)-CD3 complex and of co-stimulatory molecules including CD28, resulting in altered T cell proliferation, cytokine secretion and cell survival. We have previously shown that HIV Tg rats have defective development of T cell effector function and generation of specific effector/memory T cell subsets. Here we identify abnormalities in activated HIV-1 Tg rat CD4+ T cells that include decreased pY505 dephosphorylation of Lck (required for Lck activation), decreased CD28 function, reduced expression of the anti-apoptotic molecule Bcl-xL, decreased secretion of the mitogenic lympokine interleukin-2 (IL-2) and increased activation induced apoptosis. These events likely lead to defects in antigen-specific signaling and may help explain the disruption of Th1 responses and the generation of specific effector/memory subsets in transgenic CD4+ T cells

  11. 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.

  12. Blood group antigen studies using CdTe quantum dots and flow cytometry

    Directory of Open Access Journals (Sweden)

    Cabral Filho PE

    2015-07-01

    Full Text Available Paulo E Cabral Filho,1 Maria IA Pereira,1 Heloise P Fernandes,2 Andre A de Thomaz,3 Carlos L Cesar,3 Beate S Santos,4 Maria L Barjas-Castro,2 Adriana Fontes1 1Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Pernambuco, 2Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, 3Departamento de Eletrônica Quântica, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, Campinas, São Paulo, 4Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Abstract: New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs] as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion

  13. 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...

  14. Immunotherapy of non-Hodgkin lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells

    Science.gov (United States)

    Turtle, Cameron J.; Hanafi, Laïla-Aïcha; Berger, Carolina; Hudecek, Michael; Pender, Barbara; Robinson, Emily; Hawkins, Reed; Chaney, Colette; Cherian, Sindhu; Chen, Xueyan; Soma, Lorinda; Wood, Brent; Li, Daniel; Heimfeld, Shelly; Riddell, Stanley R.; Maloney, David G.

    2016-01-01

    CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that impact toxicity and efficacy have been difficult to define because of differences in lymphodepletion regimens and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4+:CD8+ ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had markedly increased CAR-T cell expansion and persistence, and higher response rates (50% CR, 72% ORR, n=20) than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR, n=12). The complete response (CR) rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR, n=11). Cy/Flu minimized the effects of an immune response to the murine scFv component of the CAR, which limited CAR-T cell expansion, persistence, and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥ 3 neurotoxicity were observed in 13% and 28% of all patients, respectively. Serum biomarkers one day after CAR-T cell infusion correlated with subsequent development of sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4+:CD8+ ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of a lymphodepletion regimen that improved disease response and overall and progression-free survival. PMID:27605551

  15. 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.

  16. CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens

    OpenAIRE

    Sharma, Sharad K.; Roumanes, David; Almudevar, Anthony; Mosmann, Tim R.; Pichichero, Michael E.

    2013-01-01

    We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing...

  17. Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

    Science.gov (United States)

    Greten, T F; Slansky, J E; Kubota, R; Soldan, S S; Jaffee, E M; Leist, T P; Pardoll, D M; Jacobson, S; Schneck, J P

    1998-06-23

    Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

  18. CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology

    Czech Academy of Sciences Publication Activity Database

    Engel, P.; Boumsell, L.; Balderas, R.; Gattei, V.; Hořejší, Václav; Jin, B.Q.; Malavasi, F.; Mortari, F.; Schwartz-Albiez, R.; Stockinger, H.; van Zelm, M.C.; Zola, H.; Clark, G.

    2015-01-01

    Roč. 165, č. 10 (2015), s. 4555-4563 ISSN 0022-1767 Institutional support: RVO:68378050 Keywords : CD nomenclature, , * leukocyte antigens * HLDA workshop Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.985, year: 2015

  19. 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

  20. 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.

  1. Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor.

    Directory of Open Access Journals (Sweden)

    Rachel S Leibman

    2017-10-01

    Full Text Available HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.

  2. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    OpenAIRE

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-...

  3. The diabetes type 1 locus Idd6 modulates activity of CD4+CD25+ regulatory T-cells.

    Science.gov (United States)

    Rogner, Ute Christine; Lepault, Françoise; Gagnerault, Marie-Claude; Vallois, David; Morin, Joëlle; Avner, Philip; Boitard, Christian

    2006-01-01

    The genetic locus Idd6 confers susceptibility to the spontaneous development of type 1 diabetes in the NOD mouse. Our studies on disease resistance of the congenic mouse strain NOD.C3H 6.VIII showed that Idd6 influences T-cell activities in the peripheral immune system and suggest that a major mechanism by which the Idd6 locus modifies diabetes development is via modulation of regulatory T-cell activities. Our transfer experiments using total splenocytes and purified T-cells demonstrated that the locus specifically controls the efficiency of disease protection mediated by the regulatory CD4(+)CD25(+) T-cell subset. Our data also implicate the Idd6 locus in controlling the balance between infiltrating lymphocytes and antigen-presenting cells within the pancreatic islet.

  4. The CD8+ memory T-cell state of readiness is actively maintained and reversible

    Science.gov (United States)

    Allam, Atef; Conze, Dietrich B.; Giardino Torchia, Maria Letizia; Munitic, Ivana; Yagita, Hideo; Sowell, Ryan T.; Marzo, Amanda L.

    2009-01-01

    The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G1 to the G0 cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-γ production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase–dependent manner. Consistent with these results, maintenance of G1 in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance. PMID:19617575

  5. 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.

  6. T 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.

  7. STUDIES IN DYNAMICS OF APOPTOSIS-RELATED SURFACE ANTIGEN (CD95 EXPRESSION ON NEUTROPHILS FROM CERVICAL AND VAGINAL SECRETIONS IN WOMEN WITH CHLAMIDIA INFECTION

    Directory of Open Access Journals (Sweden)

    O. A. Giesinger

    2010-01-01

    Full Text Available CD95 (Fas/APO-1 antigen expression was studied on the surface of neutrophil granulocytes from cervical secretions. Sixty-five female patients with established Chlamydia infection were found to have an increased CD95+ antigen expression following basic therapy. CD95+ receptors on neutrophils in the patients with Chlamydia infection have been shown to return to normal levels following a combined magnetic laser treatment.

  8. A novel intracellular pool of LFA-1 is critical for asymmetric CD8+ T cell activation and differentiation.

    Science.gov (United States)

    Capece, Tara; Walling, Brandon L; Lim, Kihong; Kim, Kyun-Do; Bae, Seyeon; Chung, Hung-Li; Topham, David J; Kim, Minsoo

    2017-11-06

    The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18) is a key T cell adhesion receptor that mediates stable interactions with antigen-presenting cell (APC), as well as chemokine-mediated migration. Using our newly generated CD11a-mYFP knock-in mice, we discovered that naive CD8 + T cells reserve a significant intracellular pool of LFA-1 in the uropod during migration. Intracellular LFA-1 quickly translocated to the cell surface with antigenic stimulus. Importantly, the redistribution of intracellular LFA-1 at the contact with APC was maintained during cell division and led to an unequal inheritance of LFA-1 in divided T cells. The daughter CD8 + T cells with disparate LFA-1 expression showed different patterns of migration on ICAM-1, APC interactions, and tissue retention, as well as altered effector functions. In addition, we identified Rab27 as an important regulator of the intracellular LFA-1 translocation. Collectively, our data demonstrate that an intracellular pool of LFA-1 in naive CD8 + T cells plays a key role in T cell activation and differentiation. © 2017 Capece et al.

  9. 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

  10. Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

    Science.gov (United States)

    Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

    2000-03-01

    MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

  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

    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

  12. Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.

    Directory of Open Access Journals (Sweden)

    Monique van Velzen

    2013-08-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 infection results in lifelong chronic infection of trigeminal ganglion (TG neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

  13. Mycobacterium tuberculosis Latent Antigen Rv2029c from the Multistage DNA Vaccine A39 Drives TH1 Responses via TLR-mediated Macrophage Activation

    Directory of Open Access Journals (Sweden)

    Haibo Su

    2017-11-01

    Full Text Available Targeting of Mycobacterium tuberculosis (MTB latent antigens comprises a crucial strategy for the development of alternative tuberculosis (TB vaccine(s that protects against TB reactivation. Here, we generated a multistage DNA vaccine, A39, containing the early antigens Ag85A and Rv3425 as well as the latency-associated protein Rv2029c, which conferred protective immunity in a pre-exposure mouse model. Moreover, administration of the A39 vaccination after MTB exposure inhibited reactivation and resulted in significantly lower bacterial loads in the lungs and spleen of mice, compared to those in the control population. Subsequently, we investigated the effect of Rv2029c on innate immunity and characterized the molecular details of the interaction of this protein with the host via iTRAQ proteomic and biochemical assay analyses. Rv2029c activated macrophages, triggered the production of pro-inflammatory cytokines, and promoted toll-like receptor/mitogen-activated protein kinase (TLR/MAPK-dependent macrophage apoptosis. Furthermore, Rv2029c treatment enhanced the ability of Mycobacterium bovis Bacillus Calmette-Guérin (BCG-infected macrophages to present antigens to CD4+ T cells in vitro, which correlated with an increase in MHC-II expression. Lastly, Rv2029c-treated macrophages activated T cells, effectively polarized CD4+ and CD8+ T cells to secrete IFN-γ and IL-2, and specifically expanded a population of CD44highCD62LlowCD4+/CD8+ effector/memory cells, indicating that Rv2029c, as a specific recall antigen, contributes to Th1 polarization in T cell immunity. These results suggest that Rv2029c and A39 comprise promising targets for the development of next-generation clinical TB therapeutic vaccines.

  14. 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

  15. CD4 lymphocyte counts and serum p24 antigen of no diagnostic value in monitoring HIV-infected patients with pulmonary symptoms

    DEFF Research Database (Denmark)

    Orholm, M; Nielsen, T L; Nielsen, Jens Ole

    1990-01-01

    The diagnostic value of the CD4 cell counts and the HIV p24 antigen were evaluated in a consecutive series of 105 HIV-infected patients experiencing 128 episodes of pulmonary symptoms which required bronchoscopy. One-third of patients with opportunistic infection (OI) had CD4 counts greater than 0....... In conclusion, the CD4 cell counts and the presence of p24 antigen in serum had a very limited predictive value for the presence of OI in HIV-infected patients with pulmonary symptoms....

  16. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

    Science.gov (United States)

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

  17. An inducible transgenic mouse model for immune mediated hepatitis showing clearance of antigen expressing hepatocytes by CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Marcin Cebula

    Full Text Available The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER(T2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K(b/OVA257-264-specific OT-I T cells to OVA_X_CreER(T2 mice or generated triple transgenic OVA_X CreER(T2_X_OT-I mice. OT-I T cells become activated in OVA_X_CreER(T2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER(T2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER(T2_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.

  18. 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...

  19. The CD8 and CD4 T-cell response against Kaposi's sarcoma-associated herpesvirus is skewed towards early and late lytic antigens.

    Directory of Open Access Journals (Sweden)

    Rebecca C Robey

    Full Text Available Kaposi's sarcoma-associated herpesvirus (KSHV is causally related to Kaposi's sarcoma (KS, the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs. We used these to transduce monocyte-derived dendritic cells (moDCs isolated from 14 KSHV-seropositive (12 HIV-positive and 7 KSHV-seronegative (4 HIV-positive individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle. Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1] were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37] was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1] was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.

  20. 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.

  1. Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

    Directory of Open Access Journals (Sweden)

    Shibaguchi H

    2017-08-01

    Full Text Available Hirotomo Shibaguchi,1,* Naixiang Luo,1,* Naoto Shirasu,1,* Motomu Kuroki,2 Masahide Kuroki1 1Department of Biochemistry, Faculty of Medicine, Fukuoka University, Fukuoka, Japan; 2School of Nursing, Faculty of Medicine, Fukuoka University, Fukuoka, Japan *These authors equally contributed to this work Abstract: Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy. Keywords: chimeric antigen receptor, fusion protein, human scFv, CEA, combination therapy

  2. Peptide and nucleotide sequences of rat CD4 (W3/25) antigen: evidence for derivation from a structure with four immunoglobulin-related domains

    International Nuclear Information System (INIS)

    Clark, S.J.; Jefferies, W.A.; Barclay, A.N.; Gagnon, J.; Williams, A.F.

    1987-01-01

    The rat W3/25 antigen was the first marker antigen of helper T lymphocytes to be identified. Subsequently, the human OKT4 antigen (now called CD4) was described, and cell distribution and functional data suggested that W3/25 and OKT4 antigens were homologous. This is now confirmed by the matching of peptide sequences from W3/25 antigen with sequence predicted from rat cDNA clones detected by cross-hybridization with a cDNA probe for human CD4. Analysis of the two sequences suggests an evolutionary origin from a structure with four immunoglobulin-related domains, although only domain 1 at the NH 2 terminus meets the standard criteria for an immunoglobulin-related sequence. CD4 domains 2 and 4 contain disulfide bonds but seem like truncated immunoglobulin domains, whereas domain 3 may have a pattern of β-strands like an immunoglobulin variable domain, but without the disulfide bond

  3. Chimeric Antigen Receptors to CD276 for Treating Cancer | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    This licensing opportunity from the National Cancer Institute concerns the development of CARs comprising an antigen-binding fragment derived from the MGA271 antibody. The resulting CARs can be used in adoptive cell therapy treatment for neuroblastoma and other tumors that express CD276.

  4. Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

    Science.gov (United States)

    Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

    2012-10-15

    Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. 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....

  6. Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

    Science.gov (United States)

    Lambert Emo, Kris; Hyun, Young-Min; Reilly, Emma; Barilla, Christopher; Gerber, Scott; Fowell, Deborah; Kim, Minsoo; Topham, David J

    2016-09-01

    During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

  7. Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

    Directory of Open Access Journals (Sweden)

    Kris Lambert Emo

    2016-09-01

    Full Text Available During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

  8. CD107a Expression and IFN-γ Production as Markers for Evaluation of Cytotoxic CD3+ CD8+ T Cell Response to CMV Antigen in Women with Recurrent Spontaneous Abortion.

    Science.gov (United States)

    Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz

    2014-01-01

    Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.

  9. CD4/CD8/Dendritic cell complexes in the spleen: CD8+ T cells can directly bind CD4+ T cells and modulate their response

    Science.gov (United States)

    Barinov, Aleksandr; Galgano, Alessia; Krenn, Gerald; Tanchot, Corinne; Vasseur, Florence

    2017-01-01

    CD4+ T cell help to CD8+ T cell responses requires that CD4+ and CD8+ T cells interact with the same antigen presenting dendritic cell (Ag+DC), but it remains controversial whether helper signals are delivered indirectly through a licensed DC and/or involve direct CD4+/CD8+ T cell contacts and/or the formation of ternary complexes. We here describe the first in vivo imaging of the intact spleen, aiming to evaluate the first interactions between antigen-specific CD4+, CD8+ T cells and Ag+DCs. We show that in contrast to CD4+ T cells which form transient contacts with Ag+DC, CD8+ T cells form immediate stable contacts and activate the Ag+DC, acquire fragments of the DC membranes by trogocytosis, leading to their acquisition of some of the DC properties. They express MHC class II, and become able to present the specific Marilyn peptide to naïve Marilyn CD4+ T cells, inducing their extensive division. In vivo, these CD8+ T cells form direct stable contacts with motile naïve CD4+ T cells, recruiting them to Ag+DC binding and to the formation of ternary complexes, where CD4+ and CD8+ T cells interact with the DC and with one another. The presence of CD8+ T cells during in vivo immune responses leads to the early activation and up-regulation of multiple functions by CD4+ T lymphocytes. Thus, while CD4+ T cell help is important to CD8+ T cell responses, CD8+ T cells can interact directly with naïve CD4+ T cells impacting their recruitment and differentiation. PMID:28686740

  10. 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.

  11. Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

    OpenAIRE

    Desbarats, J; Freed, J H; Campbell, P A; Newell, M K

    1996-01-01

    The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investig...

  12. Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis

    DEFF Research Database (Denmark)

    Thakur, Aneesh; Riber, Ulla; Davis, William C.

    2013-01-01

    -γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag......T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN...

  13. Flow-cytometric measurement of CD4-8- T cells bearing T-cell receptor αβ chains, 1

    International Nuclear Information System (INIS)

    Kusunoki, Yoichiro; Hirai, Yuko; Kyoizumi, Seishi; Akiyama, Mitoshi.

    1992-09-01

    In this study we detected rare, possibly abnormal, T cells bearing CD3 surface antigen and T-cell receptor (TCR) αβ chains but lacking both CD4 and CD8 antigens (viz., TCRαβ + CD4 - 8 - cells, as determined by flow cytometry). The TCRαβ + CD4 - 8 - T cells were detected at a mean frequency of 0.63 ± 0.35 % (mean ± standard deviation) in peripheral blood TCRαβ + cells of 119 normal persons. Two unusual cases besides the 119 normal persons showed extremely elevated frequencies of TCRαβ + CD4 - 8 - T cells, viz., approximately 5 % to 10 % and 14 % to 19 % in whole TCRαβ + cells. Both individuals were males who were otherwise physiologically quite normal with no history of severe illness, and these high frequencies were also observed in blood samples collected 2 or 8 years prior to the current measurements. The TCRαβ + CD4 - 8 - T cells of the two individuals were found to express mature T-cell markers such as CD2,3, and 5 antigens, as well as natural killer (NK) cell markers, viz., CD11b, 16, 56, and 57 antigens, when peripheral blood lymphocytes were subjected to three-color flow cytometry. Lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities were observed for both freshly sorted TCRαβ + CD4 - 8 - cells and in vitro established clones. Nevertheless, NK-like activity was not detected. Further, Southern blot analysis of TCRβ and γ genes revealed identical rearrangement patterns for all the TCRαβ + CD4 - 8 - clones established in vitro. These results suggest that the TCRαβ + CD4 - 8 - T cells from these two mean exhibit unique characteristics and proliferate clonally in vivo. (author)

  14. Immune Checkpoint Function of CD85j in CD8 T Cell Differentiation and Aging

    Directory of Open Access Journals (Sweden)

    Claire E. Gustafson

    2017-06-01

    Full Text Available Aging is associated with an increased susceptibility to infection and a failure to control latent viruses thought to be driven, at least in part, by alterations in CD8 T cell function. The aging T cell repertoire is characterized by an accumulation of effector CD8 T cells, many of which express the negative regulatory receptor CD85j. To define the biological significance of CD85j expression on CD8 T cells and to address the question whether presence of CD85j in older individuals is beneficial or detrimental for immune function, we examined the specific attributes of CD8 T cells expressing CD85j as well as the functional role of CD85j in antigen-specific CD8 T cell responses during immune aging. Here, we show that CD85j is mainly expressed by terminally differentiated effector (TEMRAs CD8 T cells, which increase with age, in cytomegalovirus (CMV infection and in males. CD85j+ CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j− compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57 but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 T cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of “senescent,” but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging.

  15. Rapid and sustained CD4(+) T-cell-independent immunity from adenovirus-encoded vaccine antigens

    DEFF Research Database (Denmark)

    Holst, Peter J; Bartholdy, Christina; Buus, Anette Stryhn

    2007-01-01

    -linked lymphocytic choriomeningitis virus (LCMV)-derived epitopes was long-lived and protective. Notably, in contrast to full-length protein, the response elicited with the beta(2)-microglobulin-linked LCMV-derived epitope was CD4(+) T-cell independent. Furthermore, virus-specific CD8(+) T cells primed...... in the absence of CD4(+) T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4(+) T-cell...... to that elicited with an adenovirus-encoded minimal epitope covalently linked to beta(2)-microglobulin. We demonstrate that the beta(2)-microglobulin-linked epitope induced an accelerated and augmented CD8(+) T-cell response. Furthermore, the immunity conferred by vaccination with beta(2)-microglobulin...

  16. 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.

  17. Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

    International Nuclear Information System (INIS)

    Hu Mingqian; Wang Jiongkun; Cai Jiye; Wu Yangzhe; Wang Xiaoping

    2008-01-01

    To date, nanoscale imaging of the morphological changes and adhesion force of CD4 + T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4 + T cells. The AFM images revealed that the volume of activated CD4 + T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4 + T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity

  18. Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

    Science.gov (United States)

    Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

    2008-09-12

    To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

  19. Interferon-alpha administration enhances CD8+ T cell activation in HIV infection.

    Directory of Open Access Journals (Sweden)

    Maura Manion

    Full Text Available Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.To investigate the effects of type I interferons in HIV-infection, we obtained cryopreserved peripheral blood mononuclear cell samples from 10 subjects who participated in AIDS Clinical Trials Group Study 5192, a trial investigating the activity of systemic administration of IFNα for twelve weeks to patients with untreated HIV infection. Using flow cytometry, we examined changes in cell cycle status and expression of activation antigens by circulating T cells and their maturation subsets before, during and after IFNα treatment.The proportion of CD38+HLA-DR+CD8+ T cells increased from a mean of 11.7% at baseline to 24.1% after twelve weeks of interferon treatment (p = 0.006. These frequencies dropped to an average of 20.1% six weeks after the end of treatment. In contrast to CD8+ T cells, the frequencies of activated CD4+ T cells did not change with administration of type I interferon (mean percentage of CD38+DR+ cells = 2.62% at baseline and 2.17% after 12 weeks of interferon therapy. As plasma HIV levels fell with interferon therapy, this was correlated with a "paradoxical" increase in CD8+ T cell activation (p<0.001.Administration of type I interferon increased expression of the activation markers CD38 and HLA DR on CD8+ T cells but not on CD4+ T cells of HIV+ persons. These observations suggest that type I interferons may contribute to the high levels of CD8+ T cell activation that occur during HIV infection.

  20. The early activation marker CD69 regulates the expression of chemokines and CD4 T cell accumulation in intestine.

    Directory of Open Access Journals (Sweden)

    Katarina Radulovic

    Full Text Available Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+ T cells and/or CD4(- cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/- CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/- CD4 T cell accumulation in colonic lamina propria (cLP was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/- mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/- CD45RB(high CD4 T cells into RAG(-/- hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

  1. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Directory of Open Access Journals (Sweden)

    Shweta Jain

    Full Text Available Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID, signifying class switch recombination (CSR. Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  2. CD40 signaling synergizes with TLR-2 in the BCR independent activation of resting B cells.

    Science.gov (United States)

    Jain, Shweta; Chodisetti, Sathi Babu; Agrewala, Javed N

    2011-01-01

    Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40--molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

  3. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

    Science.gov (United States)

    Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

    2017-11-09

    CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

  4. 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

  5. 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

  6. 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

  7. Risk of classical Kaposi sarcoma by plasma levels of Epstein-Barr virus antibodies, sCD26, sCD23 and sCD30

    Directory of Open Access Journals (Sweden)

    Viviano Enza

    2010-10-01

    Full Text Available Abstract Background To clarify the immunological alterations leading to classical Kaposi sarcoma (cKS among people infected with KS-associated herpesvirus (KSHV. Methods In a population-based study of 119 cKS cases, 105 KSHV-seropositive controls, and 155 KSHV-seronegative controls, we quantified plasma soluble cluster of differentiation (sCD levels and antibodies against Epstein-Barr virus nuclear antigen-1 (anti-EBNA-1 and viral capsid antigen (anti-VCA. Differences between groups in prevalence of low-tertile anti-EBNA-1 and high-tertile anti-VCA were compared by logistic regression. Continuous levels between groups and by presence of cKS co-factors among controls were compared by linear regression and Mann-Whitney-Wilcoxon methods. Results Comparisons of cKS cases to seropositive controls and of seropositive to seronegative controls revealed no significant differences. However, controls with known cKS cofactors (male sex, nonsmoking, diabetes and cortisone use had significantly lower levels of anti-EBNA (P = 0.0001 - 0.07 and anti-VCA (P = 0.0001 - 0.03. Levels of sCD26 were significantly lower for male and non-smoking controls (Padj ≤ 0.03, and they were marginally lower with older age and cortisone use (Padj ≤ 0.09. Conclusions Anti-EBV and sCD26 levels were associated with cofactors for cKS, but they did not differ between cKS cases and matched controls. Novel approaches and broader panels of assays are needed to investigate immunological contributions to cKS.

  8. Activated STAT5 promotes long-lived cytotoxic CD8+ T cells that induce regression of autochthonous melanoma.

    Science.gov (United States)

    Grange, Magali; Buferne, Michel; Verdeil, Grégory; Leserman, Lee; Schmitt-Verhulst, Anne-Marie; Auphan-Anezin, Nathalie

    2012-01-01

    Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs. ©2011 AACR.

  9. 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

  10. 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

  11. Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.

    Science.gov (United States)

    Wang, Dongrui; Aguilar, Brenda; Starr, Renate; Alizadeh, Darya; Brito, Alfonso; Sarkissian, Aniee; Ostberg, Julie R; Forman, Stephen J; Brown, Christine E

    2018-05-17

    Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

  12. CdTe QDs-based prostate-specific antigen probe for human prostate cancer cell imaging

    International Nuclear Information System (INIS)

    Dong Wei; Guo Li; Wang Meng; Xu Shukun

    2009-01-01

    L-glutathione (GSH) stabilized CdTe quantum dots (QDs) were directly prepared in aqueous solution. The as-prepared QDs were linked to prostate-specific antigen (PSA) for the direct labeling and linked to immunoglobulin G (IgG) for the indirect labeling of fixed prostate cancer cells. The results indicated that QD-based probes were ideal fluorescent markers with excellent spectral properties and photostability and much better than organic dyes making them very suitable in target detection. Meanwhile, the indirect labeling showed much better specificity than the direct labeling. Furthermore, the prepared CdTe QDs did not show detectable effect on cell growth after having cultured for three days, which suggested that the L-glutathione capped CdTe had scarcely cytotoxicity.

  13. Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high pretransplant soluble CD30.

    Science.gov (United States)

    Süsal, Caner; Pelzl, Steffen; Opelz, Gerhard

    2003-10-27

    The influence of human leukocyte antigen (HLA) matching on graft survival is greater in patients with preformed lymphocytotoxic antibodies than in nonsensitized patients. Pretransplant serum soluble CD30 (sCD30) affects graft outcome independently of presensitization status. The impact of HLA compatibility on kidney transplant survival was analyzed in 3980 nonsensitized first cadaveric kidney recipients in relation to the pretransplant serum sCD30 content. Although HLA compatibility influenced graft outcome only marginally in nonsensitized recipients with low sCD30 (at 3 years: P=0.0095; at 5 years: P=0.1033), a strong HLA matching effect was observed in nonsensitized recipients with high sCD30 (at 3 years: PsCD30 benefit from an HLA well-matched kidney. Patients should be tested for sCD30 while on the waiting list for a kidney transplant, and HLA well-matched kidneys should be allocated to patients with high sCD30.

  14. Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses.

    Science.gov (United States)

    Webb, Tonya J; Carey, Gregory B; East, James E; Sun, Wenji; Bollino, Dominique R; Kimball, Amy S; Brutkiewicz, Randy R

    2016-08-01

    Natural killer T (NKT) cells play a critical role in the host's innate immune response. CD1d-mediated presentation of glycolipid antigens to NKT cells has been established; however, the mechanisms by which NKT cells recognize infected or cancerous cells remain unclear. 5(')-AMP activated protein kinase (AMPK) is a master regulator of lipogenic pathways. We hypothesized that activation of AMPK during infection and malignancy could alter the repertoire of antigens presented by CD1d and serve as a danger signal to NKT cells. In this study, we examined the effect of alterations in metabolism on CD1d-mediated antigen presentation to NKT cells and found that an infection with lymphocytic choriomeningitis virus rapidly increased CD1d-mediated antigen presentation. Hypoxia inducible factors (HIF) enhance T-cell effector functions during infection, therefore antigen presenting cells pretreated with pharmacological agents that inhibit glycolysis, induce HIF and activate AMPK were assessed for their ability to induce NKT-cell responses. Pretreatment with 2-deoxyglucose, cobalt chloride, AICAR and metformin significantly enhanced CD1d-mediated NKT-cell activation. In addition, NKT cells preferentially respond to malignant B cells and B-cell lymphomas express HIF-1α. These data suggest that targeting cellular metabolism may serve as a novel means of inducing innate immune responses. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Antigen smuggling in tuberculosis.

    Science.gov (United States)

    Hudrisier, Denis; Neyrolles, Olivier

    2014-06-11

    The importance of CD4 T lymphocytes in immunity to M. tuberculosis is well established; however, how dendritic cells activate T cells in vivo remains obscure. In this issue of Cell Host & Microbe, Srivastava and Ernst (2014) report a mechanism of antigen transfer for efficient activation of antimycobacterial T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody-Activated Chimeric Antigen Receptor-Modified T Cells.

    Science.gov (United States)

    Schmueck-Henneresse, Michael; Omer, Bilal; Shum, Thomas; Tashiro, Haruko; Mamonkin, Maksim; Lapteva, Natalia; Sharma, Sandhya; Rollins, Lisa; Dotti, Gianpietro; Reinke, Petra; Volk, Hans-Dieter; Rooney, Cliona M

    2017-07-01

    The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO + CCR7 - effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28-activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell-derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro-expanded T cells. Copyright © 2017 by The American Association of Immunologists, Inc.

  17. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling

    NARCIS (Netherlands)

    Chu, D. H.; Spits, H.; Peyron, J. F.; Rowley, R. B.; Bolen, J. B.; Weiss, A.

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs.

  18. Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Xu-Monette, Zijun Y; Li, Ling; Byrd, John C

    2016-01-01

    CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its...

  19. Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

    Science.gov (United States)

    Venkataswamy, Manjunatha M; Ng, Tony W; Kharkwal, Shalu S; Carreño, Leandro J; Johnson, Alison J; Kunnath-Velayudhan, Shajo; Liu, Zheng; Bittman, Robert; Jervis, Peter J; Cox, Liam R; Besra, Gurdyal S; Wen, Xiangshu; Yuan, Weiming; Tsuji, Moriya; Li, Xiangming; Ho, David D; Chan, John; Lee, Sunhee; Frothingham, Richard; Haynes, Barton F; Panas, Michael W; Gillard, Geoffrey O; Sixsmith, Jaimie D; Korioth-Schmitz, Birgit; Schmitz, Joern E; Larsen, Michelle H; Jacobs, William R; Porcelli, Steven A

    2014-01-01

    Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

  20. Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

    Directory of Open Access Journals (Sweden)

    Manjunatha M Venkataswamy

    Full Text Available Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag. We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

  1. 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.

  2. Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure.

    Directory of Open Access Journals (Sweden)

    Sophie Roetynck

    Full Text Available Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

  3. 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.

  4. Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies.

    Science.gov (United States)

    Png, Yi Tian; Vinanica, Natasha; Kamiya, Takahiro; Shimasaki, Noriko; Coustan-Smith, Elaine; Campana, Dario

    2017-11-28

    Effective immunotherapies for T-cell malignancies are lacking. We devised a novel approach based on chimeric antigen receptor (CAR)-redirected T lymphocytes. We selected CD7 as a target because of its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL samples), median CD7 expression was >99%; CD7 expression remained high at relapse (n = 14), and during chemotherapy (n = 54). We targeted CD7 with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes caused fratricide due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control fratricide, we applied a new method (protein expression blocker [PEBL]), based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain. Transduction of anti-CD7 PEBL resulted in virtually instantaneous abrogation of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7 + vs 98.1% ± 1.5% of mock-transduced T cells (n = 5; P < .0001). PEBL expression did not impair T-cell proliferation, interferon-γ and tumor necrosis factor-α secretion, or cytotoxicity, and eliminated CAR-mediated fratricide. PEBL-CAR T cells were highly cytotoxic against CD7 + leukemic cells in vitro and were consistently more potent than CD7 + T cells spared by fratricide. They also showed strong anti-leukemic activity in cell line- and patient-derived T-ALL xenografts. The strategy described in this study fits well with existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.

  5. 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....

  6. 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

  7. Naive T lymphocytes traffic to inflamed central nervous system, but require antigen recognition for activation

    DEFF Research Database (Denmark)

    Krakowski, M L; Owens, T

    2000-01-01

    Organ-specific autoimmune diseases may be induced by infiltration of the target tissue by CD4(+) T cells with specificity for self antigen(s). As disease progresses, T cells of other specificities appear in the tissue. Traffic of naive, antigen-inexperienced T cells to target tissues has not been...

  8. Rh antigen expression during erythropoeisis: Comparison of cord and adult derived CD34 + cells

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    Gupta Namita

    2008-01-01

    Full Text Available Objectives: Concentrations of O 2 and CO 2 in the fetal circulation differ to that in maternal blood. Previous studies done in algae demonstrate the functional role of Rh antigen as CO 2 transporter. As a preliminary study, it was the aim of this project to compare the expression of Rh polypeptides on cord and adult red blood cell progenitors during ex vivo proliferation and differentiation of CD34 + cells during erythropoeisis. Materials and Methods: CD34 positive hematopoeitic progenitor cells were isolated from umbilical cord blood and adult peripheral blood using an immunomagnetic system and cultured in serum free medium containing erythropoietin in order to compel them along the erythroid lineage. Cultured cells were analyzed for cell surface marker expression by flow cytometry, using monoclonal antibodies to RhAG, Glycophorin A, Rh polypeptides, CD47 and Band 3. Cytospin analysis was also done to study the morphology of cultured cells. Results: The appearance of cell surface markers analyzed on different days of culture varied slightly between samples. There was no evidence to suggest that RhAG, GPA, CD47 and Band 3 expression was any different between adult and cord derived cells. Nevertheless, the results of Rh antigenic expression suggest a reasonable difference between the two groups with adult sample derived cells showing higher and earlier expression than cord blood derived cells. These preliminary findings require further investigation. Conclusion: Comparing the expression of cell surface markers especially Rh polypeptides between adult and cord blood derived erythroid progenitors might assist in discerning their functions and could be valuable in the study of erythropoeisis.

  9. Relationships between Th1 or Th2 iNKT cell activity and structures of CD1d-antigen complexes: meta-analysis of CD1d-glycolipids dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Xavier Laurent

    2014-11-01

    Full Text Available A number of potentially bioactive molecules can be found in nature. In particular, marine organisms are a valuable source of bioactive compounds. The activity of an α-galactosylceramide was first discovered in 1993 via screening of a Japanese marine sponge (Agelas mauritanius. Very rapidly, a synthetic glycololipid analogue of this natural molecule was discovered, called KRN7000. Associated with the CD1d protein, this α-galactosylceramide 1 (KRN7000 interacts with the T-cell antigen receptor to form a ternary complex that yields T helper (Th 1 and Th2 responses with opposing effects. In our work, we carried out molecular dynamics simulations (11.5 µs in total involving eight different ligands (conducted in triplicate in an effort to find out correlation at the molecular level, if any, between chemical modulation of 1 and the orientation of the known biological response, Th1 or Th2. Comparative investigations of human versus mouse and Th1 versus Th2 data have been carried out. A large set of analysis tools was employed including free energy landscapes. One major result is the identification of a specific conformational state of the sugar polar head, which could be correlated, in the present study, to the biological Th2 biased response. These theoretical tools provide a structural basis for predicting the very different dynamical behaviors of α-glycosphingolipids in CD1d and might aid in the future design of new analogues of 1.

  10. Identification of a Novel CD8 T Cell Epitope Derived from Plasmodium berghei Protective Liver-Stage Antigen

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    Alexander Pichugin

    2018-01-01

    Full Text Available We recently identified novel Plasmodium berghei (Pb liver stage (LS genes that as DNA vaccines significantly reduce Pb LS parasite burden (LPB in C57Bl/6 (B6 mice through a mechanism mediated, in part, by CD8 T cells. In this study, we sought to determine fine antigen (Ag specificities of CD8 T cells that target LS malaria parasites. Guided by algorithms for predicting MHC class I-restricted epitopes, we ranked sequences of 32 Pb LS Ags and selected ~400 peptides restricted by mouse H-2Kb and H-2Db alleles for analysis in the high-throughput method of caged MHC class I-tetramer technology. We identified a 9-mer H-2Kb restricted CD8 T cell epitope, Kb-17, which specifically recognized and activated CD8 T cell responses in B6 mice immunized with Pb radiation-attenuated sporozoites (RAS and challenged with infectious sporozoites (spz. The Kb-17 peptide is derived from the recently described novel protective Pb LS Ag, PBANKA_1031000 (MIF4G-like protein. Notably, immunization with the Kb-17 epitope delivered in the form of a minigene in the adenovirus serotype 5 vector reduced LPB in mice infected with spz. On the basis of our results, Kb-17 peptide was available for CD8 T cell activation and recall following immunization with Pb RAS and challenge with infectious spz. The identification of a novel MHC class I-restricted epitope from the protective Pb LS Ag, MIF4G-like protein, is crucial for advancing our understanding of immune responses to Plasmodium and by extension, toward vaccine development against malaria.

  11. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    International Nuclear Information System (INIS)

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2015-01-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  12. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Energy Technology Data Exchange (ETDEWEB)

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: t-takai@juntendo.ac.jp [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)

    2015-05-01

    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  13. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  14. 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...

  15. Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

    Directory of Open Access Journals (Sweden)

    Robert Moot

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs. VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

  16. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    Directory of Open Access Journals (Sweden)

    Radhika Thokala

    Full Text Available Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML. CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL, and has been an effective target for T cells expressing chimeric antigen receptors (CARs. The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb, coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  17. Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

    Science.gov (United States)

    Kielian, T. L.; Ross, C. R.; McVey, D. S.; Chapes, S. K.; Blecha, F.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

  18. Vaccination Expands Antigen-Specific CD4+ Memory T Cells and Mobilizes Bystander Central Memory T Cells

    Science.gov (United States)

    Li Causi, Eleonora; Parikh, Suraj C.; Chudley, Lindsey; Layfield, David M.; Ottensmeier, Christian H.; Stevenson, Freda K.; Di Genova, Gianfranco

    2015-01-01

    CD4+ T helper memory (Thmem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Thmem cells as expected, but was accompanied by parallel increase of Thmem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Thmem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Thmem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Thmem cells were activated (CD38High HLA-DR+), cycling or recently divided (Ki-67+), and apparently vulnerable to death (IL-7RαLow and Bcl-2 Low). In contrast, bystander Thmem cells were resting (CD38Low HLA-DR- Ki-67-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Thmem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines. PMID:26332995

  19. THE ANTIGEN-SPECIFIC CELL IN VITRO TESTS FOR POST-VACCINATION ANTIPLAGUE IMMUNITY FORMATION

    Directory of Open Access Journals (Sweden)

    A. N. Kulichenko

    2017-01-01

    Full Text Available The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+ and late (CD45+CD3+HLA-DR+ lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37% and at 3 (47.41% months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days and late (6 months periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.

  20. Shaping the CD4+ memory immune response against tuberculosis: the role of antigen persistence, location and multi-functionality.

    Science.gov (United States)

    Ancelet, Lindsay; Kirman, Joanna

    2012-02-01

    Abstract Effective vaccination against intracellular pathogens, such as tuberculosis (TB), relies on the generation and maintenance of CD4 memory T cells. An incomplete understanding of the memory immune response has hindered the rational design of a new, more effective TB vaccine. This review discusses how the persistence of antigen, the location of memory cells, and their multifunctional ability shape the CD4 memory T cell response against TB.

  1. Complexity of type-specific 56 kDa antigen CD4 T-cell epitopes of Orientia tsutsugamushi strains causing scrub typhus in India.

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    Arunachalam Ramaiah

    Full Text Available Orientia tsutsugamushi (Ots is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III and three spacer conserved domains (SVDI, SVDII/III, SVDIII. The total number (152 of antigenic types (amino acid variants varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435 of the predicted CD4 T-cell epitopes (TCEs from all the six domains had high binding affinities (HBA to at least one of the prevalent Indian human leukocyte antigen (HLA alleles. A surprisingly high proportion (61% of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.

  2. Cytotoxic human CD4(+) T cells

    NARCIS (Netherlands)

    van de Berg, Pablo J.; van Leeuwen, Ester M.; ten Berge, Ineke J.; van Lier, Rene

    2008-01-01

    The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate naïve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the

  3. Using reduced graphene oxide-Ca:CdSe nanocomposite to enhance photoelectrochemical activity of gold nanoparticles functionalized tungsten oxide for highly sensitive prostate specific antigen detection.

    Science.gov (United States)

    Wang, Xueping; Xu, Rui; Sun, Xu; Wang, Yaoguang; Ren, Xiang; Du, Bin; Wu, Dan; Wei, Qin

    2017-10-15

    An ultrasensitive sandwich-type photoelectrochemical (PEC) immunosensor was constructed for the detection of prostate specific antigen (PSA). In this work, Au-nanoparticle-loaded tungsten oxide (WO 3 -Au) hybrid composites was applied as PEC sensing platform, while Ca ions doped CdSe equipped on the conducting framework of reduced graphene oxide (rGO-Ca:CdSe) nanocomposites were employed as the signal amplification probe. As for WO 3 -Au, massive Au nanoparticles were formed on the surface of WO 3 without any additional reducing agent, providing a novel nanocarriers for anchoring plenty of the primary antibodies due to the large specific surface area and good biocompatibility by chemical bonding between Au nanoparticles and -NH 2 of antibodies. Besides, the incorporation of the rGO and the doping of Ca ions could improve the conductivity and hinder the recombination of electron-hole pairs of CdSe nanoparticles effectively, thereby enhancing the photocurrent conversion efficiency. Based on the sandwich immunoreaction, the primary antibody was immobilized onto WO 3 -Au substrate, after the formed rGO-Ca:CdSe labels were captured onto the electrode surface via the specific antibody-antigen interaction, the photocurrent intensity could be further enhanced due to the sensitization effect. Under the optimal conditions, the proposed PEC immunosensor shows a linear relationship between photocurrent variation and the logarithm of PSA concentration in the wide range of 5pgmL -1 to 50ngmL -1 with a low detection limit of 2.6pgmL -1 (S/N=3). Moreover, it also presented good stability and acceptable specificity, indicating the potential applications in clinical diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1996-01-01

    by a defect in the cytotoxic capacity of the individual CD4+ lymphocyte after antigen stimulation, and it could not be explained by a reduction in the total number of CD4+ cells before antigen stimulation. The antigen-specific cytotoxic activity was, however, closely related to the ability of the CD4+ T cells...

  5. 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

  6. CD19-Chimeric Antigen Receptor T Cells for Treatment of Chronic Lymphocytic Leukaemia and Acute Lymphoblastic Leukaemia

    DEFF Research Database (Denmark)

    Lorentzen, C L; thor Straten, Per

    2015-01-01

    Adoptive cell therapy (ACT) for cancer represents a promising new treatment modality. ACT based on the administration of cytotoxic T cells genetically engineered to express a chimeric antigen receptor (CAR) recognizing CD19 expressed by B cell malignancies has been shown to induce complete lasting...

  7. 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

  8. 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.

  9. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    Science.gov (United States)

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-γ on either protein or mRNA levels. The anergic state of CD4+CD25+ T cells is not reversible by the addition of anti-CD28, anti–CTLA-4, anti–transforming growth factor β, or anti–IL-10 antibody. However, the refractory state of CD4+CD25+ T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties. PMID:11390435

  10. In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33+ Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    A. Dutour

    2012-01-01

    Full Text Available Genetic engineering of T cells with chimeric T-cell receptors (CARs is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV- specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

  11. 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

  12. Antigen and Memory CD8 T Cells: Were They Both Right?

    Directory of Open Access Journals (Sweden)

    Epelman Slava

    2007-06-01

    Full Text Available Picture yourself as a researcher in immunology. To begin your project, you ask a question: Do CD8 T cells require antigen to maintain a memory response? This question is of prime importance to numerous medical fields. In chronologic order, you digest the literature, but unfortunately, you hit a major stumbling block in the 1990s. The crux of the problem is that which so often happens in science: two well-recognized, capable groups emerge with diametrically opposed conclusions, leaving you pondering which set of wellcontrolled data to believe. Fortunately, years later, a surprising group of articles sheds light on this mystery and subtly reconciles these two positions.

  13. 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.

  14. Inhibitory receptor expression depends more dominantly on differentiation and activation than exhaustion of human CD8 T cells

    Directory of Open Access Journals (Sweden)

    Amandine eLegat

    2013-12-01

    Full Text Available Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed exhaustion. Expression of inhibitory Receptors (iRs is often regarded as a hallmark of exhaustion. Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160 and KLRG-1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term (chronic antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking upregulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

  15. 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.

  16. T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

    OpenAIRE

    Roberts, Joseph L.; Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

    2007-01-01

    CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD...

  17. Conformational plasticity at the IgE-binding site of the B-cell receptor CD23.

    Science.gov (United States)

    Dhaliwal, Balvinder; Pang, Marie O Y; Yuan, Daopeng; Yahya, Norhakim; Fabiane, Stella M; McDonnell, James M; Gould, Hannah J; Beavil, Andrew J; Sutton, Brian J

    2013-12-01

    IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response.

    Science.gov (United States)

    Xue, Qiong; Bettini, Emily; Paczkowski, Patrick; Ng, Colin; Kaiser, Alaina; McConnell, Timothy; Kodrasi, Olja; Quigley, Máire F; Heath, James; Fan, Rong; Mackay, Sean; Dudley, Mark E; Kassim, Sadik H; Zhou, Jing

    2017-11-21

    It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high

  19. IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

    Science.gov (United States)

    Tran, Giang T; Hodgkinson, Suzanne J; Carter, Nicole M; Verma, Nirupama D; Plain, Karren M; Boyd, Rochelle; Robinson, Catherine M; Nomura, Masaru; Killingsworth, Murray; Hall, Bruce M

    2012-05-10

    Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

  20. Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines.

    Directory of Open Access Journals (Sweden)

    Troels R Petersen

    2011-03-01

    Full Text Available Transfer of antigen between antigen-presenting cells (APCs is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs, were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs, suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.

  1. Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle

    DEFF Research Database (Denmark)

    Hart, Jane; MacHugh, Niall D.; Sheldrake, Tara

    2017-01-01

    candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens...... cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV...

  2. Study of CD69 antigen expression and integrity of leukocyte cellular membrane in stored platelet concentrates following irradiation and treatment with Mirasol® PRT System.

    Science.gov (United States)

    Lachert, Elżbieta; Woźniak, Jolanta; Antoniewicz-Papis, Jolanta; Krzywdzińska, Agnieszka; Kubis, Jolanta; Mikołowska, Agata; Letowska, Magdalena

    2017-01-01

    Leukocytes in transfused blood components, particularly residual lymphocytes, have been shown to contribute to the occurrence of various adverse reactions. One of the most severe is transfusionassociated graft versus host disease (TA-GvHD) following transfusion of blood components contaminated with immunocompetent T lymphocytes. Irradiation is a routine method for protection against TA-GvHD. According to the literature, some pathogen reduction methods have also been proven effective for the inactivation of T lymphocytes, and so they may be considered as an alternative to irradiation. Comparison of CD69 antigen expression and the integrity of the leukocyte cellular membrane in stored platelet concentrates (PCs) following irradiation with the Gammacell 3000 Elan (Nordion Inc., Ottawa, Canada) and treatment with the Mirasol® Pathogen Reduction Technology (PRT) System (Terumo BCT, Lakewood, USA). The study included seven experiments. For each experiment we used 3 PCs, for Mirasol® PRT System treatment (M), for Gammacell 3000 Elan irradiation (R), and for the control (C). 7-amino-actinomycin D (7-AAD, Becton Dickinson, Franklin Lakes, USA) permeability was used to determine lymphocyte viability while CD69 antigen expression was the marker of lymphocyte activation. Analyses of 7-AAD and CD69 antigen expression were performed in a FACS Canto I flow cytometer (Becton Dickinson, USA). During 6 storage days, viable lymphocyte count decreased to 28% (p = 0.001) in the Mirasol® PRT System treated PCs and to 65% (p = 0.004) in the irradiated PCs. A statistically significant increase in CD69 expression in the irradiated PCs was observed; 1.3-fold on day 3 and 1.5-fold on day 6. In the Mirasol ® PRT System treated PCs, no statistically significant increase was observed. The in vitro results suggest that the Mirasol® PRT System is as effective as irradiation due to donor leukocyte inactivation capacity.

  3. Antigen-driven focal inflammatory death of malaria liver stages

    Directory of Open Access Journals (Sweden)

    Ganchimeg eBayarsaikhan

    2015-02-01

    Full Text Available Multiple immunizations using live irradiated sporozoites, the infectious plasmodial stage delivered into the host skin during a mosquito bite, can elicit sterile immunity to malaria. CD8+ T cells seem to play an essential role in this protective immunity, since their depletion consistently abolishes sterilizing protection in several experimental models. So far, only a few parasite antigens are known to induce CD8+ T cell-dependent protection, but none of them can reach the levels of protection afforded by live attenuated parasites. Systematic attempts to identify novel antigens associated with this efficient cellular protection were so far unsuccessful. In addition, the precise mechanisms involved in the recognition and elimination of parasitized hepatocytes in vivo by CD8+ T cells still remain obscure. Recently, it has been shown that specific effector CD8+ T cells, after recognition of parasitized hepatocytes, recruit specific and non-specific activated CD8+ T cells to the site of infection, resulting in the formation of cellular clusters around and in the further elimination of intracellular parasites. The significance of this finding is discussed in the perspective of a general mechanism of antigen-dependent focalized inflammation and its consequences for the elimination of malaria liver stages.

  4. CD4+CD25hiFOXP3+ cells in cord blood of neonates born from filaria infected mother are negatively associated with CD4+Tbet+ and CD4+RORγt+ T cells.

    Science.gov (United States)

    Ateba-Ngoa, Ulysse; Mombo-Ngoma, Ghyslain; Zettlmeissl, Eva; van der Vlugt, Luciën E P M; de Jong, Sanne E; de Jong, Sanne; Matsiegui, Pierre-Blaise; Ramharter, Michael; Kremsner, Peter G; Yazdanbakhsh, Maria; Adegnika, Ayola Akim

    2014-01-01

    Children who have been exposed in utero to maternal filarial infection are immunologically less responsive to filarial antigens, have less pathology, and are more susceptible to acquire infection than offspring of uninfected mothers. Moreover children from filaria infected mothers have been shown to be less responsive to vaccination as a consequence of an impairment of their immune response. However, it is not well known how in utero exposure to parasite antigens affects cellular immune responses. Here, 30 pregnant women were examined for the presence of microfilaria of Loa loa and Mansonella perstans in peripheral blood. At delivery, cord blood mononuclear cells (CBMC) were obtained and the CD4+T cells were phenotyped by expression of the transcription factors Tbet, RORγt, and FOXP3. No significant difference was observed between newborns from infected versus uninfected mothers in the frequencies of total CD4+T cells and CD4+T cells subsets including CD4+Tbet+, CD4+RORγt+ T and CD4+CD25hiFOXP3+ T cells. However, there was a negative association between CD4+CD25hiFOXP3+T cells and CD4+Tbet+ as well as CD4+RORγt+ T cells in the infected group only (B = -0.242, P = 0.002; B = -0.178, P = 0.013 respectively). Our results suggest that filarial infection during pregnancy leads to an expansion of functionally active regulatory T cells that keep TH1 and TH17 in check.

  5. Different thresholds of T cell activation regulate FIV infection of CD4+CD25+ and CD4+CD25- cells

    International Nuclear Information System (INIS)

    Joshi, Anjali; Garg, Himanshu; Tompkins, Mary B.; Tompkins, Wayne A.

    2005-01-01

    Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4 + CD25 + T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4 + CD25 + and CD4 + CD25 - cells under different stimulation conditions. Both CD4 + CD25 + and CD4 + CD25 - cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4 + CD25 + cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4 + CD25 - T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4 + CD25 - cells to replicate FIV, it induces apoptosis in a high percentage of CD4 + CD25 + T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4 + CD25 - cells. These results suggest that CD4 + CD25 + and CD4 + CD25 - T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4 + CD25 + and CD4 + CD25 - cells to serve as potential reservoirs of a productive and latent FIV infection

  6. 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

  7. CD4+ CD25+ CD127low Regulatory T Cells as Indicator of Rheumatoid Arthritis Disease Activity.

    Science.gov (United States)

    Khattab, Sahar S; El-Saied, Amany M; Mohammed, Rehab A; Mohamed, Eman E

    2016-06-01

    Rheumatoid arthritis (RA) is an autoimmune disease characterized by disturbed immune regulation, inducing a progressive cartilage and bone destruction. Despite enrichment of T regulatory cell (T-regs) in synovial fluid, conflicting results are reported concerning T-regs in peripheral blood (PB) of RA patients. To determine possible correlation between the frequency of PB CD4+ CD25+CD127low (T-regs) with RA disease activity. Forty females with RA, classified according to the Disease Activity Score 28 (DAS-28), as highly active, mild-moderate or low disease activity; and 20 age and sex matched healthy controls, were enrolled to study CD4+ CD25+ CD127low T- regs in PB by flow cytometry. Active RA patients had lower frequency of the CD4+ CD25+ CD127low T- regs compared to those with mild-moderate or low disease activity (P <0.001). The frequencies of the T- regs showed negative correlation with the DAS-28 (P<0.01). In conclusion, CD4+ CD25+ CD127low T-regs is significantly lower in highly active RA patients compared to patients with lower activity or controls. Copyright© by the Egyptian Association of Immunologists.

  8. Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

    Science.gov (United States)

    Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

    2015-03-19

    Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

  9. Prolonged activation of virus-specific CD8+T cells after acute B19 infection.

    Directory of Open Access Journals (Sweden)

    Adiba Isa

    2005-12-01

    Full Text Available Human parvovirus B19 (B19 is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously.The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%-0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low.This is the first example to our knowledge of an "acute" human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation--analogous to that for cytomegalovirus infection--is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development.

  10. Prolonged activation of virus-specific CD8+T cells after acute B19 infection.

    Directory of Open Access Journals (Sweden)

    2005-12-01

    Full Text Available BACKGROUND: Human parvovirus B19 (B19 is a ubiquitous and clinically significant pathogen, causing erythema infectiosum, arthropathy, transient aplastic crisis, and intrauterine fetal death. The phenotype of CD8+ T cells in acute B19 infection has not been studied previously. METHODS AND FINDINGS: The number and phenotype of B19-specific CD8+ T cell responses during and after acute adult infection was studied using HLA-peptide multimeric complexes. Surprisingly, these responses increased in magnitude over the first year post-infection despite resolution of clinical symptoms and control of viraemia, with T cell populations specific for individual epitopes comprising up to 4% of CD8+ T cells. B19-specific T cells developed and maintained an activated CD38+ phenotype, with strong expression of perforin and CD57 and downregulation of CD28 and CD27. These cells possessed strong effector function and intact proliferative capacity. Individuals tested many years after infection exhibited lower frequencies of B19-specific cytotoxic T lymphocytes, typically 0.05%-0.5% of CD8+ T cells, which were perforin, CD38, and CCR7 low. CONCLUSION: This is the first example to our knowledge of an "acute" human viral infection inducing a persistent activated CD8+ T cell response. The likely explanation--analogous to that for cytomegalovirus infection--is that this persistent response is due to low-level antigen exposure. CD8+ T cells may contribute to the long-term control of this significant pathogen and should be considered during vaccine development.

  11. Zinc supplementation induces CD4+CD25+Foxp3+ antigen-specific regulatory T cells and suppresses IFN-γ production by upregulation of Foxp3 and KLF-10 and downregulation of IRF-1.

    Science.gov (United States)

    Maywald, Martina; Rink, Lothar

    2017-08-01

    The essential trace element zinc plays a fundamental role in immune function and regulation since its deficiency is associated with autoimmunity, allergies, and transplant rejection. Thus, we investigated the influence of zinc supplementation on the Th1-driven alloreaction in mixed lymphocyte cultures (MLC), on generation of antigen-specific T cells, and analyzed underlying molecular mechanisms. Cell proliferation and pro-inflammatory cytokine production were monitored by [ 3 H]-thymidine proliferation assay and ELISA, respectively. Analysis of surface and intracellular T cell marker was performed by flow cytometry. Western blotting and mRNA analysis were used for Foxp3, KLF-10, and IRF-1 expression. Zinc supplementation on antigen-specific T cells in physiological doses (50 µM) provokes a significant amelioration of cell proliferation and pro-inflammatory cytokine production after reactivation compared to untreated controls. Zinc administration on MLC results in an increased induction and stabilization of CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + T cells (p zinc-induced upregulation of Foxp3 and KLF-10 and downregulation of IRF-1. However, in resting lymphocytes zinc increases IRF-1. In summary, zinc is capable of ameliorating the allogeneic immune reaction by enhancement of antigen-specific iTreg cells due to modulation of essential molecular targets: Foxp3, KLF-10, and IRF-1. Thus, zinc can be seen as an auspicious tool for inducing tolerance in adverse immune reactions.

  12. 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

  13. Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo

    Directory of Open Access Journals (Sweden)

    Yoshihito Minoda

    2017-10-01

    Full Text Available Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These “humanized” mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs in mice are categorized into cDC1, which mediate T helper (Th1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study

  14. IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8+ T cells.

    Science.gov (United States)

    Stark, Regina; Hartung, Anett; Zehn, Dietmar; Frentsch, Marco; Thiel, Andreas

    2013-06-01

    CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. The administration route is decisive for the ability of the vaccine adjuvant CAF09 to induce antigen-specific CD8(+) T-cell responses

    DEFF Research Database (Denmark)

    Schmidt, Signe Tandrup; Khadke, Swapnil; Korsholm, Karen Smith

    2016-01-01

    A prerequisite for vaccine-mediated induction of CD8(+) T-cell responses is the targeting of dendritic cell (DC) subsets specifically capable of cross-presenting antigen epitopes to CD8(+) T cells. Administration of a number of cationic adjuvants via the intraperitoneal (i.p.) route has been show...

  16. Downregulation of cathepsin G reduces the activation of CD4+ T cells in murine autoimmune diabetes.

    Science.gov (United States)

    Zou, Fang; Lai, Xiaoyang; Li, Jing; Lei, Shuihong; Hu, Lei

    2017-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease due to progressive injury of islet cells mediated by T lymphocytes (T cells). Our previous studies have shown that only cathepsin G (CatG), not other proteases, is involved in the antigen presentation of proinsulin, and if the presentation is inhibited, the activation of CD4+ T cells induced by proinsulin is alleviated in T1DM patients, and CatG-specific inhibitor reduces the activation of CD4+ cells induced by proinsulin in T1DM patients. Therefore, we hypothesize that CatG may play an important role in the activation of CD4+ T cells in T1DM. To this end, mouse studies were conducted to demonstrate that CatG impacts the activation of CD4+ T cells in non-obese diabetic (NOD) mice. CatG gene expression and the activation of CD4+ T cells were examined in NOD mice. The effect of CatG inhibitor was investigated in NOD mice on the activation of CD4+ T cells, islet β cell function, islet inflammation and β-cell apoptosis. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on the activation status of CD4+ T cells and the progression of diabetes. During the pathogenesis of diabetes, the expression level of CatG in NOD mice gradually increased and the CD4+ T cells were gradually activated, resulting in more TH1 cells and less TH2 and Treg cells. Treatment with CatG-specific inhibitor reduced the blood glucose level, improved the function of islet β cells and reduced the activation of CD4+ T cells. Early application of CatG siRNA improved the function of islet β cells, reduced islet inflammation and β cell apoptosis, and lowered the activation level of CD4+ T cells, thus slowing down the progression of diabetes.

  17. 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.

  18. Focal radiation therapy combined with 4-1BB activation and CTLA-4 blockade yields long-term survival and a protective antigen-specific memory response in a murine glioma model.

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    Zineb Belcaid

    Full Text Available Glioblastoma (GBM is the most common malignant brain tumor in adults and is associated with a poor prognosis. Cytotoxic T lymphocyte antigen -4 (CTLA-4 blocking antibodies have demonstrated an ability to generate robust antitumor immune responses against a variety of solid tumors. 4-1BB (CD137 is expressed by activated T lymphocytes and served as a co-stimulatory signal, which promotes cytotoxic function. Here, we evaluate a combination immunotherapy regimen involving 4-1BB activation, CTLA-4 blockade, and focal radiation therapy in an immune-competent intracranial GBM model.GL261-luciferace cells were stereotactically implanted in the striatum of C57BL/6 mice. Mice were treated with a triple therapy regimen consisted of 4-1BB agonist antibodies, CTLA-4 blocking antibodies, and focal radiation therapy using a small animal radiation research platform and mice were followed for survival. Numbers of brain-infiltrating lymphocytes were analyzed by FACS analysis. CD4 or CD8 depleting antibodies were administered to determine the relative contribution of T helper and cytotoxic T cells in this regimen. To evaluate the ability of this immunotherapy to generate an antigen-specific memory response, long-term survivors were re-challenged with GL261 glioma en B16 melanoma flank tumors.Mice treated with triple therapy had increased survival compared to mice treated with focal radiation therapy and immunotherapy with 4-1BB activation and CTLA-4 blockade. Animals treated with triple therapy exhibited at least 50% long-term tumor free survival. Treatment with triple therapy resulted in a higher density of CD4+ and CD8+ tumor infiltrating lymphocytes. Mechanistically, depletion of CD4+ T cells abrogated the antitumor efficacy of triple therapy, while depletion of CD8+ T cells had no effect on the treatment response.Combination therapy with 4-1BB activation and CTLA-4 blockade in the setting of focal radiation therapy improves survival in an orthotopic mouse

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

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    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.

  20. Activation-Induced TIM-4 Expression Identifies Differential Responsiveness of Intestinal CD103+ CD11b+ Dendritic Cells to a Mucosal Adjuvant.

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    Kerry L Hilligan

    Full Text Available Macrophage and dendritic cell (DC populations residing in the intestinal lamina propria (LP are highly heterogeneous and have disparate yet collaborative roles in the promotion of adaptive immune responses towards intestinal antigen. Under steady-state conditions, macrophages are efficient at acquiring antigen but are non-migratory. In comparison, intestinal DC are inefficient at antigen uptake but migrate to the mesenteric lymph nodes (mLN where they present antigen to T cells. Whether such distinction in the roles of DC and macrophages in the uptake and transport of antigen is maintained under immunostimulatory conditions is less clear. Here we show that the scavenger and phosphatidylserine receptor T cell Immunoglobulin and Mucin (TIM-4 is expressed by the majority of LP macrophages at steady-state, whereas DC are TIM-4 negative. Oral treatment with the mucosal adjuvant cholera toxin (CT induces expression of TIM-4 on a proportion of CD103+ CD11b+ DC in the LP. TIM-4+ DC selectively express high levels of co-stimulatory molecules after CT treatment and are detected in the mLN a short time after appearing in the LP. Importantly, intestinal macrophages and DC expressing TIM-4 are more efficient than their TIM-4 negative counterparts at taking up apoptotic cells and soluble antigen ex vivo. Taken together, our results show that CT induces phenotypic changes to migratory intestinal DC that may impact their ability to take up local antigens and in turn promote the priming of mucosal immunity.

  1. Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

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    Maria de Lourdes Palermo

    2012-12-01

    Full Text Available Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts. We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1, which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

  2. CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

    Science.gov (United States)

    Wong, Kuan Y; Baron, Rebecca; Seldon, Therese A; Jones, Martina L; Rice, Alison M; Munster, David J

    2018-05-15

    Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83 + human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83 + B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83 - ) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells. Copyright © 2018 by The American Association of Immunologists, Inc.

  3. TLR1/2 activation during heterologous prime-boost vaccination (DNA-MVA enhances CD8+ T Cell responses providing protection against Leishmania (Viannia.

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    Asha Jayakumar

    2011-06-01

    Full Text Available Leishmania (Viannia parasites present particular challenges, as human and murine immune responses to infection are distinct from other Leishmania species, indicating a unique interaction with the host. Further, vaccination studies utilizing small animal models indicate that modalities and antigens that prevent infection by other Leishmania species are generally not protective.Using a newly developed mouse model of chronic L. (Viannia panamensis infection and the heterologous DNA prime - modified vaccinia virus Ankara (MVA boost vaccination modality, we examined whether the conserved vaccine candidate antigen tryparedoxin peroxidase (TRYP could provide protection against infection/disease.Heterologous prime - boost (DNA/MVA vaccination utilizing TRYP antigen can provide protection against disease caused by L. (V. panamensis. However, protection is dependent on modulating the innate immune response using the TLR1/2 agonist Pam3CSK4 during DNA priming. Prime-boost vaccination using DNA alone fails to protect. Prior to infection protectively vaccinated mice exhibit augmented CD4 and CD8 IFNγ and memory responses as well as decreased IL-10 and IL-13 responses. IL-13 and IL-10 have been shown to be independently critical for disease in this model. CD8 T cells have an essential role in mediating host defense, as CD8 depletion reversed protection in the vaccinated mice; vaccinated mice depleted of CD4 T cells remained protected. Hence, vaccine-induced protection is dependent upon TLR1/2 activation instructing the generation of antigen specific CD8 cells and restricting IL-13 and IL-10 responses.Given the general effectiveness of prime-boost vaccination, the recalcitrance of Leishmania (Viannia to vaccine approaches effective against other species of Leishmania is again evident. However, prime-boost vaccination modality can with modulation induce protective responses, indicating that the delivery system is critical. Moreover, these results suggest that

  4. CD4+ T cell effects on CD8+ T cell location defined using bioluminescence.

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    Mitra Azadniv

    2011-01-01

    Full Text Available T lymphocytes of the CD8+ class are critical in delivering cytotoxic function and in controlling viral and intracellular infections. These cells are "helped" by T lymphocytes of the CD4+ class, which facilitate their activation, clonal expansion, full differentiation and the persistence of memory. In this study we investigated the impact of CD4+ T cells on the location of CD8+ T cells, using antibody-mediated CD4+ T cell depletion and imaging the antigen-driven redistribution of bioluminescent CD8+ T cells in living mice. We documented that CD4+ T cells influence the biodistribution of CD8+ T cells, favoring their localization to abdominal lymph nodes. Flow cytometric analysis revealed that this was associated with an increase in the expression of specific integrins. The presence of CD4+ T cells at the time of initial CD8+ T cell activation also influences their biodistribution in the memory phase. Based on these results, we propose the model that one of the functions of CD4+ T cell "help" is to program the homing potential of CD8+ T cells.

  5. Systemic immunological tolerance to ocular antigens is mediated by TRAIL-expressing CD8+ T cells.

    Science.gov (United States)

    Griffith, Thomas S; Brincks, Erik L; Gurung, Prajwal; Kucaba, Tamara A; Ferguson, Thomas A

    2011-01-15

    Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.

  6. Predominant cerebral cytokine release syndrome in CD19-directed chimeric antigen receptor-modified T cell therapy

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    Yongxian Hu

    2016-08-01

    Full Text Available Abstract Chimeric antigen receptor-modified (CAR T cells targeting CD19 (CART19 have shown therapeutical activities in CD19+ malignancies. However, the etiological nature of neurologic complications remains a conundrum. In our study, the evidence of blood-brain barrier (BBB-penetrating CAR T cells as a culprit was revealed. A patient with acute lymphocytic leukemia developed sustained pyrexia with tremors about 6 h after CART19 infusion, followed by a grade 2 cytokine release syndrome (CRS and neurological symptoms in the next 3 days. Contrast-enhanced magnetic resonance showed signs of intracranial edema. Lumbar puncture on day 5 showed an over 400-mmH2O cerebrospinal pressure. The cerebrospinal fluid (CSF contained 20 WBCs/μL with predominant CD3+ T cells. qPCR analysis for CAR constructs showed 3,032,265 copies/μg DNA in CSF and 988,747 copies/μg DNA in blood. Cytokine levels including IFN-γ and IL-6 in CSF were extremely higher than those in the serum. Methyprednisone was administrated and the symptoms relieved gradually. The predominance of CART19 in CSF and the huge discrepancies in cytokine distributions indicated the development of a cerebral CRS, presumably featured as CSF cytokines largely in situ produced by BBB-penetrating CAR T cells. For the first time, we reported the development of cerebral CRS triggered by BBB-penetrating CAR T cells. Trial registration: ChiCTR-OCC-15007008 .

  7. Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

    Science.gov (United States)

    Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

    2013-01-01

    Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

  8. Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

    Science.gov (United States)

    Kwun, Jean; Farris, Alton B; Song, Hyunjin; Mahle, William T; Burlingham, William J; Knechtle, Stuart J

    2015-12-01

    Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

  9. PERSPECTIVE OF IN VITRO LYMPHOCYTES ANTIGENICITY EVALUATION FOR THE DIAGNOSTICS OF ACUTE BRUCELLOSIS

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    M. V. Kostyuchenko

    2017-01-01

    Full Text Available The brucellosis remains to one of the most urgent dangerous infections in regions with developed livestock production. An exclusive polymorphism of symptoms, variety of forms of a disease, small informational content of results of routine laboratory all-clinical inspection, quite often leads to diagnostic mistakes at a pre-hospital stage. Improvement of a complex of laboratory diagnosis of a brucellous infection demands development of the modern padding methods of verification based on cell-like factors of immunity as leaders in an immunogenesis and a pathogenesis of a brucellosis. Considering the leading role of cell-like immunity in formation of protection against the majority of bacteriemic especially dangerous infections, studying of cell-like reaction in response to antigenic stimulation, it is necessary to consider the most informative (marker and objective at assessment of immunologic reorganization of an organism at a disease or vaccination. The following markers (receptors of activation of lymphocytes can act as perspective indexes of a specific cell-like antigenreactivity: CD25 — a high-affine receptor of interleukin 2 (IL-2Ra, a marker of early activation of Tlymphocytes; HLA-DR — an antigen of the main complex of a histocompatibility of a class II, an expression of a marker is associated not only with late, but also long-lived activation of lymphocytes; CD95 (Fas, APO-1 — a receptor of an induction of an apoptosis (“cell death”, a marker of “late” activation (CD4+ lymphocytes is presented mainly and Fas L (CD178 — a receptor of an induction of an apoptosis, expresses generally on CD8+ cages. The work purpose — to estimate an opportunity and prospects of use of technology of a flowing cytofluorometry and the in vitro cell tests for diagnosis of a acute brucellosis. 35 people with the diagnosis “Acute brucellosis” and 12 people — not the patients who did not have a brucellosis, are not vaccinated

  10. 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...

  11. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis.

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    Alessandra Citro

    Full Text Available CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control. The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients.

  12. B Cell Help by CD1d-Rectricted NKT Cells

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    Livia Clerici

    2015-10-01

    Full Text Available B cell activation and antibody production against foreign antigens is a central step of host defense. This is achieved via highly regulated multi-phase processes that involve a variety of cells of both innate and adaptive arms of the immune system. MHC class II-restricted CD4+ T cells specific for peptide antigens, which acquire professional follicular B cell helper functions, have been long recognized as key players in this process. Recent data, however, challenge this paradigm by showing the existence of other helper cell types. CD1d restricted NKT cells specific for lipid antigens are one such new player and can coopt bona fide follicular helper phenotypes. Their role in helping antigen-specific B cell response to protein antigens, as well as to the so called “help-less” antigens that cannot be recognized by T follicular helper cells, is being increasingly elucidated, highlighting their potential pathophysiological impact on the immune response, as well as on the design of improved vaccine formulations.

  13. Demonstration of strong enterobacterial reactivity of CD4+CD25- T cells from conventional and germ-free mice which is counter-regulated by CD4+CD25+ T cells

    DEFF Research Database (Denmark)

    Gad, Monika; Pedersen, Anders Elm; Kristensen, Nanna N

    2004-01-01

    Unfractionated CD4+ T cells from the gut-associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25- T...

  14. CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent.

    Science.gov (United States)

    D'Souza, Lucas; Gupta, Sneh Lata; Bal, Vineeta; Rath, Satyajit; George, Anna

    2017-12-01

    B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM + cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73 + IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory. © 2017 John Wiley & Sons Ltd.

  15. Bispecific antibodies targeting human CD73

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen.......The present invention relates to a bispecific antibody targeting CD73. In particular, the present invention relates to a bispecific antibody targeting different epitopes on CD73 or a bispecific antibody targeting an epitope on CD73 and an epitope on a different antigen....

  16. 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.

  17. Anti-CD20 B-cell depletion enhances monocyte reactivity in neuroimmunological disorders

    Directory of Open Access Journals (Sweden)

    Hohlfeld Reinhard

    2011-10-01

    Full Text Available Abstract Background Clinical trials evaluating anti-CD20-mediated B-cell depletion in multiple sclerosis (MS and neuromyelitis optica (NMO generated encouraging results. Our recent studies in the MS model experimental autoimmune encephalomyelitis (EAE attributed clinical benefit to extinction of activated B-cells, but cautioned that depletion of naïve B-cells may be undesirable. We elucidated the regulatory role of un-activated B-cells in EAE and investigated whether anti-CD20 may collaterally diminish regulatory B-cell properties in treatment of neuroimmunological disorders. Methods Myelin oligodendrocyte glycoprotein (MOG peptide-immunized C57Bl/6 mice were depleted of B-cells. Functional consequences for regulatory T-cells (Treg and cytokine production of CD11b+ antigen presenting cells (APC were assessed. Peripheral blood mononuclear cells from 22 patients receiving anti-CD20 and 23 untreated neuroimmunological patients were evaluated for frequencies of B-cells, T-cells and monocytes; monocytic reactivity was determined by TNF-production and expression of signalling lymphocytic activation molecule (SLAM. Results We observed that EAE-exacerbation upon depletion of un-activated B-cells closely correlated with an enhanced production of pro-inflammatory TNF by CD11b+ APC. Paralleling this pre-clinical finding, anti-CD20 treatment of human neuroimmunological disorders increased the relative frequency of monocytes and accentuated pro-inflammatory monocyte function; when reactivated ex vivo, a higher frequency of monocytes from B-cell depleted patients produced TNF and expressed the activation marker SLAM. Conclusions These data suggest that in neuroimmunological disorders, pro-inflammatory APC activity is controlled by a subset of B-cells which is eliminated concomitantly upon anti-CD20 treatment. While this observation does not conflict with the general concept of B-cell depletion in human autoimmunity, it implies that its safety and

  18. Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

    Science.gov (United States)

    Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

    1997-09-15

    Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

  19. 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.

  20. Ebola Virus Disease Is Characterized by Poor Activation and Reduced Levels of Circulating CD16+ Monocytes.

    Science.gov (United States)

    Lüdtke, Anja; Ruibal, Paula; Becker-Ziaja, Beate; Rottstegge, Monika; Wozniak, David M; Cabeza-Cabrerizo, Mar; Thorenz, Anja; Weller, Romy; Kerber, Romy; Idoyaga, Juliana; Magassouba, N'Faly; Gabriel, Martin; Günther, Stephan; Oestereich, Lisa; Muñoz-Fontela, César

    2016-10-15

    A number of previous studies have identified antigen-presenting cells (APCs) as key targets of Ebola virus (EBOV), but the role of APCs in human Ebola virus disease (EVD) is not known. We have evaluated the phenotype and kinetics of monocytes, neutrophils, and dendritic cells (DCs) in peripheral blood of patients for whom EVD was diagnosed by the European Mobile Laboratory in Guinea. Acute EVD was characterized by reduced levels of circulating nonclassical CD16 + monocytes with a poor activation profile. In survivors, CD16 + monocytes were activated during recovery, coincident with viral clearance, suggesting an important role of this cell subset in EVD pathophysiology. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  1. CD8αα expression marks terminally differentiated human CD8+ T cells expanded in chronic viral infection

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    Lucy Jane Walker

    2013-08-01

    Full Text Available The T cell co-receptor CD8αβ enhances T cell sensitivity to antigen, however studies indicate CD8αα has the converse effect and acts as a co-repressor. Using a combination of Thymic Leukaemia antigen (TL tetramer, which directly binds CD8αα, anti-CD161 and anti-Vα7.2 antibodies we have been able for the first time to clearly define CD8αα expression on human CD8 T cells subsets. In healthy controls CD8αα is most highly expressed by CD161 bright (CD161++ mucosal associated invariant T (MAIT cells, with CD8αα expression highly restricted to the TCR Vα7.2+ cells of this subset. We also identified CD8αα-expressing populations within the CD161 mid (CD161+ and negative (CD161- non-MAIT CD8 T cell subsets and show TL-tetramer binding to correlate with expression of CD8β at low levels in the context of maintained CD8α expression (CD8α+CD8βlow. In addition, we found CD161-CD8α+CD8βlow populations to be significantly expanded in the peripheral blood of HIV-1 and hepatitis B (mean of 47% and 40% of CD161- T cells respectively infected individuals. Such CD8αα expressing T cells are an effector-memory population (CD45RA-, CCR7-, CD62L- that express markers of activation and maturation (HLA-DR+, CD28-, CD27-, CD57+ and are functionally distinct, expressing greater levels of TNF-α and IFN-γ on stimulation and perforin at rest than their CD8α+CD8βhigh counterparts. Antigen-specific T cells in HLA-B*4201+HIV-1 infected patients are found within both the CD161-CD8α+CD8βhigh and CD161-CD8α+CD8βlow populations. Overall we have clearly defined CD8αα expressing human T cell subsets using the TL-tetramer, and have demonstrated CD161-CD8α+CD8βlow populations, highly expanded in disease settings, to co-express CD8αβ and CD8αα. Co-expression of CD8αα on CD8αβ T cells may impact on their overall function in-vivo and contribute to the distinctive phenotype of highly differentiated populations in HBV and HIV-1 infection.

  2. Further phenotypic characterization of the primitive lineage− CD34+CD38−CD90+CD45RA− hematopoietic stem cell/progenitor cell sub-population isolated from cord blood, mobilized peripheral blood and patients with chronic myelogenous leukemia

    International Nuclear Information System (INIS)

    Wisniewski, D; Affer, M; Willshire, J; Clarkson, B

    2011-01-01

    The most primitive hematopoietic stem cell (HSC)/progenitor cell (PC) population reported to date is characterized as being Lin−CD34+CD38−CD90+CD45R. We have a long-standing interest in comparing the characteristics of hematopoietic progenitor cell populations enriched from normal subjects and patients with chronic myelogenous leukemia (CML). In order to investigate further purification of HSCs and for potential targetable differences between the very primitive normal and CML stem/PCs, we have phenotypically compared the normal and CML Lin−CD34+CD38−CD90+CD45RA− HSC/PC populations. The additional antigens analyzed were HLA-DR, the receptor tyrosine kinases c-kit and Tie2, the interleukin-3 cytokine receptor, CD33 and the activation antigen CD69, the latter of which was recently reported to be selectively elevated in cell lines expressing the Bcr-Abl tyrosine kinase. Notably, we found a strikingly low percentage of cells from the HSC/PC sub-population isolated from CML patients that were found to express the c-kit receptor (<1%) compared with the percentages of HSC/PCs expressing the c-kitR isolated from umbilical cord blood (50%) and mobilized peripheral blood (10%). Surprisingly, Tie2 receptor expression within the HSC/PC subset was extremely low from both normal and CML samples. Using in vivo transplantation studies, we provide evidence that HLA-DR, c-kitR, Tie2 and IL-3R may not be suitable markers for further partitioning of HSCs from the Lin−CD34+CD38−CD90+CD45RA− sub-population

  3. 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

  4. Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

    NARCIS (Netherlands)

    Heijnen, I.; Barnett, D.; Arroz, M.J.; Barry, S.M.; Bonneville, M.; Brando, B.; D'Hautcourt, J.L.; Kern, F.; Totterman, T.H.; Marijt, E.W.; Bossy, D.; Preijers, F.W.M.B.; Rothe, G.; Gratama, J.W.

    2004-01-01

    BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic

  5. Blood feeding by the Rocky Mountain spotted fever vector, Dermacentor andersoni, induces interleukin-4 expression by cognate antigen responding CD4+ T cells

    Directory of Open Access Journals (Sweden)

    Wikel Stephen K

    2009-10-01

    Full Text Available Abstract Background Tick modulation of host defenses facilitates both blood feeding and pathogen transmission. Several tick species deviate host T cell responses toward a Th2 cytokine profile. The majority of studies of modulation of T cell cytokine expression by ticks were performed with lymphocytes from infested mice stimulated in vitro with polyclonal T cell activators. Those reports did not examine tick modulation of antigen specific responses. We report use of a transgenic T cell receptor (TCR adoptive transfer model reactive with influenza hemagglutinin peptide (110-120 to examine CD4+ T cell intracellular cytokine responses during infestation with the metastriate tick, Dermacentor andersoni, or exposure to salivary gland extracts. Results Infestation with pathogen-free D. andersoni nymphs or administration of an intradermal injection of female or male tick salivary gland extract induced significant increases of IL-4 transcripts in skin and draining lymph nodes of BALB/c mice as measured by quantitative real-time RT-PCR. Furthermore, IL-10 transcripts were significantly increased in skin while IL-2 and IFN-γ transcripts were not significantly changed by tick feeding or intradermal injection of salivary gland proteins, suggesting a superimposed Th2 response. Infestation induced TCR transgenic CD4+ T cells to divide more frequently as measured by CFSE dilution, but more notably these CD4+ T cells also gained the capacity to express IL-4. Intracellular levels of IL-4 were significantly increased. A second infestation administered 14 days after a primary exposure to ticks resulted in partially reduced CFSE dilution with no change in IL-4 expression when compared to one exposure to ticks. Intradermal inoculation of salivary gland extracts from both male and female ticks also induced IL-4 expression. Conclusion This is the first report of the influence of a metastriate tick on the cytokine profile of antigen specific CD4+ T cells. Blood feeding

  6. CD4dullCD8bright double-positive T-lymphocytes have a phenotype of granzyme Bpos CD8pos memory T-lymphocytes

    NARCIS (Netherlands)

    Rentenaar, R. J.; Wever, P. C.; van Diepen, F. N.; Schellekens, P. T.; Wertheim, P. M.; ten Berge, I. J.

    1999-01-01

    BACKGROUND: T-lymphocytes that co-express CD4 and CD8 antigens may be found in small percentages in the peripheral blood of healthy individuals, and have a CD4brightCD8dull phenotype. CD4dullCD8bright T-lymphocytes have been found only in temporal association with some viral infections. METHODS:

  7. Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

    Science.gov (United States)

    He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

    2013-10-17

    Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. 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.

  9. Soluble CD30 and ELISA-detected human leukocyte antigen antibodies for the prediction of acute rejection in pediatric renal transplant recipients.

    Science.gov (United States)

    Billing, Heiko; Sander, Anja; Süsal, Caner; Ovens, Jörg; Feneberg, Reinhard; Höcker, Britta; Vondrak, Karel; Grenda, Ryszard; Friman, Stybjorn; Milford, David V; Lucan, Mihai; Opelz, Gerhard; Tönshoff, Burkhard

    2013-03-01

    Biomarker-based post-transplant immune monitoring for the prediction of impending graft rejection requires validation in specific patient populations. Serum of 28 pediatric renal transplant recipients within the framework of a well-controlled prospective randomized trial was analyzed pre- and post-transplant for soluble CD30 (sCD30), a biomarker reflecting mainly T-cell reactivity, and anti-human leukocyte antigen (anti-HLA) antibody reactivity, a biomarker for B-cell activation. A sCD30 concentration ≥40.3 U/ml on day 14 was able to discriminate between patients with or without biopsy-proven acute rejection (BPAR) with a sensitivity of 100% and a specificity of 76%. Six of seven patients (86%) with BPAR showed a sCD30 above this cut-off, whereas only 3/21 patients (14%) without BPAR had a sCD30 above this cut-off (P = 0.004). For pre- and post-transplant anti-HLA class II reactivities by enzyme-linked immunosorbent assay, a cut-off value of 140 optical density was able to discriminate rejecters from nonrejecters with a sensitivity of 86% or 71% and a specificity of 81% or 90%, respectively. Withdrawal of steroids was associated with a approximately twofold higher serum sCD30 compared to controls, but did not affect anti-HLA reactivities. An increased post-transplant sCD30 serum concentration and positive pre- and post-transplant anti-HLA class II reactivities are informative biomarkers for impending BPAR in pediatric renal transplant recipients. (TWIST, Clinical Trial No: FG-506-02-43). © 2012 The Authors Transplant International © 2012 European Society for Organ Transplantation. Published by Blackwell Publishing Ltd.

  10. 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.

  11. Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

    Science.gov (United States)

    Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

    2017-01-01

    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

  12. Fullerene (C{sub 60})/CdS nanocomposite with enhanced photocatalytic activity and stability

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qiang [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Hu, Zhuofeng, E-mail: st04hzhf@gmail.com [Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Zhang, Qian; Li, Boyuan [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Shen, Zhurui, E-mail: shenzhurui@tju.edu.cn [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

    2017-05-01

    Highlights: • C{sub 60}/CdS nanocomposite has been fabricated as a novel visible-light-driven photocatalyst. • It exhibits enhanced photocatalytic activity and photostability than that of pure CdS reference. • The C{sub 60} improved the charge separation and transfer of nanocomposite due to its high electron affinity. - Abstract: Herein, the fullerene (C{sub 60})/CdS nanocomposite has been fabricated by a facile one-pot hydrothermal method. Its photocatatlytic hydrogen (H{sub 2}) evolution rate and degradation efficiency of Rhodamine B (Rh B) are evaluated under visible light irradiation (λ ≥ 420 nm). The content of C{sub 60} has been changed from 0.4 wt% to 8 wt%, and the optimal value for photocatalytic activity is determined to be 0.4 wt%. The H{sub 2} evolution rate over this optimal sample reaches 1.73 mmol h{sup −1} g{sup −1} and its apparent degradation rate of Rh B is 0.089 min{sup −1} (degradation efficiency of 97% within 40 min), which is 2.3 times and 1.5 times compared to that of pure CdS reference. Moreover, the photocorrosion of CdS in composite is effectively suppressed, and its photocatalytic activity can be well maintained after three recycles (97.8% retaining for composite vs. 84.4% retaining for CdS). Then, the enhanced photocatalytic activity and stability of C{sub 60}/CdS nanocomposite are further studied by spectroscopic and electrochemical methods. Results show that the C{sub 60} species covering on the surface of CdS can efficiently accelerate the separation and transfer of photoexcited charge carriers, which can improve its activity, and reduce the photocorrosion of CdS.

  13. 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.

  14. Quantitative imaging by pixel-based contrast-enhanced ultrasound reveals a linear relationship between synovial vascular perfusion and the recruitment of pathogenic IL-17A-F+IL-23+ CD161+ CD4+ T helper cells in psoriatic arthritis joints.

    Science.gov (United States)

    Fiocco, Ugo; Stramare, Roberto; Martini, Veronica; Coran, Alessandro; Caso, Francesco; Costa, Luisa; Felicetti, Mara; Rizzo, Gaia; Tonietto, Matteo; Scanu, Anna; Oliviero, Francesca; Raffeiner, Bernd; Vezzù, Maristella; Lunardi, Francesca; Scarpa, Raffaele; Sacerdoti, David; Rubaltelli, Leopoldo; Punzi, Leonardo; Doria, Andrea; Grisan, Enrico

    2017-02-01

    To develop quantitative imaging biomarkers of synovial tissue perfusion by pixel-based contrast-enhanced ultrasound (CEUS), we studied the relationship between CEUS synovial vascular perfusion and the frequencies of pathogenic T helper (Th)-17 cells in psoriatic arthritis (PsA) joints. Eight consecutive patients with PsA were enrolled in this study. Gray scale CEUS evaluation was performed on the same joint immediately after joint aspiration, by automatic assessment perfusion data, using a new quantification approach of pixel-based analysis and the gamma-variate model. The set of perfusional parameters considered by the time intensity curve includes the maximum value (peak) of the signal intensity curve, the blood volume index or area under the curve, (BVI, AUC) and the contrast mean transit time (MTT). The direct ex vivo analysis of the frequencies of SF IL17A-F + CD161 + IL23 + CD4 + T cells subsets were quantified by fluorescence-activated cell sorter (FACS). In cross-sectional analyses, when tested for multiple comparison setting, a false discovery rate at 10%, a common pattern of correlations between CEUS Peak, AUC (BVI) and MTT parameters with the IL17A-F + IL23 + - IL17A-F + CD161 + - and IL17A-F + CD161 + IL23 + CD4 + T cells subsets, as well as lack of correlation between both peak and AUC values and both CD4 + T and CD4 + IL23 + T cells, was observed. The pixel-based CEUS assessment is a truly measure synovial inflammation, as a useful tool to develop quantitative imaging biomarker for monitoring target therapeutics in PsA.

  15. 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.

  16. Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

    Science.gov (United States)

    Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

    1994-04-01

    The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO

  17. 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.

  18. 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

  19. Construction of a new anti-CD19 chimeric antigen receptor and the anti-leukemia function study of the transduced T cells

    Science.gov (United States)

    An, Na; Tao, Zhongfei; Li, Saisai; Xing, Haiyan; Tang, Kejing; Tian, Zheng; Rao, Qing; Wang, Min; Wang, Jianxiang

    2016-01-01

    Chimeric antigen receptor (CAR) transduced T cells have been used to efficiently kill the target tumor cells depending on the single chain variable fragment (scFv) against the specific tumor associated antigen. Here we show the high specific cytotoxicity of the CAR-T cells with very low effector to target cell (E:T) ratio owing to the CD19-scFv, which was constructed in our laboratory and proved to be highly effective in our previous study. Four plasmids containing three generation of CAR were constructed by cloning the CD19-CAR fragment into the lentiviral vector pCDH. CD3 positive T cells were successfully transduced and the CAR protein expression was confirmed by flow cytometry and Western blot. When cocultured with CD19 positive leukemia cell line Nalm-6 cells, CAR-T cells showed specific cytotoxicity: the percentage of target cells decreased to 0 in 24 hours; IL-2, IFN-γ and TNF-α produced in cocultured supernatants increased obviously; and the cytotoxicity reached more than 80%, still remarkable even when the E:T ratio was as low as 1:4. Dynamic change of cell interaction between CAR-T and leukemia cells was visually tracked by using living cells workstation for the first time. A NOD/SCID B-ALL murine model was established using Nalm-6 cells inoculation with a morbidity rate of 100%, and the survival time was prolonged statistically with CAR-T cell treatment. These data demonstrate that the CAR-T cells we prepared could be a promising treatment strategy for CD19 positive tumor diseases. PMID:26840021

  20. Circumvention of regulatory CD4(+) T cell activity during cross-priming strongly enhances T cell-mediated immunity.

    Science.gov (United States)

    Heit, Antje; Gebhardt, Friedemann; Lahl, Katharina; Neuenhahn, Michael; Schmitz, Frank; Anderl, Florian; Wagner, Hermann; Sparwasser, Tim; Busch, Dirk H; Kastenmüller, Kathrin

    2008-06-01

    Immunization with purified antigens is a safe and practical vaccination strategy but is generally unable to induce sustained CD8(+) T cell-mediated protection against intracellular pathogens. Most efforts to improve the CD8(+) T cell immunogenicity of these vaccines have focused on co-administration of adjuvant to support cross-presentation and dendritic cell maturation. In addition, it has been shown that CD4(+) T cell help during the priming phase contributes to the generation of protective CD8(+) memory T cells. In this report we demonstrate that the depletion of CD4(+) T cells paradoxically enhances long-lasting CD8-mediated protective immunity upon protein vaccination. Functional and genetic in vivo inactivation experiments attribute this enhancement primarily to MHC class II-restricted CD4(+) regulatory T cells (Treg), which appear to physiologically suppress the differentiation process towards long-living effector memory T cells. Since, in functional terms, this suppression by Treg largely exceeds the positive effects of conventional CD4(+) T cell help, even the absence of all CD4(+) T cells or lack of MHC class II-mediated interactions on priming dendritic cells result in enhanced CD8(+) T cell immunogenicity. These findings have important implications for the improvement of vaccines against intracellular pathogens or tumors, especially in patients with highly active Treg.

  1. Molecular mechanism and function of CD40/CD40L engagement in the immune system.

    Science.gov (United States)

    Elgueta, Raul; Benson, Micah J; de Vries, Victor C; Wasiuk, Anna; Guo, Yanxia; Noelle, Randolph J

    2009-05-01

    During the generation of a successful adaptive immune response, multiple molecular signals are required. A primary signal is the binding of cognate antigen to an antigen receptor expressed by T and B lymphocytes. Multiple secondary signals involve the engagement of costimulatory molecules expressed by T and B lymphocytes with their respective ligands. Because of its essential role in immunity, one of the best characterized of the costimulatory molecules is the receptor CD40. This receptor, a member of the tumor necrosis factor receptor family, is expressed by B cells, professional antigen-presenting cells, as well as non-immune cells and tumors. CD40 binds its ligand CD40L, which is transiently expressed on T cells and other non-immune cells under inflammatory conditions. A wide spectrum of molecular and cellular processes is regulated by CD40 engagement including the initiation and progression of cellular and humoral adaptive immunity. In this review, we describe the downstream signaling pathways initiated by CD40 and overview how CD40 engagement or antagonism modulates humoral and cellular immunity. Lastly, we discuss the role of CD40 as a target in harnessing anti-tumor immunity. This review underscores the essential role CD40 plays in adaptive immunity.

  2. 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.

  3. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

    Directory of Open Access Journals (Sweden)

    Aniuska Becerra-Artiles

    Full Text Available Most of humanity is chronically infected with human herpesvirus 6 (HHV-6, with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48 and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune

  4. The cell biology of T-dependent B cell activation

    DEFF Research Database (Denmark)

    Owens, T; Zeine, R

    1989-01-01

    The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

  5. B Cells Negatively Regulate the Establishment of CD49b(+)T-bet(+) Resting Memory T Helper Cells in the Bone Marrow.

    Science.gov (United States)

    Hojyo, Shintaro; Sarkander, Jana; Männe, Christian; Mursell, Mathias; Hanazawa, Asami; Zimmel, David; Zhu, Jinfang; Paul, William E; Fillatreau, Simon; Löhning, Max; Radbruch, Andreas; Tokoyoda, Koji

    2016-01-01

    During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs) to the bone marrow (BM) in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b(+)T-bet(+) antigen-experienced CD4 T cells in the spleen. CD49b(+)T-bet(+) antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b(+)T-bet(+) precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

  6. Interleukin 1-induced down-regulation of antibody binding to CD4 molecules on human lymphocytes

    DEFF Research Database (Denmark)

    Tvede, N; Christensen, L D; Ødum, Niels

    1988-01-01

    Interleukin 1 (IL-1) is involved in the early activation of T lymphocytes. The CD4 antigen, described as a phenotypic marker of helper T cells, is also important in early T-cell activation by its ability to bind to MHC class II molecules on antigen-presenting cells, and to transmit positive (and...

  7. In vitro propagation and dynamics of T cells from skin biopsies by methods using interleukins-2 and -4 or anti-CD3/CD28 antibody-coated microbeads

    DEFF Research Database (Denmark)

    Hashizume, Hideo; Hansen, Anker; Poulsen, Lars K

    2010-01-01

    In order to explore the mechanisms of inflammatory skin disorders, we established two methods of expanding skin-derived lymphocytes, one using high levels of interleukin (IL)-2 and IL-4 (method A) and the other using low levels of cytokines and anti-CD3/CD28 microbeads (method B). Both methods...... provide advantages for functional studies. With either of these two, we could obtain more than 10(7) cells/ from a 3 mm skin biopsy in 21 days from 23 out of 26 biopsies of various skin diseases. The relevance of these cells was confirmed by shifted T-cell receptor beta chain variable region (TCR......-Vbeta) repertoire and antigen-dependent proliferation in antigen-driven skin disorders. The propagation of skin-resident lymphocytes, seen especially in method A, seems to be mediated by a functional defect of regulatory T cells residing in skin sequentially expanding under the conditions of our methods....

  8. Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

    Science.gov (United States)

    Tsujimura, Kunio; Takahashi, Toshitada; Morita, Akimichi; Hasegawa-Nishiwaki, Hitomi; Iwase, Shigeru; Obata, Yuichi

    1996-01-01

    To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla a -3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from H-2K b/T3 b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tlaa-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8+ and 6 were CD4−CD8− double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8+ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of

  9. 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.

  10. Immunogenicity is unrelated to protective immunity when induced by soluble and particulate antigens from Nocardia brasiliensis in BALB/c mice.

    Science.gov (United States)

    Salinas-Carmona, Mario C; Ramos, Alma I; Pérez-Rivera, Isabel

    2006-08-01

    Cell-mediated immunity plays a major role in protection against intracellular microbes. Nocardia brasiliensis is a facultative intracellular pathogen that causes chronic actinomycetoma. In this work, we injected BALB/c mice with soluble P24 and particulate antigens from N. brasiliensis. A higher antibody titer and lymphocyte proliferation was induced by the particulate antigen than by the soluble antigen. However, five months after antigen injection, antibody concentration and lymphocyte proliferation were similar. An increase in CD45R and CD4 T cells was unrelated to protective immunity. Active immunization with soluble or particulate antigens induced complete protection during the primary immune response. This protective response was IgM mediated. The higher immunogenicity was not related to protective immunity since the particulate antigen induced protection similar to the soluble antigen. Using particulate antigens for vaccination guarantees a stronger immune response, local and systemic side effects, but not necessarily protection.

  11. Poly-functional and long-lasting anticancer immune response elicited by a safe attenuated Pseudomonas aeruginosa vector for antigens delivery

    Directory of Open Access Journals (Sweden)

    Xavier Chauchet

    2016-01-01

    Full Text Available Live-attenuated bacterial vectors for antigens delivery have aroused growing interest in the field of cancer immunotherapy. Their potency to stimulate innate immunity and to promote intracellular antigen delivery into antigen-presenting cells could be exploited to elicit a strong and specific cellular immune response against tumor cells. We previously described genetically-modified and attenuated Pseudomonas aeruginosa vectors able to deliver in vivo protein antigens into antigen-presenting cells, through Type 3 secretion system of the bacteria. Using this approach, we managed to protect immunized mice against aggressive B16 melanoma development in both a prophylactic and therapeutic setting. In this study, we further investigated the antigen-specific CD8+ T cell response, in terms of phenotypic and functional aspects, obtained after immunizations with a killed but metabolically active P. aeruginosa attenuated vector. We demonstrated that P. aeruginosa vaccine induces a highly functional pool of antigen-specific CD8+ T cell able to infiltrate the tumor. Furthermore, multiple immunizations allowed the development of a long-lasting immune response, represented by a pool of predominantly effector memory cells which protected mice against late tumor challenge. Overall, killed but metabolically active P. aeruginosa vector is a safe and promising approach for active and specific antitumor immunotherapy.

  12. Immunization against HTLV-I with chitosan and tri-methylchitosan nanoparticles loaded with recombinant env23 and env13 antigens of envelope protein gp46.

    Science.gov (United States)

    Amirnasr, Maryam; Fallah Tafti, Tannan; Sankian, Mojtaba; Rezaei, Abdorrahim; Tafaghodi, Mohsen

    2016-08-01

    To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P nanoparticles showed good immunoadjuvant potential. Env23 antigen was a better candidate for vaccination against HTLV-I, as it induced higher cellular immune responses, compared with env13. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Platelets Promote Metastasis via Binding Tumor CD97 Leading to Bidirectional Signaling that Coordinates Transendothelial Migration

    Directory of Open Access Journals (Sweden)

    Yvona Ward

    2018-04-01

    Full Text Available Summary: Tumor cells initiate platelet activation leading to the secretion of bioactive molecules, which promote metastasis. Platelet receptors on tumors have not been well-characterized, resulting in a critical gap in knowledge concerning platelet-promoted metastasis. We identify a direct interaction between platelets and tumor CD97 that stimulates rapid bidirectional signaling. CD97, an adhesion G protein-coupled receptor (GPCR, is an overexpressed tumor antigen in several cancer types. Purified CD97 extracellular domain or tumor cell-associated CD97 stimulated platelet activation. CD97-initiated platelet activation led to granule secretion, including the release of ATP, a mediator of endothelial junction disruption. Lysophosphatidic acid (LPA derived from platelets induced tumor invasiveness via proximal CD97-LPAR heterodimer signaling, coupling coincident tumor cell migration and vascular permeability to promote transendothelial migration. Consistent with this, CD97 was necessary for tumor cell-induced vascular permeability in vivo and metastasis formation in preclinical models. These findings support targeted blockade of tumor CD97 as an approach to ameliorate metastatic spread. : Tumor-initiated platelet activation promotes tissue invasion of cancer cells and metastasis. Ward et al. demonstrate that a common tumor-associated antigen, CD97, accounts for platelet activation and participates directly in LPA-mediated signal transduction leading to tumor cell invasion. CD97 promotes vascular extravasation and metastasis in pre-clinical models. Keywords: platelets, metastasis, transendothelial migration, circulating tumor cells, CD97, adhesion GPCR, LPA

  14. Humoral and cellular immune response in mice induced by the classical swine fever virus E2 protein fused to the porcine CD154 antigen.

    Science.gov (United States)

    Sordo, Yusmel; Suárez, Marisela; Caraballo, Rosalina; Sardina, Talía; Brown, Emma; Duarte, Carlos; Lugo, Joanna; Gil, Lázaro; Perez, Danny; Oliva, Ayme; Vargas, Milagros; Santana, Elaine; Valdés, Rodolfo; Rodríguez, María Pilar

    2018-03-01

    The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 μg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 μg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine. Copyright © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  15. T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

    Science.gov (United States)

    Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

    2011-05-01

    T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. The leukocyte common antigen (CD45) on human pre-B leukemia cells: variant glycoprotein form expression during the cell exposure to phorbol ester is blocked by a nonselective protein kinase inhibitor H7

    International Nuclear Information System (INIS)

    Duraj, J.; Sedlak, J.; Chorvath, B.; Rauko, P.

    1997-01-01

    The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted, CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI∼6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium meta-periodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor iso-quinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells. (author)

  17. CD1d-Restricted Type II NKT Cells Reactive With Endogenous Hydrophobic Peptides.

    Science.gov (United States)

    Nishioka, Yusuke; Masuda, Sakiko; Tomaru, Utano; Ishizu, Akihiro

    2018-01-01

    NKT cells belong to a distinct subset of T cells that recognize hydrophobic antigens presented by major histocompatibility complex class I-like molecules, such as CD1d. Because NKT cells stimulated by antigens can activate or suppress other immunocompetent cells through an immediate production of a large amount of cytokines, they are regarded as immunological modulators. CD1d-restricted NKT cells are classified into two subsets, namely, type I and type II. CD1d-restricted type I NKT cells express invariant T cell receptors (TCRs) and react with lipid antigens, including the marine sponge-derived glycolipid α-galactosylceramide. On the contrary, CD1d-restricted type II NKT cells recognize a wide variety of antigens, including glycolipids, phospholipids, and hydrophobic peptides, by their diverse TCRs. In this review, we focus particularly on CD1d-restricted type II NKT cells that recognize endogenous hydrophobic peptides presented by CD1d. Previous studies have demonstrated that CD1d-restricted type I NKT cells usually act as pro-inflammatory cells but sometimes behave as anti-inflammatory cells. It has been also demonstrated that CD1d-restricted type II NKT cells play opposite roles to CD1d-restricted type I NKT cells; thus, they function as anti-inflammatory or pro-inflammatory cells depending on the situation. In line with this, CD1d-restricted type II NKT cells that recognize type II collagen peptide have been demonstrated to act as anti-inflammatory cells in diverse inflammation-induction models in mice, whereas pro-inflammatory CD1d-restricted type II NKT cells reactive with sterol carrier protein 2 peptide have been demonstrated to be involved in the development of small vessel vasculitis in rats.

  18. Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34+ cells of juvenile myelomonocytic leukemia

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    Yozo Nakazawa

    2016-03-01

    Full Text Available Abstract Background Juvenile myelomonocytic leukemia (JMML is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF receptor (GMR, CD116 signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR for JMML. Methods We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7, and normal CD34+ cells. Results GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38− cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth. Conclusions Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.

  19. 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.

  20. Lysosomal-associated transmembrane protein 5 (LAPTM5 is a molecular partner of CD1e.

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    Catherine Angénieux

    Full Text Available The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

  1. An MHC II-Dependent Activation Loop between Adipose Tissue Macrophages and CD4+ T Cells Controls Obesity-Induced Inflammation

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    Kae Won Cho

    2014-10-01

    Full Text Available An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4+ T cells by unclear mechanisms. We have examined whether interactions between adipose tissue macrophages (ATMs and CD4+ T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen-dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHC II showed protection from diet-induced obesity. Deletion of MHC II expression in macrophages led to an adipose tissue-specific decrease in the effector/memory CD4+ T cells, attenuation of CD11c+ ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c+ ATMs in obese mice. These studies demonstrate the importance of MHCII-restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.

  2. Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

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    Emily E I M Mouser

    Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

  3. 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.

  4. 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

  5. B cells negatively regulate the establishment of CD49b+T-bet+ resting memory T helper cells in the bone marrow

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    Shintaro eHojyo

    2016-02-01

    Full Text Available During an immune reaction, some antigen-experienced CD4 T cells relocate from secondary lymphoid organs (SLOs to the bone marrow (BM in a CD49b-dependent manner and reside and rest there as professional memory CD4 T cells. However, it remains unclear how the precursors of BM memory CD4 T cells are generated in the SLOs. While several studies have so far shown that B cell depletion reduces the persistence of memory CD4 T cells in the spleen, we here show that B cell depletion enhances the establishment of memory CD4 T cells in the BM and that B cell transfer conversely suppresses it. Interestingly, the number of antigen-experienced CD4 T cells in the BM synchronizes the number of CD49b+T-bet+ antigen-experienced CD4 T cells in the spleen. CD49b+T-bet+ antigen-experienced CD4 T cells preferentially localize in the red pulp area of the spleen and the BM in a T-bet-independent manner. We suggest that B cells negatively control the generation of CD49b+T-bet+ precursors of resting memory CD4 T cells in the spleen and may play a role in bifurcation of activated effector and resting memory CD4 T cell lineages.

  6. CD28-CD80 interactions control regulatory T cell motility and immunological synapse formation1,2

    Science.gov (United States)

    Thauland, Timothy J.; Koguchi, Yoshinobu; Dustin, Michael L.; Parker, David C.

    2014-01-01

    Regulatory T cells (Tregs) are essential for tolerance to self and environmental antigens, acting in part by downmodulating costimulatory molecules on the surface of dendritic cells (DCs) and altering naïve CD4 T cell-DC interactions. Here, we show that Tregs form stable conjugates with DCs before, but not after, they decrease surface expression of the costimulatory molecule CD80 on the DCs. We use supported planar bilayers to show that Tregs dramatically slow down, but maintain a highly polarized and motile phenotype after recognizing antigen in the absence of costimulation. These motile cells are characterized by distinct accumulations of LFA-1-ICAM-1 in the lamella and TCR-MHC in the uropod, consistent with a motile immunological synapse or ‘kinapse’. However, in the presence of high, but not low, concentrations of CD80, Tregs form stationary, symmetrical synapses. Using blocking antibodies, we show that, while CTLA-4 is required for CD80 downmodulation, CD28-CD80 interactions are critical for modulating Treg motility in the presence of antigen. Together, these results support the hypothesis that Tregs are tuned to alter their motility depending on costimulatory signals. PMID:25355918

  7. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  8. Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells

    Science.gov (United States)

    Backer, Ronald; Schwandt, Timo; Greuter, Mascha; Oosting, Marije; Jüngerkes, Frank; Tüting, Thomas; Boon, Louis; O’Toole, Tom; Kraal, Georg; Limmer, Andreas; den Haan, Joke M. M.

    2009-01-01

    The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8+ DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8+ DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8+ T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity. PMID:20018690

  9. In vitro induction of functional allergen-specific CD4+ CD25high Treg cells in horses affected with insect bite hypersensitivity.

    Science.gov (United States)

    Hamza, E; Akdis, C A; Wagner, B; Steinbach, F; Marti, E

    2013-08-01

    Insect bite hypersensitivity (IBH) is a recurrent allergic dermatitis of horses with similarities to human atopic eczema, caused by bites of insects of the genus Culicoides. Previous studies suggested a dysregulated T cell tolerance to Culicoides allergen in IBH-affected horses. We have investigated whether the suppressive function of CD4(+) CD25(high) cells is impaired in IBH-affected horses and possible ways to restore it. CD4(+) CD25(-) cells sorted from peripheral blood mononuclear cells (PBMC) were stimulated with irradiated autologous PBMC pulsed with Culicoides or tetanus toxoid as control antigen, in the presence of CD4(+) CD25(high) cells. Furthermore, Culicoides-specific CD4(+) CD25(high) regulatory cells were expanded or induced from CD4(+) CD25(-) cells in vitro in the presence of a combination of rIL-2 and rTGF-β1 (rIL-2/rTGF-β1) or of retinoic acid and rapamycin (RetA/Rapa). Proliferation was determined by [(3) H] thymidine incorporation and cytokine production measured by flow cytometry. The ability of Culicoides- but not tetanus-stimulated CD4(+) CD25(high) cells to suppress proliferation of CD4(+) CD25(-) cells was significantly lower in IBH-affected horses (28%) than in healthy controls (86%). The decreased suppression in IBH-affected horses was associated with a significantly higher proportion of IL-4(+) cells and a lower percentage of FoxP3(+) IL-10(+) compared to controls. Addition of rIL-2/rTGF-β1 or of RetA/Rapa to Culicoides-stimulated CD4(+) CD25(high) cells from IBH-affected horses significantly increased the proportion of FoxP3(+) IL-10(+) cells. We also found that RetA/Rapa induced a more significant decrease in the frequency of IL-4(+) cells than rIL-2/rTGF-β1. Moreover, the suppressive activity of Culicoides-stimulated CD4(+) CD25(high) cells was significantly restored by both rIL-2/rTGF-β1and RetA/Rapa, albeit in an antigen-unspecific manner. In contrast, in vitro induced Culicoides-specific CD4(+) CD25(high) cells suppressed

  10. Evaluation of CA-125 and soluble CD-23 in patients with pelvic endometriosis: a case-control study Avaliação do CA-125 e CD-23 solúvel em pacientes com endometriose pélvica: estudo de caso-controle

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    Ivana Maria de Luna Ramos

    2012-02-01

    Full Text Available OBJECTIVE: To evaluate serum concentrations of CA-125 and soluble CD-23 and to correlate them with clinical symptoms, localization and stage of pelvic endometriosis and histological classification of the disease. METHODS: Blood samples were collected from 44 women with endometriosis and 58 without endometriosis, during the first three days (1st sample and during the 7th, 8th and 9th day (2nd sample of the menstrual cycle. Measurements of CA-125 and soluble CD-23 were performed by ELISA. Mann-Whitney U test was used for age, pain evaluations (visual analog scale and biomarkers concentrations. RESULTS: Serum levels of CA125 were higher in endometriosis patients when compared to the control group during both periods of the menstrual cycle evaluated in the study. This marker was also elevated in women with chronic pelvic pain, deep dyspareunia (2nd sample, dysmenorrhea (both samples and painful defecation during the menstrual flow (2nd sample. CA-125 concentration was higher in advanced stages of the disease in both samples and also in women with ovarian endometrioma. Concerning CD-23, no statistically significant differences were observed between groups. CONCLUSION: The concentrations of CA-125 were higher in patients with endometriosis than in patients without the disease. No significantly differences were observed for soluble CD-23 levels between groups.OBJETIVO: Avaliar as concentrações séricas de CA-125 e CD-23 solúvel e correlacioná-los com sintomas clínicos, localização e estádio da endometriose pélvica e classificação histológica da doença. MÉTODOS: Amostras de sangue foram coletadas de 44 mulheres com endometriose e 58 sem endometriose durante os primeiros três dias (1ª amostra e durante o sétimo, o oitavo e o nono dia (2ª amostra do ciclo menstrual. As dosagens de CA-125 e CD-23 solúvel foram realizadas por ELISA. O teste U de Mann-Whitney foi usado para idade, avaliação de dor (escala analógica visual e para a

  11. 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.

  12. Down regulation of the TCR complex CD3 ζ-chain on CD3+ T cells: a potential mechanism for helminth mediated immune modulation

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    Laura Jane Appleby

    2015-02-01

    Full Text Available The CD3ζ forms part of the T cell receptor (TCR where it plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways leading to T cell effector functions. Down regulation of CD3ζ leads to impairment of immune responses including reduced cell proliferation and cytokine production. In experimental models helminth parasites have been shown to modulate immune responses directed against them and unrelated antigens, so called bystander antigens, but there is a lack of studies validating these observations in humans. This study focused on investigated the relationship between expression levels of the TCR CD3ζ chain with lymphocyte cell proliferation during human infection with the helminth parasite, Schistosoma haematobium which causes uro-genital schistosomiasis. Using flow cytometry, peripheral blood mononuclear cells (PBMCs from individuals naturally exposed to S. haematobium in rural Zimbabwe were phenotyped, and expression levels of CD3ζ on T cells were related to intensity of infection. In this population, parasite infection intensity was inversely related to CD3ζ expression levels (p<0.05, consistent with down-regulation of CD3ζ expression during helminth infection. Furthermore, PBMC proliferation was positively related to expression levels of CD3ζ (p<0.05 after allowing for confounding variables (host age, sex, infection level. CD3ζ expression levels had a differing relationship between immune correlates of susceptibility and immunity, measured by antibody responses, indicating a complex relationship between immune activation status and immunity. The relationships between the CD3ζ chain of the TCR and schistosome infection, PBMC proliferation and schistosome-specific antibody responses have not previously been reported, and these results may indicate a mechanism for the impaired T cell proliferative responses observed during human schistosome infection.

  13. Percentages of CD4+CD161+ and CD4−CD8−CD161+ T Cells in the Synovial Fluid Are Correlated with Disease Activity in Rheumatoid Arthritis

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    Jinlin Miao

    2015-01-01

    Full Text Available Objective. CD161 has been identified as a marker of human IL-17-producing T cells that are implicated in the pathogenesis of rheumatoid arthritis (RA. This study aimed to investigate the potential link between the percentage of CD161+ T cells and disease activity in RA patients. Methods. Peripheral blood (PB from 54 RA patients and 21 healthy controls was evaluated. Paired synovial fluid (SF (n = 17 was analyzed. CD161 expression levels on CD4+, CD8+, and CD4−CD8− T cells were assessed by flow cytometry. Results. The percentage of CD4+CD161+ T cells in RA SF was higher than RA PB, and it was positively correlated with DAS28, erythrocyte sedimentation rate (ESR, and C-reactive protein (CRP. CD4−CD8−CD161+ T cell percentage was decreased in RA PB and was further reduced in RA SF, and its level in SF was inversely correlated with DAS28, ESR, and CRP. However, CD8+CD161+ T cell percentage was neither changed in RA PB and SF nor correlated with disease activity indices. Conclusion. An increased CD4+CD161+ T cell percentage and a decreased CD4−CD8−CD161+ T cell percentage are present in RA SF and are associated with disease activity, and the accumulation of CD4+CD161+ T cells in SF may contribute to the local inflammation of RA.

  14. Expression of alpha V integrin is modulated by Epstein-Barr virus nuclear antigen 3C and the metastasis suppressor Nm23-H1 through interaction with the GATA-1 and Sp1 transcription factors

    International Nuclear Information System (INIS)

    Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke; Robertson, Erle S.

    2006-01-01

    Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C

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

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    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

  16. Active self-healing encapsulation of vaccine antigens in PLGA microspheres

    Science.gov (United States)

    Desai, Kashappa-Goud H.; Schwendeman, Steven P.

    2013-01-01

    Herein, we describe the detailed development of a simple and effective method to microencapsulate vaccine antigens in poly(lactic-co-glycolic acid) (PLGA) by simple mixing of preformed active self-microencapsulating (SM) PLGA microspheres in a low concentration aqueous antigen solution at modest temperature (10-38 °C). Co-encapsulating protein-sorbing vaccine adjuvants and polymer plasticizers were used to “actively” load the protein in the polymer pores and facilitate polymer self-healing at temperature > hydrated polymer glass transition temperature, respectively. The microsphere formulation parameters and loading conditions to provide optimal active self-healing microencapsulation of vaccine antigen in PLGA was investigated. Active self-healing encapsulation of two vaccine antigens, ovalbumin and tetanus toxoid (TT), in PLGA microspheres was adjusted by preparing blank microspheres containing different vaccine adjuvant (aluminum hydroxide (Al(OH)3) or calcium phosphate). Active loading of vaccine antigen in Al(OH)3-PLGA microspheres was found to: a) increase proportionally with an increasing loading of Al(OH)3 (0.88-3 wt%) and addition of porosigen, b) decrease when the inner Al(OH)3/trehalose phase to 1 mL outer oil phase and size of microspheres was respectively > 0.2 mL and 63 μm, and c) change negligibly by PLGA concentration and initial incubation (loading) temperature. Encapsulation of protein sorbing Al(OH)3 in PLGA microspheres resulted in suppression of self-healing of PLGA pores, which was then overcome by improving polymer chain mobility, which in turn was accomplished by coincorporating hydrophobic plasticizers in PLGA. Active self-healing microencapsulation of manufacturing process-labile TT in PLGA was found to: a) obviate micronization- and organic solvent-induced TT degradation, b) improve antigen loading (1.4-1.8 wt% TT) and encapsulation efficiency (~ 97%), c) provide nearly homogeneous distribution and stabilization of antigen in polymer

  17. Detection of circulating tumor lysate-reactive CD4+ T cells in melanoma patients

    DEFF Research Database (Denmark)

    Ladekarl, Morten; Agger, Ralf; Fleischer, Charlotte C

    2004-01-01

    PURPOSE: We wanted to study whether an allogeneic melanoma lysate would be a feasible stimulatory antigen source for detection of a peripheral CD4+ T-cell immune response in patients with medically untreated malignant melanoma. The lysate was produced from a melanoma cell line (FM3.29) which...... was found in 1 of 4 patients radically operated for localized disease, whereas no responders were seen among 7 healthy donors. The fraction of circulating lysate-activated T cells ranged from 0.0037% to 0.080% of the CD4+ population. A negative result of the assay was found occasionally, especially...... expresses high amounts of melanoma antigens. METHODS: Fresh peripheral blood was incubated with and without lysate for 6 h in the presence of anti-CD28/anti-CD49d MoAb (for costimulation). After flow cytometric estimation of the frequency of CD69+/IFN-gamma+ cells in the CD4+ population, the response...

  18. Control of CD56 expression and tumor cell cytotoxicity in human Vγ2Vδ2 T cells

    Directory of Open Access Journals (Sweden)

    Focaccetti Chiara

    2009-09-01

    Full Text Available Abstract Background In lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1 correlates with cytotoxic effector activity. For cells bearing the Vγ2Vδ2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and proliferation, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. Our goal was to show whether CD56 expression was regulated stochastically, similar to conventional activation antigens, or whether CD56 defined a lineage of cells committed to the cytotoxic phenotype. Results Tracking individual cell clones defined by their Vγ2 chain CDR3 region sequences, we found that CD56 was expressed on precursor cytotoxic T cells already present in the population irrespective of their capacity to proliferate after antigen stimulation. Public T cell receptor sequences found in the CD56+ subset from one individual might appear in the CD56- subset of another donor. The commitment of individual clones to CD56+ or CD56- lineages was stable for each donor over a 1 year interval. Conclusion The ability to express CD56 was not predicted by TCR sequence or by the strength of signal received by the TCR. For γδ T cells, cytotoxic effector function is acquired when cytotoxic precursors within the population are stimulated to proliferate and express CD56. Expression of CD56 defines a committed lineage to the cytotoxic phenotype.

  19. Indoleamine 2,3-dioxygenase-expressing leukemic dendritic cells impair a leukemia-specific immune response by inducing potent T regulatory cells.

    Science.gov (United States)

    Curti, Antonio; Trabanelli, Sara; Onofri, Chiara; Aluigi, Michela; Salvestrini, Valentina; Ocadlikova, Darina; Evangelisti, Cecilia; Rutella, Sergio; De Cristofaro, Raimondo; Ottaviani, Emanuela; Baccarani, Michele; Lemoli, Roberto M

    2010-12-01

    The immunoregulatory enzyme indoleamine 2,3-dioxygenase, which catalyzes the conversion of tryptophan into kynurenine, is expressed in a significant subset of patients with acute myeloid leukemia, resulting in the inhibition of T-cell proliferation and the induction of regulatory T cells. Acute myeloid leukemia cells can be differentiated into dendritic cells, which have increased immunogenicity and have been proposed as vaccines against leukemia. Leukemic dendritic cells were generated from acute myeloid leukemia cells and used as stimulators in functional assays, including the induction of regulatory T cells. Indoleamine 2,3-dioxygenase expression in leukemic dendritic cells was evaluated at molecular, protein and enzymatic levels. We demonstrate that, after differentiation into dendritic cells, both indoleamine 2,3-dioxygenase-negative and indoleamine 2,3-dioxygenase-positive acute myeloid leukemia samples show induction and up-regulation of indoleamine 2,3-dioxygenase gene and protein, respectively. Indoleamine 2,3-dioxygenase-positive acute myeloid leukemia dendritic cells catabolize tryptophan into kynurenine metabolite and inhibit T-cell proliferation through an indoleamine 2,3-dioxygenase-dependent mechanism. Moreover, indoleamine 2,3-dioxygenase-positive leukemic dendritic cells increase the number of allogeneic and autologous CD4(+)CD25(+) Foxp3(+) T cells and this effect is completely abrogated by the indoleamine 2,3-dioxygenase-inhibitor, 1-methyl tryptophan. Purified CD4(+)CD25(+) T cells obtained from co-culture with indoleamine 2,3-dioxygenase-positive leukemic dendritic cells act as regulatory T cells as they inhibit naive T-cell proliferation and impair the complete maturation of normal dendritic cells. Importantly, leukemic dendritic cell-induced regulatory T cells are capable of in vitro suppression of a leukemia-specific T cell-mediated immune response, directed against the leukemia-associated antigen, Wilms' tumor protein. These data identify

  20. Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.

    Science.gov (United States)

    Tremblay-McLean, Alexandra; Bruneau, Julie; Lebouché, Bertrand; Lisovsky, Irene; Song, Rujun; Bernard, Nicole F

    2017-10-12

    Natural Killer (NK) cell responses to HIV-infected CD4 T cells (iCD4) depend on the integration of signals received through inhibitory (iNKR) and activating NK receptors (aNKR). iCD4 activate NK cells to inhibit HIV replication. HIV infection-dependent changes in the human leukocyte antigen (HLA) ligands for iNKR on iCD4 are well documented. By contrast, less is known regarding the HIV infection related changes in ligands for aNKR on iCD4. We examined the aNKR ligand profiles HIV p24⁺ HIV iCD4s that maintained cell surface CD4 (iCD4⁺), did not maintain CD4 (iCD4 - ) and uninfected CD4 (unCD4) T cells for expression of unique long (UL)-16 binding proteins-1 (ULBP-1), ULBP-2/5/6, ULBP-3, major histocompatibility complex (MHC) class 1-related (MIC)-A, MIC-B, CD48, CD80, CD86, CD112, CD155, Intercellular adhesion molecule (ICAM)-1, ICAM-2, HLA-E, HLA-F, HLA-A2, HLA-C, and the ligands to NKp30, NKp44, NKp46, and killer immunoglobulin-like receptor 3DS1 (KIR3DS1) by flow cytometry on CD4 T cells from 17 HIV-1 seronegative donors activated and infected with HIV. iCD4⁺ cells had higher expression of aNKR ligands than did unCD4. However, the expression of aNKR ligands on iCD4 where CD4 was downregulated (iCD4 - ) was similar to (ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80, CD86 and CD155) or significantly lower than (MIC-B, CD112 and ICAM-2) what was observed on unCD4. Thus, HIV infection can be associated with increased expression of aNKR ligands or either baseline or lower than baseline levels of aNKR ligands, concomitantly with the HIV-mediated downregulation of cell surface CD4 on infected cells.

  1. Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes

    International Nuclear Information System (INIS)

    Ozsahin, Mahmut; Ozsahin, Huelya; Yuquan, Shi; Larsson, Boerje; Wuergler, Friedrich E.; Crompton, Nigel E. A.

    1997-01-01

    Purpose: An assay for radiosensitivity has numerous applications in the clinic. Avoidance of acute responses, prediction of normal tissue toxicity, and individualization of patient radiotherapy are included among these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radiation-induced apoptosis. Methods and Materials: Fresh blood samples (1-2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8-Gy X rays at a dose rate of approximately 3 Gy/min. Following irradiation, the cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available with the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte types were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of these cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor variation in a cohort of 45 healthy adult blood donors and 5 children [one had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor variation was assessed with 10 different experiments from a single donor. Results: CD4 and CD8 T-lymphocyte radiosensitivities were correlated (r 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r 0.77 for both). Interdonor variation was significantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen

  2. 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

  3. Scaffold protein JLP mediates TCR-initiated CD4+T cell activation and CD154 expression.

    Science.gov (United States)

    Yan, Qi; Yang, Cheng; Fu, Qiang; Chen, Zhaowei; Liu, Shan; Fu, Dou; Rahman, Rahmat N; Nakazato, Ryota; Yoshioka, Katsuji; Kung, Sam K P; Ding, Guohua; Wang, Huiming

    2017-07-01

    CD4 + T-cell activation and its subsequent induction of CD154 (CD40 ligand, CD40L) expression are pivotal in shaping both the humoral and cellular immune responses. Scaffold protein JLP regulates signal transduction pathways and molecular trafficking inside cells, thus represents a critical component in maintaining cellular functions. Its role in regulating CD4 + T-cell activation and CD154 expression, however, is unclear. Here, we demonstrated expression of JLP in mouse tissues of lymph nodes, thymus, spleen, and also CD4 + T cells. Using CD4+ T cells from jlp-deficient and jlp-wild-type mice, we demonstrated that JLP-deficiency impaired T-cell proliferation, IL-2 production, and CD154 induction upon TCR stimulations, but had no impacts on the expression of other surface molecules such as CD25, CD69, and TCR. These observed impaired T-cell functions in the jlp-/- CD4 + T cells were associated with defective NF-AT activation and Ca 2 + influx, but not the MAPK, NF-κB, as well as AP-1 signaling pathways. Our findings indicated that, for the first time, JLP plays a critical role in regulating CD4 + T cells response to TCR stimulation partly by mediating the activation of TCR-initiated Ca 2+ /NF-AT. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Memory phenotype CD4 T cells undergoing rapid, nonburst-like, cytokine-driven proliferation can be distinguished from antigen-experienced memory cells.

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    Souheil-Antoine Younes

    2011-10-01

    Full Text Available Memory phenotype (CD44(bright, CD25(negative CD4 spleen and lymph node T cells (MP cells proliferate rapidly in normal or germ-free donors, with BrdU uptake rates of 6% to 10% per day and Ki-67 positivity of 18% to 35%. The rapid proliferation of MP cells stands in contrast to the much slower proliferation of lymphocytic choriomeningitis virus (LCMV-specific memory cells that divide at rates ranging from <1% to 2% per day over the period from 15 to 60 days after LCMV infection. Anti-MHC class II antibodies fail to inhibit the in situ proliferation of MP cells, implying a non-T-cell receptor (TCR-driven proliferation. Such proliferation is partially inhibited by anti-IL-7Rα antibody. The sequence diversity of TCRβ CDR3 gene segments is comparable among the proliferating and quiescent MP cells from conventional and germ-free mice, implying that the majority of proliferating MP cells have not recently derived from a small cohort of cells that expand through multiple continuous rounds of cell division. We propose that MP cells constitute a diverse cell population, containing a subpopulation of slowly dividing authentic antigen-primed memory cells and a majority population of rapidly proliferating cells that did not arise from naïve cells through conventional antigen-driven clonal expansion.

  5. CD4~+Foxp3~+ regulatory T cells converted by rapamycin from peripheral CD4~+ CD25~-naive T cells display more potent regulatory ability in vitro

    Institute of Scientific and Technical Information of China (English)

    CHEN Jian-fei; GAO Jie; ZHANG Dong; WANG Zi-han; ZHU Ji-ye

    2010-01-01

    Background Rapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells. Methods Peripheral CD4~+CD25~- naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4~+CD25~- T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4~+CD25~+CD127~- subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4~+CD25~+ T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1×10~5 carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4~+CD25~- T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25×10~5/well) in 96-well round-bottom plates for 6 days. Then 1×10~5 or 0.25×10~5 converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4~+CD25~- T cells using flow cytometry. Data were analyzed using CellQuest software.Results We found that RAPA can convert peripheral CD4~+CD25~- naive T Cells to CD4~+Foxp3~+ Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity. Conclusion Our findings provide evidence that RAPA induces Treg cell conversion from Teff cells and

  6. Memory CD8+ T Cells: Orchestrators and Key Players of Innate Immunity?

    Directory of Open Access Journals (Sweden)

    Grégoire Lauvau

    2016-09-01

    Full Text Available Over the past decades, the dichotomy between innate and adaptive immune responses has largely dominated our understanding of immunology. Upon primary encounter with microbial pathogens, differentiation of adaptive immune cells into functional effectors usually takes several days or even longer, making them contribute to host protection only late during primary infection. However, once generated, antigen-experienced T lymphocytes can persist in the organism and constitute a pool of memory cells that mediate fast and effective protection to a recall infection with the same microbial pathogen. Herein, we challenge this classical paradigm by highlighting the "innate nature" of memory CD8+ T cells. First, within the thymus or in the periphery, naïve CD8+ T cells may acquire phenotypic and functional characteristics of memory CD8+ T cells independently of challenge with foreign antigens. Second, both the "unconventional" and the "conventional" memory cells can rapidly express protective effector functions in response to sets of inflammatory cytokines and chemokines signals, independent of cognate antigen triggering. Third, memory CD8+ T cells can act by orchestrating the recruitment, activation, and licensing of innate cells, leading to broad antimicrobial states. Thus, collectively, memory CD8+ T cells may represent important actors of innate immune defenses.

  7. The differentiation and protective function of cytolytic CD4 T cells in influenza infection

    Science.gov (United States)

    CD4 T cells that recognize peptide antigen in the context of Class II MHC can differentiate into various subsets that are characterized by their helper functions. However, increasing evidence indicates that CD4 cells with direct cytolytic activity play a role in chronic, as well as, acute infections...

  8. CD1d expression and invariant NKT cell responses in herpesvirus infections

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    Rusung eTan

    2015-06-01

    Full Text Available Invariant natural killer T (iNKT cells are a highly conserved subset of unconventional T lymphocytes that express a canonical, semi-invariant T cell receptor (TCR and surface markers shared with the natural killer cell lineage. iNKT cells recognize exogenous and endogenous glycolipid antigens restricted by non-polymorphic CD1d molecules, and are highly responsive to the prototypical agonist, α-galactosylceramide. Upon activation, iNKT cells rapidly coordinate signaling between innate and adaptive immune cells through the secretion of proinflammatory cytokines, leading to the maturation of antigen-presenting cells and expansion of antigen-specific CD4+ and CD8+ T cells. Because of their potent immunoregulatory properties, iNKT cells have been extensively studied and are known to play a pivotal role in mediating immune responses against microbial pathogens including viruses. Here, we review evidence that herpesviruses manipulate CD1d expression to escape iNKT cell surveillance and establish lifelong latency in humans. Collectively, published findings suggest that iNKT cells play critical roles in anti-herpesvirus immune responses and could be harnessed therapeutically to limit viral infection and viral-associated disease.

  9. Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

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    Markus Haug

    2018-04-01

    Full Text Available Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses.

  10. Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions.

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    Paola Lorena Smaldini

    Full Text Available The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-β were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-β-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-β-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

  11. CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

    Science.gov (United States)

    Marusina, Alina I; Ono, Yoko; Merleev, Alexander A; Shimoda, Michiko; Ogawa, Hiromi; Wang, Elizabeth A; Kondo, Kayo; Olney, Laura; Luxardi, Guillaume; Miyamura, Yoshinori; Yilma, Tilahun D; Villalobos, Itzel Bustos; Bergstrom, Jennifer W; Kronenberg, Daniel G; Soulika, Athena M; Adamopoulos, Iannis E; Maverakis, Emanual

    2017-02-01

    It is widely accepted that central and effector memory CD4 + T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4 + T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4 + T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4 + T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4 + T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4 + T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones. Published by

  12. 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/

  13. Metformin inhibits proliferation and cytotoxicity and induces apoptosis via AMPK pathway in CD19-chimeric antigen receptor-modified T cells

    Directory of Open Access Journals (Sweden)

    Mu Q

    2018-04-01

    Full Text Available Qian Mu,1,2,* Miao Jiang,1,* Yuzhu Zhang,1 Fei Wu,1 Hui Li,1 Wen Zhang,1 Fang Wang,1 Jiang Liu,1 Liang Li,1 Dongshan Wang,3 Wenjuan Wang,1 Shiwu Li,1 Haibo Song,4 Dongqi Tang1 1Gene and Immunotherapy Center, The Second Hospital of Shandong University, Jinan, People’s Republic of China; 2Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3Health Management Center, The Second Hospital of Shandong University, Jinan, People’s Republic of China; 4Central Research Laboratory, Zibo Maternal and Child Health Hospital, Affiliated to Shandong Academy of Medical Science, Zibo, People’s Republic of China *These authors contributed equally to this work Background: CD19-chimericantigen receptor (CAR modified T cells (CD19-CAR T cells have been well documented to possess potent anti-tumor properties against CD19-expressingleukemia cells. As a traditional medicine, metformin has been widely used to treat type II diabetes mellitus and more recently has become a candidate for the treatment of cancer. However, no report has revealed the direct effect of metformin on CD19-CAR T cell biological function and its underling mechanisms. Purpose: The purpose of this research was to explore the effect of metformin on CD19-CAR T cell biological function and the mechanisms involved. Methods: CD19-CAR T cells proliferation, apoptosis and cytotoxicity were mainly tested by CCK-8 assay, flow cytometry and ELISA. The detection of mechanism primarily used western blot. Bioluminescence imaging is the main application technology of animal studies. Results: In the current study, it was found that metformin inhibited CD19-CAR T cell proliferation and cytotoxicity and induced apoptosis. Furthermore, our study revealed that metformin activated AMPK and suppressed mTOR and HIF1α expression. By using an AMPK inhibitor, compound C, we demonstrated the crucial roles of AMPK in CD19

  14. 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.

  15. 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.

  16. Reactivity of naive CD4+CD25- T cells against gut microflora in healthy mice

    DEFF Research Database (Denmark)

    Gad, Monika; Lundsgaard, Dorthe; Kjellev, Stine

    2006-01-01

    We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under...

  17. 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

  18. Selective enrichment of Th1 CD45RBlow CD4+ T cells in autoimmune infiltrates in experimental allergic encephalomyelitis

    DEFF Research Database (Denmark)

    Renno, T; Zeine, R; Girard, J M

    1994-01-01

    The cytokine effector status of CD4+ T cells from lymph nodes (LN) and the central nervous system (CNS) of SJL/J mice immunized with autoantigen in adjuvant for the induction of experimental allergic encephalomyelitis (EAE) was compared. CD4+ T cells were FACS sorted based on the levels...... in the sorted populations. CD45RBlow cells constituted a minority of CD4+ cells in the LN and expressed elevated levels of IL-2, IFN-gamma, and IL-4 mRNA, whereas the CD45RBlow CD4+ population did not express detectable message for these cytokines under linear PCR conditions. By contrast to the LN, CD4+ cells...... of expression of the activation marker CD45RB. Low levels of expression of this surface marker are induced by antigen recognition and are associated with 'effector' T cell function. Reverse transcriptase polymerase chain reaction (PCR) was used to analyze the expression of different T cell cytokine genes...

  19. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells.

    Science.gov (United States)

    Huang, Yu-Hsuan; Tsai, Kevin; Tan, Sara Y; Kang, Sohyeong; Ford, Mandy L; Harder, Kenneth W; Priatel, John J

    2017-01-01

    Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8 + T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a - / - CD8 + T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a - / - CD8 + T cells responded equivalently to wild-type CD8 + T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8 + T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8 + T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8 + T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

  20. 2B4-SAP signaling is required for the priming of naive CD8+ T cells by antigen-expressing B cells and B lymphoma cells

    Science.gov (United States)

    2017-01-01

    ABSTRACT Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein–Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8+ T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a−/− CD8+ T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a−/− CD8+ T cells responded equivalently to wild-type CD8+ T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8+ T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8+ T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8+ T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas. PMID:28344876

  1. Neem leaf glycoprotein promotes dual generation of central and effector memory CD8(+) T cells against sarcoma antigen vaccine to induce protective anti-tumor immunity.

    Science.gov (United States)

    Ghosh, Sarbari; Sarkar, Madhurima; Ghosh, Tithi; Guha, Ipsita; Bhuniya, Avishek; Saha, Akata; Dasgupta, Shayani; Barik, Subhasis; Bose, Anamika; Baral, Rathindranath

    2016-03-01

    We have previously shown that Neem Leaf Glycoprotein (NLGP) mediates sustained tumor protection by activating host immune response. Now we report that adjuvant help from NLGP predominantly generates CD44(+)CD62L(high)CCR7(high) central memory (TCM; in lymph node) and CD44(+)CD62L(low)CCR7(low) effector memory (TEM; in spleen) CD8(+) T cells of Swiss mice after vaccination with sarcoma antigen (SarAg). Generated TCM and TEM participated either to replenish memory cell pool for sustained disease free states or in rapid tumor eradication respectively. TCM generated after SarAg+NLGP vaccination underwent significant proliferation and IL-2 secretion following SarAg re-stimulation. Furthermore, SarAg+NLGP vaccination helps in greater survival of the memory precursor effector cells at the peak of the effector response and their maintenance as mature memory cells, in comparison to single modality treatment. Such response is corroborated with the reduced phosphorylation of FOXO in the cytosol and increased KLF2 in the nucleus associated with enhanced CD62L, CCR7 expression of lymph node-resident CD8(+) T cells. However, spleen-resident CD8(+) T memory cells show superior efficacy for immediate memory-to-effector cell conversion. The data support in all aspects that SarAg+NLGP demonstrate superiority than SarAg vaccination alone that benefits the host by rapid effector functions whenever required, whereas, central-memory cells are thought to replenish the memory cell pool for ultimate sustained disease free survival till 60 days following post-vaccination tumor inoculation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The CD8+ memory T-cell state of readiness is actively maintained and reversible

    OpenAIRE

    Allam, Atef; Conze, Dietrich B.; Giardino Torchia, Maria Letizia; Munitic, Ivana; Yagita, Hideo; Sowell, Ryan T.; Marzo, Amanda L.; Ashwell, Jonathan D.

    2009-01-01

    The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G1 to the G0 cell-cy...

  3. Characterization of the antigen-specific CD4+ T cell response induced by prime-boost strategies with CAF01 and CpG adjuvants administered by the intranasal and subcutaneous routes

    Directory of Open Access Journals (Sweden)

    Annalisa eCiabattini

    2015-08-01

    Full Text Available The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1821 adjuvants, administered by the parenteral and nasal routes. By using peptide-MHC class II tetramers, antigen-specific CD4+ T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4+ tetramer-positive T cells in the spleen, however only parenterally primed cells responded to booster immunization. Subcutaneous priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4+ tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4+ T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that subcutaneous priming is fundamental for eliciting CD4+ T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.

  4. 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.

  5. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    Energy Technology Data Exchange (ETDEWEB)

    Ohnuma, Kei [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Uchiyama, Masahiko [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Computational Intelligence and System Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503 (Japan); Hatano, Ryo; Takasawa, Wataru; Endo, Yuko [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Dang, Nam H. [Department of Hematologic Malignancies, Nevada Cancer Institute, Las Vegas, NV 89135 (United States); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  6. 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...

  7. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

    Directory of Open Access Journals (Sweden)

    Tim J Schuijt

    2011-01-01

    Full Text Available Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

  8. [Evaluation of percentage of lymphocytes B with expression of co-receptors CD 40, CD22 and CD72 in hypertrophied adenoid at children with otitis media with effusion].

    Science.gov (United States)

    Wysocka, Jolanta; Zelazowska-Rutkowska, Beata; Ratomski, Karol; Skotnicka, Bozena; Hassmann-Poznańska, Elzbieta

    2009-01-01

    In hypertrophied adenoid lymphocytes B make up about 60% all lymphocytes. When the lymphocytes B come in interaction with antigens this membranes signal be passed through their receptor (BCR) to interior of cell. This signal affect modulation on gene expression, activation from which depends activation, anergy or apoptosis of lymphocyte B. Accompany BCR co-receptors regulate his functions influence stimulate or inhibitive. To the most important co-receptors stepping out on lymphocyte B belong: CD40, CD22, CD72. The aim of study was evaluation of lymphocytes B (CD19) with co-expression with CD72 and CD40 receptors in hypertrophied adenoid with at children with otitis media with effusion. An investigation was executed in hypertrophied adenoids with or without otitis media with effusion. By flow cytometry percentage of lymphocytes B with co-receptors CD 40, CD22 and CD72 in was analyzed. The percentages of CD19+CD72+ lymphocytes in the group of children with adenoid hypertrophy and exudative otitis media were lower as compared to the reference group. However, the percentages of CD19+CD22+, CD19+CD40+ in the study group was approximate to the reference group. The lower percentage of lymphocytes B CD72 + near approximate percentages of lymphocytes B CD40+ and BCD22+ at children with otitis media with effusion can be the cause of incorrect humoral response in hypertrophied adenoid at children. Maybe it is cause reduced spontaneous production IgA and IgG through lymphocyte at children with otitis media with effusion.

  9. Regulatory function of cytomegalovirus-specific CD4+CD27-CD28- T cells

    International Nuclear Information System (INIS)

    Tovar-Salazar, Adriana; Patterson-Bartlett, Julie; Jesser, Renee; Weinberg, Adriana

    2010-01-01

    CMV infection is characterized by high of frequencies of CD27 - CD28 - T cells. Here we demonstrate that CMV-specific CD4 + CD27 - CD28 - cells are regulatory T cells (T R ). CD4 + CD27 - CD28 - cells sorted from CMV-stimulated PBMC of CMV-seropositive donors inhibited de novo CMV-specific proliferation of autologous PBMC in a dose-dependent fashion. Compared with the entire CMV-stimulated CD4 + T-cell population, higher proportions of CD4 + CD27 - CD28 - T R expressed FoxP3, TGFβ, granzyme B, perforin, GITR and PD-1, lower proportions expressed CD127 and PD1-L and similar proportions expressed CD25, CTLA4, Fas-L and GITR-L. CMV-CD4 + CD27 - CD28 - T R expanded in response to IL-2, but not to CMV antigenic restimulation. The anti-proliferative effect of CMV-CD4 + CD27 - CD28 - T R significantly decreased after granzyme B or TGFβ inhibition. The CMV-CD4 + CD27 - CD28 - T R of HIV-infected and uninfected donors had similar phenotypes and anti-proliferative potency, but HIV-infected individuals had higher proportions of CMV-CD4 + CD27 - CD28 - T R . The CMV-CD4 + CD27 - CD28 - T R may contribute to the downregulation of CMV-specific and nonspecific immune responses of CMV-infected individuals.

  10. Effects of dendritic cell vaccine activated with protein components of toxoplasma gondii on tumor specific CD8+ T-cells

    Directory of Open Access Journals (Sweden)

    Amari A

    2009-12-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Dendritic Cell (DC is an important antigen-presenting cell that present tumor antigen to CD8+ and CD4+ T- Lymphocytes and induce specific anti-tumor immunity. In order to induce effective anti-tumor response, an option is increasing the efficiency of antigen presentation of dendritic cells and T cell activation capacity. The aim of the present study was to investigate the effect of dendritic cell maturation with protein components of toxoplasma gondii on cytotoxic T lymphocyte activity and their infiltration in to the tumor."n"nMethods: For DC generation, bone marrow cells were cultured in the presence of GM-CSF and IL-4 for five days. After that, LPS, protein components and whole extract of toxoplasma gondii were added to the culture media and incubated for another two days for DC maturation. To generate tumor, mices were injected subcutaneously with WEHI-164 cell line. For immunotherapy 106 DCs matured with different compounds were injected around the tumor site. Infiltration of CD8+ T cells were determined by flow cytometry and cytotoxic activity was measured by LDH detection kit."n"nResults: Immunotherapy with DCs treated with protein components of toxoplasma gondii led to a significant increase in the

  11. 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.

  12. 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

  13. Food supplement 20070721-GX may increase CD34+ stem cells and telomerase activity.

    Science.gov (United States)

    Lin, Po-Cheng; Chiou, Tzyy-Wen; Liu, Po-Yen; Chen, Shee-Ping; Wang, Hsin-I; Huang, Pi-Chun; Lin, Shinn-Zong; Harn, Horng-Jyh

    2012-01-01

    Few rejuvenation and antiaging markers are used to evaluate food supplements. We measured three markers in peripheral blood to evaluate the antiaging effects of a food supplement containing placental extract. Samples were evaluated for CD34(+) cells, insulin-like growth factor 1 (IGF1), and telomerase activity, which are all markers related to aging. To control the quality of this food supplement, five active components were monitored. In total, we examined 44 individuals who took the food supplement from 1.2 months to 23 months; the average number of CD34(+) cells was almost 6-fold higher in the experimental group compared with the control group. Food supplement intake did not change serum IGF1 levels significantly. Finally, the average telomerase activity was 30% higher in the subjects taking this food supplement. In summary, our results suggest that the placental extract in the food supplement might contribute to rejuvenation and antiaging.

  14. How, with whom and when: an overview of CD147-mediated regulatory networks influencing matrix metalloproteinase activity

    Science.gov (United States)

    Grass, G. Daniel; Toole, Bryan P.

    2015-01-01

    Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent enzymes involved in various pathologic and physiologic processes. In cancer, MMPs contribute to processes from tumour initiation to establishment of distant metastases. Complex signalling and protein transport networks regulate MMP synthesis, cell surface presentation and release. Earlier attempts to disrupt MMP activity in patients have proven to be intolerable and with underwhelming clinical efficacy; thus targeting ancillary proteins that regulate MMP activity may be a useful therapeutic approach. Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally characterized as a factor present on lung cancer cells, which stimulated collagenase (MMP-1) production in fibroblasts. Subsequent studies demonstrated that EMMPRIN was identical with several other protein factors, including basigin (Bsg), all of which are now commonly termed CD147. CD147 modulates the synthesis and activity of soluble and membrane-bound [membrane-type MMPs (MT-MMPs)] in various contexts via homophilic/heterophilic cell interactions, vesicular shedding or cell-autonomous processes. CD147 also participates in inflammation, nutrient and drug transporter activity, microbial pathology and developmental processes. Despite the hundreds of manuscripts demonstrating CD147-mediated MMP regulation, the molecular underpinnings governing this process have not been fully elucidated. The present review summarizes our present knowledge of the complex regulatory systems influencing CD147 biology and provides a framework to understand how CD147 may influence MMP activity. PMID:26604323

  15. How, with whom and when: an overview of CD147-mediated regulatory networks influencing matrix metalloproteinase activity.

    Science.gov (United States)

    Grass, G Daniel; Toole, Bryan P

    2015-11-24

    Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent enzymes involved in various pathologic and physiologic processes. In cancer, MMPs contribute to processes from tumour initiation to establishment of distant metastases. Complex signalling and protein transport networks regulate MMP synthesis, cell surface presentation and release. Earlier attempts to disrupt MMP activity in patients have proven to be intolerable and with underwhelming clinical efficacy; thus targeting ancillary proteins that regulate MMP activity may be a useful therapeutic approach. Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally characterized as a factor present on lung cancer cells, which stimulated collagenase (MMP-1) production in fibroblasts. Subsequent studies demonstrated that EMMPRIN was identical with several other protein factors, including basigin (Bsg), all of which are now commonly termed CD147. CD147 modulates the synthesis and activity of soluble and membrane-bound [membrane-type MMPs (MT-MMPs)] in various contexts via homophilic/heterophilic cell interactions, vesicular shedding or cell-autonomous processes. CD147 also participates in inflammation, nutrient and drug transporter activity, microbial pathology and developmental processes. Despite the hundreds of manuscripts demonstrating CD147-mediated MMP regulation, the molecular underpinnings governing this process have not been fully elucidated. The present review summarizes our present knowledge of the complex regulatory systems influencing CD147 biology and provides a framework to understand how CD147 may influence MMP activity. © 2016 Authors.

  16. Activation of the aryl hydrocarbon receptor affects activation and function of human monocyte-derived dendritic cells.

    Science.gov (United States)

    Wang, C; Ye, Z; Kijlstra, A; Zhou, Y; Yang, P

    2014-08-01

    Aryl hydrocarbon receptor (AhR) is well known for mediating the toxic effects of dioxin-containing pollutants, but has also been shown to be involved in the natural regulation of the immune response. In this study, we investigated the effect of AhR activation by its endogenous ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) on the differentiation, maturation and function of monocyte-derived DCs in Behçet's disease (BD) patients. In this study, we showed that AhR activation by FICZ and ITE down-regulated the expression of co-stimulatory molecules including human leucocyte antigen D-related (HLA-DR), CD80 and CD86, while it had no effect on the expression of CD83 and CD40 on DCs derived from BD patients and normal controls. Lipopolysaccharide (LPS)-treated dendritic cells (DCs) from active BD patients showed a higher level of interleukin (IL)-1β, IL-6, IL-23 and tumour necrosis factor (TNF)-α production. FICZ or ITE significantly inhibited the production of IL-1β, IL-6, IL-23 and TNF-α, but induced IL-10 production by DCs derived from active BD patients and normal controls. FICZ or ITE-treated DCs significantly inhibited the T helper type 17 (Th17) and Th1 cell response. Activation of AhR either by FICZ or ITE inhibits DC differentiation, maturation and function. Further studies are needed to investigate whether manipulation of the AhR pathway may be used to treat BD or other autoimmune diseases. © 2014 British Society for Immunology.

  17. Human Blood CD1c+ Dendritic Cells Promote Th1 and Th17 Effector Function in Memory CD4+ T Cells.

    Science.gov (United States)

    Leal Rojas, Ingrid M; Mok, Wai-Hong; Pearson, Frances E; Minoda, Yoshihito; Kenna, Tony J; Barnard, Ross T; Radford, Kristen J

    2017-01-01

    Dendritic cells (DC) initiate the differentiation of CD4 + helper T cells into effector cells including Th1 and Th17 responses that play an important role in inflammation and autoimmune disease pathogenesis. In mice, Th1 and Th17 responses are regulated by different conventional (c) DC subsets, with cDC1 being the main producers of IL-12p70 and inducers of Th1 responses, while cDC2 produce IL-23 to promote Th17 responses. The role that human DC subsets play in memory CD4 + T cell activation is not known. This study investigated production of Th1 promoting cytokine IL-12p70, and Th17 promoting cytokines, IL-1β, IL-6, and IL-23, by human blood monocytes, CD1c + DC, CD141 + DC, and plasmacytoid DC and examined their ability to induce Th1 and Th17 responses in memory CD4 + T cells. Human CD1c + DC produced IL-12p70, IL-1β, IL-6, and IL-23 in response to R848 combined with LPS or poly I:C. CD141 + DC were also capable of producing IL-12p70 and IL-23 but were not as proficient as CD1c + DC. Activated CD1c + DC were endowed with the capacity to promote both Th1 and Th17 effector function in memory CD4 + T cells, characterized by high production of interferon-γ, IL-17A, IL-17F, IL-21, and IL-22. These findings support a role for CD1c + DC in autoimmune inflammation where Th1/Th17 responses play an important role in disease pathogenesis.

  18. Phosphine-free synthesis of high-quality reverse type-I ZnSe/CdSe core with CdS/Cd{sub x}Zn{sub 1-x}S/ZnS multishell nanocrystals and their application for detection of human hepatitis B surface antigen

    Energy Technology Data Exchange (ETDEWEB)

    Shen Huaibin; Niu Jin Zhong; Xu Shasha; Zhou Changhua; Li Linsong [Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004 (China); Yuan Hang; Ma Lan, E-mail: malan@sz.tsinghua.edu.cn, E-mail: lsli@henu.edu.cn [Life Science Division, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055 (China)

    2011-09-16

    Highly photoluminescent (PL) reverse type-I ZnSe/CdSe nanocrystals (NCs) and ZnSe/CdSe/CdS/Cd{sub x}Zn{sub 1-x}S/ZnS core/multishell NCs were successfully synthesized by a phosphine-free method. By this low-cost, 'green' synthesis route, more than 10 g of high-quality ZnSe/CdSe/CdS/Cd{sub x}Zn{sub 1-x}S/ZnS NCs were synthesized in a large scale synthesis. After the overgrowth of a CdS/Cd{sub x}Zn{sub 1-x}S/ZnS multishell on ZnSe/CdSe cores, the PL quantum yields (QYs) increased from 28% to 75% along with the stability improvement. An amphiphilic oligomer was used as a surface coating agent to conduct a phase transfer experiment, core/multishell NCs were dissolved in water by such surface modification and the QYs were still kept above 70%. The as-prepared water dispersible ZnSe/CdSe/CdS/Cd{sub x}Zn{sub 1-x}S/ZnS core/multishell NCs not only have high fluorescence QYs but also are extremely stable in various physiological conditions. Furthermore, a biosensor system (lateral flow immunoassay system, LFIA) for the detection of human hepatitis B surface antigen (HBsAg) was developed by using this water-soluble core/multishell NCs as a fluorescent label and a nitrocellulose filter membrane for lateral flow. The result showed that such ZnSe/CdSe/CdS/Cd{sub x}Zn{sub 1-x}S/ZnS core/multishell NCs were excellent fluorescent labels to detect HBsAg. The sensitivity of HBsAg detection could reach as high as 0.05 ng ml{sup -1}.

  19. 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.

  20. Urinary Soluble CD163 in Active Renal Vasculitis.

    Science.gov (United States)

    O'Reilly, Vincent P; Wong, Limy; Kennedy, Claire; Elliot, Louise A; O'Meachair, Shane; Coughlan, Alice Marie; O'Brien, Eoin C; Ryan, Michelle M; Sandoval, Diego; Connolly, Emma; Dekkema, Gerjan J; Lau, Jiaying; Abdulahad, Wayel H; Sanders, Jan-Stephan F; Heeringa, Peter; Buckley, Colm; O'Brien, Cathal; Finn, Stephen; Cohen, Clemens D; Lindemeyer, Maja T; Hickey, Fionnuala B; O'Hara, Paul V; Feighery, Conleth; Moran, Sarah M; Mellotte, George; Clarkson, Michael R; Dorman, Anthony J; Murray, Patrick T; Little, Mark A

    2016-09-01

    A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3 ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV. Copyright © 2016 by the American Society of Nephrology.

  1. Neonatal CD8+ T-cell differentiation is dependent on interleukin-12.

    LENUS (Irish Health Repository)

    McCarron, Mark J

    2012-02-01

    Neonatal CD8(+) T-cell activation is significantly impaired compared with that in adults. Recent studies have demonstrated that interleukin (IL)-12 is necessary as a third signal, in addition to antigen and co-stimulation, to authorize the differentiation of naive CD8(+) T cells. We examined whether human neonatal CD8(+) T cells, which possess an exclusively naive T-cell phenotype, required a third signal to authorize a productive T-cell response. IL-12 enhanced activated naive CD8(+) T-cell survival, expansion, CD25 expression, and IL-2 production. Activated CD8(+) T cells produced interferon-gamma and intracellular granzyme B and were cytotoxic only in the presence of IL-12. Sustained IL-12 signaling for 72 hours was required for optimal interferon-gamma production. IL-12, in concert with T cell receptor (TCR) stimulation, sustained late-stage (48-72 hours) intracellular phosphorylation and particularly total protein levels of the proximal TCR components, Lck, and CD3xi. The requirement for a third signal for productive human neonatal CD8(+) T-cell differentiation may have implications for neonatal vaccination strategies.

  2. Interleukin-7 (IL-7) enhances class switching to IgE and IgG4 in the presence of T cells via IL-9 and sCD23.

    Science.gov (United States)

    Jeannin, P; Delneste, Y; Lecoanet-Henchoz, S; Gretener, D; Bonnefoy, J Y

    1998-02-15

    Interleukin-7 (IL-7) is a B-cell growth factor produced by both bone marrow stroma cells and follicular dendritic cells (FDCs) located in primary lymphoid follicles and germinal centers. In this study, we have evaluated the role of IL-7 on human Ig class switching. IL-7 was added to peripheral blood mononuclear cells (PBMCs) or tonsillar B cells in the absence or presence of IL-4 and/or anti-CD40 monoclonal antibody (MoAb). Alone, IL-7 did not affect Ig production by PBMCs or by anti-CD40 MoAb-stimulated B cells. Rather, IL-7 potentiated IL-4-induced IgE and IgG4 production by PBMCs. In parallel, IgG3 production was also enhanced but to a lesser extent, whereas the production of the other isotypes was unaltered. The activity of IL-2, IL-9, or IL-15, which share usage of the common gamma chain for signaling, was also assessed. IL-9, like IL-7, potentiated mainly IgE and IgG4 production by IL-4-stimulated PBMCs. IL-15, in contrast, was ineffective, whereas IL-2 enhanced the production of all isotypes. More precisely, IL-7 potentiation of IgE and IgG4 production required the presence of T cells and was accompanied by an increase of the expression of two soluble molecules favoring preferentially IgE and IgG4 synthesis: CD23 (sCD23) and IL-9. Moreover, neutralizing anti-CD23 and anti-IL-9 antibodies partly inhibited the increase of IgE synthesis induced by IL-7. Thus, IL-7 produced locally in the germinal centers by FDCs may interact with T cells and potentiate human IgE and IgG4 switching by favoring IL-9 and sCD23 production.

  3. CD4 down regulation and raft dissociation by the non-depleting YTS177 antibody hinder murine T helper cell activities

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng-Jang [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093 (United States); Lu, Chun-Hao [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Chen, Li-Chen [Division of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan (China); Nguyen, Duc T. [Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093 (United States); Huang, Yi-Shu [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Lin, Hsi-Hsien [Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Chang Gung Immunology Consortium, Chang Gung Memorial Hospital and Chang Gung University, Tao-Yuan, Taiwan (China); Department of Anatomic Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan (China); Lin, Chun-Yen [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Chang Gung Immunology Consortium, Chang Gung Memorial Hospital and Chang Gung University, Tao-Yuan, Taiwan (China); Department of Hepatogastroenterology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan (China); Kuo, Ming-Ling, E-mail: mingling@mail.cgu.edu.tw [Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan (China); Division of Allergy, Asthma, and Rheumatology, Department of Pediatrics, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan (China); Chang Gung Immunology Consortium, Chang Gung Memorial Hospital and Chang Gung University, Tao-Yuan, Taiwan (China)

    2016-05-13

    Non-depleting YTS177 anti-CD4 monoclonal antibody (MoAb) has been reported to lead to antigen-specific immunotolerance in allograft transplantation and autoimmune diabetes, as well as possibly to inhibition of allergic inflammation in mice. However, the molecular mechanisms underlying hyporesponsive T cell responses induced by YTS177 MoAb remain elusive. Herein, we demonstrate that the YTS177 MoAb increases the levels of anergy factors p27{sup kip1} and Cbl-b, inhibits IL-2 production, and impairs calcium mobilization in activated T cells in vitro. YTS177 MoAb suppresses OVA-driven proliferation of DO11.10 CD4{sup +} T cells in vivo as well. Mechanistically, YTS177 MoAb induces tolerance by causing CD4 down-regulation through clathrin-dependent and raft dissociation. The results obtained in this study lead us to propose novel protective or curative approaches to CD4 T cell-mediated diseases.

  4. CD4 down regulation and raft dissociation by the non-depleting YTS177 antibody hinder murine T helper cell activities

    International Nuclear Information System (INIS)

    Wu, Cheng-Jang; Lu, Chun-Hao; Chen, Li-Chen; Nguyen, Duc T.; Huang, Yi-Shu; Lin, Hsi-Hsien; Lin, Chun-Yen; Kuo, Ming-Ling

    2016-01-01

    Non-depleting YTS177 anti-CD4 monoclonal antibody (MoAb) has been reported to lead to antigen-specific immunotolerance in allograft transplantation and autoimmune diabetes, as well as possibly to inhibition of allergic inflammation in mice. However, the molecular mechanisms underlying hyporesponsive T cell responses induced by YTS177 MoAb remain elusive. Herein, we demonstrate that the YTS177 MoAb increases the levels of anergy factors p27"k"i"p"1 and Cbl-b, inhibits IL-2 production, and impairs calcium mobilization in activated T cells in vitro. YTS177 MoAb suppresses OVA-driven proliferation of DO11.10 CD4"+ T cells in vivo as well. Mechanistically, YTS177 MoAb induces tolerance by causing CD4 down-regulation through clathrin-dependent and raft dissociation. The results obtained in this study lead us to propose novel protective or curative approaches to CD4 T cell-mediated diseases.

  5. Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

    Science.gov (United States)

    Hus, I; Schmitt, M; Tabarkiewicz, J; Radej, S; Wojas, K; Bojarska-Junak, A; Schmitt, A; Giannopoulos, K; Dmoszyńska, A; Roliński, J

    2008-05-01

    Recently, we described that vaccination with allogeneic dendritic cells (DCs) pulsed with tumor cell lysate generated specific CD8+ T cell response in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the potential of autologous DCs pulsed ex vivo with tumor cell lysates to stimulate antitumor immunity in patients with B-CLL in early stages was evaluated. Twelve patients at clinical stage 0-2 as per Rai were vaccinated intradermally up to eight times with a mean number of 7.4 x 10(6) DCs pulsed with B-CLL cell lysate. We observed a decrease of peripheral blood leukocytes and CD19+/CD5+ leukemic cells in five patients, three patients showed a stable disease and four patients progressed despite DC vaccination. A significant increase of specific cytotoxic CD8+ T lymphocytes against the leukemia-associated antigens RHAMM or fibromodulin was detected in four patients after DC vaccination. In patients with a clinical response, an increase of interleukin 12 (IL-12) serum levels and a decrease of the frequency of CD4+CD25(+)FOXP3+ T regulatory cells were observed. Taken together, the study demonstrated that vaccination with autologous DC in CLL patients is feasible and safe. Immunological and to some extend hematological responses could be noted, justifying further investigation on this immunotherapeutical approach.

  6. Profiling the Targets of Protective CD8+ T Cell Responses to Infection

    Directory of Open Access Journals (Sweden)

    Joseph T. Bruder

    2017-12-01

    Full Text Available T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

  7. 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.

  8. Immunogenicity and Immunological Memory Induced by the 13-Valent Pneumococcal Conjugate Followed by the 23-Valent Polysaccharide Vaccine in HIV-Infected Adults.

    Science.gov (United States)

    Farmaki, Paraskevi F; Chini, Maria C; Mangafas, Nikolaos M; Tzanoudaki, Marianna T; Piperi, Christina P; Lazanas, Marios Z; Spoulou, Vana S

    2018-05-02

    Vaccine-induced memory B-cell (MBC) subsets have distinct roles in the establishment of protective immunity; MBCs expressing nonswitched immunoglobulin M (IgM+ MBCs) replenish the MBC pool, whereas MBCs expressing isotype-switched immunoglobulin (sIg+ MBCs) differentiate into plasma cells upon antigen reencounter. We investigated immunogenicity and MBCs induced by combined 13-valent pneumococcal conjugate vaccine (PCV13) and 23-valent pneumococcal polysaccharide vaccine (PPV23) in human immunodeficiency virus (HIV)-infected adults. Forty HIV-seropositive adults receiving ART with undetectable viral loads were enrolled. Seventeen had a CD4+ T-cell count of ≥400 cells/μL (group A), and 23 had a CD4+ T-cell count of 200-399 cells/μL (group B). All adults received PCV13 and, 1 year later, PPV23. Levels of IgM+ MBCs (defined as polysaccharide [PS]-specific CD19+CD10-CD27+CD21++IgM+ MBCs) and sIg+ MBCs (defined as PS-specific CD19+CD10-CD27+CD21++IgM- MBCs) and antibodies against PS14 and PS3 were measured prior and 1 month after each vaccination. Immunization caused a significant increase in PS antibodies, compared with levels at baseline (P < .001). Group B achieved significantly lower titers than group A (P < .05 for both PS14 and PS3). After receipt of PCV13, levels of IgM+ MBCs were unchanged, whereas levels of sIg+ MBCs increased significantly (P < .05 for PS14 and P < .001 for PS3). In contrast, following PPV23 receipt, levels of IgM+ MBCs were significantly reduced, and levels of sIg+ MBCs remained stable. A positive correlation was observed between baseline IgM+ and sIg+ MBC counts 1 month after PCV13 receipt but not after PPV23 receipt. PPV23 receipt 12 months after PCV13 receipt improved PCV13 immunogenicity. The reduction in the IgM+ MBC count observed after PPV23 receipt suggests that PPV23 has a depleting effect on PCV13-associated immunological memory. NCT03041051.

  9. Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

    Science.gov (United States)

    Gust, Juliane; Hay, Kevin A; Hanafi, Laïla-Aïcha; Li, Daniel; Myerson, David; Gonzalez-Cuyar, Luis F; Yeung, Cecilia; Liles, W Conrad; Wurfel, Mark; Lopez, Jose A; Chen, Junmei; Chung, Dominic; Harju-Baker, Susanna; Özpolat, Tahsin; Fink, Kathleen R; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

    2017-12-01

    Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19 + cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity. Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR. See related commentary by Mackall and Miklos, p. 1371 This article is highlighted in the In This Issue feature, p. 1355 . ©2017 American Association for Cancer Research.

  10. Effect of mammals’ excretory function on aspartate aminotransferase activity in Glechoma hederacea leaves in conditions of Cd pollution

    Directory of Open Access Journals (Sweden)

    O. M. Vasilyuk

    2014-07-01

    Full Text Available The paper includes analysis of research of Cd impact on the activity of the enzyme of aspartate aminotransferase (AST nitrogen metabolism and the content of water-soluble protein fraction (albumin in Glechoma hederacea L. leaves, which dominated in the research area (in natural floodplain oak forest with Stellaria holostea L.. Cd was introduced in the form of salts of Cd(NO32 in the range of concentrations of: 0.25, 1.25, 2.5 g/m2, equivalent to the inclusion of Cd in 1, 5, 10 doses of MAC. Increase (P < 0.05 in the activity of AST 2.6–3.0 times (with adding Cd salts at a dose of 1 and 5 МAС and albumin content by 37% (with adding Cd salts at a dose of 10 МAС compared to control (the area without Cd pollution and excretory activity of mammals was shown. Using of excreta of some representatives of mammals (for example, Capreolus capreolus L. contributed to reduction of Cd toxic effects and restoring of the functional metabolic activity of AST by 23% (with Cd 1 МAС and by 34% (Cd 5 МAС. It is the evidence of protective function of mammals and their normalization effect at the above concentrations of Cd. Whereas the adding of Cd salts at a dose of 10 МAС led to 3 times’ inhibition of AST activity, the toxic effect of metal by excretory function of mammals was not reduced. Observations revealed the albumin content normalization by 22% in the presence of Cd 1MAC respectively (with the introduction of C. capreolus excreta and to the control level (the area without Cd pollution and excretory activity of mammals with the excreta of Sus scrofa L. in the setting of Cd 10 MAC. It proves the need to use the different mammal species for integrated and comprehensive normalization of ecosystems under conditions of uncontrolled anthropogenic pollution.

  11. Cellular endocytic compartment localization of expressed canine CD1 molecules

    DEFF Research Database (Denmark)

    Schjærff, Mette; Keller, Stefan M.; Affolter, Verena K.

    2016-01-01

    CD1 molecules are glycoproteins present primarily on dendritic cells (DCs), which recognize and presenta variety of foreign- and self-lipid antigens to T-cells. Humans have five different CD1 isoforms that sur-vey distinct cellular compartments allowing for recognition of a large repertoire...... onlya diminished GFP expression. In conclusion, canine CD1 transfectants show distinct localization patternsthat are similar to human CD1 proteins with the exception of the canine CD1d isoform, which most likelyis non-functional. These findings imply that canine CD1 localization overall resembles human...... CD1 traf-ficking patterns. This knowledge is important for the understanding of lipid antigen-receptor immunityin the dog....

  12. The immune system strikes back: cellular immune responses against indoleamine 2,3-dioxygenase

    DEFF Research Database (Denmark)

    Sørensen, Rikke Baek; Berge-Hansen, Linda; Junker, Niels

    2009-01-01

    BACKGROUND: The enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance...... to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses. METHODS AND FINDINGS: The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T...... of the major immune suppressive cell populations. CONCLUSION: IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals...

  13. CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

    Science.gov (United States)

    Solstad, Therese; Bains, Simer Jit; Landskron, Johannes; Aandahl, Einar Martin; Thiede, Bernd; Taskén, Kjetil; Torgersen, Knut Martin

    2011-11-10

    Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

  14. GM-CSF production allows the identification of immunoprevalent antigens recognized by human CD4+ T cells following smallpox vaccination.

    Directory of Open Access Journals (Sweden)

    Valeria Judkowski

    Full Text Available The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a "T cell-driven" methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

  15. 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.

  16. HIV-specific cytotoxic T lymphocyte precursors exist in a CD28-CD8+ T cell subset and increase with loss of CD4 T cells.

    Science.gov (United States)

    Lewis, D E; Yang, L; Luo, W; Wang, X; Rodgers, J R

    1999-06-18

    To determine whether the CD28-CD8+ T cells that develop during HIV infection contain HIV-specific cytotoxic precursor cells. CD8 subpopulations from six asymptomatic HIV-positive adults, with varying degrees of CD4 T cell loss, were sorted by flow cytometry and HIV-specific precursor cytotoxic T lymphocyte frequencies were measured. Three populations of CD8 T cells were tested: CD28+CD5-- T cells, CD28-CD57+ T cells (thought to be memory cells) and CD28-CD57- T cells (function unknown). Sorted CD8 subsets were stimulated with antigen presenting cells expressing HIV-1 Gag/Pol molecules. Cytotoxic T cell assays on Gag/Pol expressing 51Cr-labeled Epstein-Barr virus transformed autologous B cells lines or control targets were performed after 2 weeks. Specific lysis and precursor frequencies were calculated. Both CD28 positive and CD28-CD57+ populations contained appreciable numbers of precursors (9-1720 per 10(6) CD8+ T cells). However, the CD28-CD57- population had fewer precursors in five out of six people studied. More CD28 positive HIV-specific cytotoxic T lymphocyte precursors were found in patients with CD4:CD8 ratios > 1, whereas more CD28-CD57+ precursors were found in patients whose CD4:CD8 ratios were < 1 (r2, 0.68). Memory HIV-specific precursor cytotoxic T lymphocytes are found in both CD28 positive and CD28-CD8+ cells, however, a CD28-CD57- subpopulation had fewer. Because CD28-CD57+ cells are antigen-driven with limited diversity, the loss of CD28 on CD8 T cells during disease progression may reduce the response to new HIV mutations; this requires further testing.

  17. CD4 T cell activation and disease activity at onset of multiple sclerosis

    DEFF Research Database (Denmark)

    Jensen, J; Langkilde, Annika Reynberg; Fenst, C

    2004-01-01

    We studied CD4 T cell activation in patients with clinically isolated syndromes (CIS) suggesting an initial attack of multiple sclerosis. The percentage of blood CD26+ CD4 T cells was increased in these patients, and correlated with magnetic resonance imaging disease activity and clinical disease...... severity. In contrast, the percentage of CD25+ CD4 T cells in cerebrospinal fluid correlated negatively with the cerebrospinal fluid concentration of myelin basic protein and the presence of IgG oligoclonal bands. These results suggest that distinct systemic and intrathecal T cell activation states...

  18. Heat Shock Proteins 60 and 70 Specific Proinflammatory and Cytotoxic Response of CD4+CD28null Cells in Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Ashok K. Yadav

    2013-01-01

    Full Text Available Background. CD4+CD28null T cells are expanded in peripheral blood of patients with chronic kidney disease and associated with subclinical atherosclerosis. However, triggers for the oligoclonal expansion and activation of these cells are not clear. Methods. We investigated twenty-five stage V-IV chronic kidney disease (CKD patients and eight healthy subjects (HC. Peripheral mononuclear cells were isolated and incubated with heat shock protein- (HSP 60 and 70. CD4+CD28null and CD4+CD28+ cells were sorted by flowcytometry and antigen specific response was assessed by the mRNA and protein expression of interferon (IFN-γ, perforin, and granzyme B using qRT-PCR and Elispot. Results. The basal mRNA expression of IFN-γ, perforin, and granzyme B in CD4+CD28null cells was higher in subjects with CKD compared to that in HC (P<0.0001. Subjects with CKD also showed expression of IFN-γ, perforin, and granzyme B in the CD4+CD28+ subset, but this was much weaker than that seen in the CD4+CD28null population (P<0.0001. We did not note the expression of these molecules at mRNA or protein level in either subset of CD4 cells in HC. After incubation with HSP60 and HSP70, CD4+CD28null cells showed increased expression at mRNA (P<0.001 and protein level (P<0.001. CD4+CD28+ cells also showed a weak increase in expression. No antigen-specific response was noted in HC. Conclusion. These data show that CD4+CD28null cells in subjects with CKD react with HSP60 and HSP70 by upregulating the expression of IFN-γ, perforin and granzyme B. Increased circulating level of HSP60 and HSP70 might play a role in initiation and/or progression of atherosclerosis in CKD subjects through perturbation of CD4+CD28null cells.

  19. Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig

    Science.gov (United States)

    Morgan, Sophie B.; Attaf, Meriem; Szomolay, Barbara; Miles, John J.; Townsend, Alain; Bailey, Mick; Charleston, Bryan; Tchilian, Elma

    2018-01-01

    There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory

  20. Food Supplement 20070721-GX May Increase CD34+ Stem Cells and Telomerase Activity

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    Po-Cheng Lin

    2012-01-01

    Full Text Available Few rejuvenation and antiaging markers are used to evaluate food supplements. We measured three markers in peripheral blood to evaluate the antiaging effects of a food supplement containing placental extract. Samples were evaluated for CD34+ cells, insulin-like growth factor 1 (IGF1, and telomerase activity, which are all markers related to aging. To control the quality of this food supplement, five active components were monitored. In total, we examined 44 individuals who took the food supplement from 1.2 months to 23 months; the average number of CD34+ cells was almost 6-fold higher in the experimental group compared with the control group. Food supplement intake did not change serum IGF1 levels significantly. Finally, the average telomerase activity was 30% higher in the subjects taking this food supplement. In summary, our results suggest that the placental extract in the food supplement might contribute to rejuvenation and antiaging.

  1. Changing of expression level of fas-antigen (CD95), cytokines synthesis and production after irradiation in low doses

    International Nuclear Information System (INIS)

    Kalinina, N.M.; Solntceva, O.S.; Bytchkova, N.V.; Nikiforov, A.M.

    1997-01-01

    It is known that bone marrow progenitor (CD34+), tymocytes and peripheral blood lymphocytes (PBL) are most radiosensitive than other cell types. Even low doses of radiation induce apoptosis. The investigators suggest that it is possible relationship between synthesis and production of cytokines and apoptotic process. With the purpose to determine correlation between expression of Fas-antigen and synthesis of cytokines after low doses irradiation the experiments by irradiation PBL of healthy persons in vitro were held. Cells were X-irradiated by 12,5, 25 and 50 cGy. In consequence of the experiments increasing of Fas-antigen was revealed. This increasing correlated with changing in synthesis and production of cytokines. Also the Chernobyl's accident liquidators (CAL) were investigated. After comparison data in the group CAL (I) with data in the control group (II) increasing of Fas-antigen expression was revealed. Also in I group was discovered increasing of the cell number sinthesied interleukine-4 (IL-4) and interleukine-6 (IL-6). Interleukine-lβ (IL-1 β) producing pell were decreased. These changes have been correlated with degree of immunodeficiency at CAL. These data allow to consider the apoptosis as cell mechanism included in pathogenesis of diseases, which can be showed later long time after irradiation. (author)

  2. B Cell Help by CD1d-Rectricted NKT Cells

    OpenAIRE

    Livia Clerici; Giulia Casorati; Paolo Dellabona

    2015-01-01

    B cell activation and antibody production against foreign antigens is a central step of host defense. This is achieved via highly regulated multi-phase processes that involve a variety of cells of both innate and adaptive arms of the immune system. MHC class II-restricted CD4+ T cells specific for peptide antigens, which acquire professional follicular B cell helper functions, have been long recognized as key players in this process. Recent data, however, challenge this paradigm by showing th...

  3. MWCNT/CdS hybrid nanocomposite for enhanced photocatalytic activity

    International Nuclear Information System (INIS)

    Chaudhary, Deepti; Khare, Neeraj; Vankar, V. D.

    2016-01-01

    Multi-walled carbon nanotubes (MWCNT)/CdS hybrid nanocomposite were synthesized by one step hydrothermal method. MWCNTs were used as a substrate for the growth of CdS nanoparticles. MWCNT/CdS nanocomposite and pure CdS were characterized by XRD, TEM, UV-vis and photoluminescence spectroscopy. HRTEM study confirms the intimate contact of CdS with MWCNT. The photocatalytic activity of nanocomposite was studied for the degradation of methylene blue dye under UV irradiation. The enhanced photocatalytic activity of MWCNT/CdS nanocomposite as compared to pure CdS has been attributed to reduced recombination of photogenerated charge carriers due to interfacial electron transfer from CdS to MWCNT.

  4. Systemic immunological tolerance to ocular antigens is mediated by TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD8+ T cells*

    Science.gov (United States)

    Griffith, Thomas S.; Brincks, Erik L.; Gurung, Prajwal; Kucaba, Tamara A.; Ferguson, Thomas A.

    2010-01-01

    Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required FasL-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8+ regulatory T cells. The present study examined the mechanism by which these CD8+ regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4+ T cells, and led to increased TRAIL expression by splenic CD8+ T cells. Unlike wildtype mice, Trail−/− or Dr5−/− mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8+ T cells from Trail−/− mice that were first injected AC with Ag were unable to transfer tolerance to naïve recipient wildtype mice, but CD8+ T cells from AC-injected wildtype or Dr5−/− mice could transfer tolerance. Importantly, the transferred wildtype (Trail+/+) CD8+ T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail−/− CD8+ T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that “helpless” CD8+ regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye. PMID:21169546

  5. The exhausted CD4+CXCR5+ T cells involve the pathogenesis of human tuberculosis disease.

    Science.gov (United States)

    Bosco, Munyemana Jean; Wei, Ming; Hou, Hongyan; Yu, Jing; Lin, Qun; Luo, Ying; Sun, Ziyong; Wang, Feng

    2018-06-21

    The CD4 + CXCR5 + T cells have been previously established. However, their decreased frequency during tuberculosis (TB) disease is partially understood. The aim of this study was to explore the depletion of CD4 + CXCR5 + T cells in human TB. The frequency and function of CD4 + CXCR5 + T cells were evaluated in active TB (ATB) patients and healthy control (HC) individuals. The function of CD4 + CXCR5 + T cells was determined after blockade of inhibitory receptors. The frequency of CD4 + CXCR5 + T cells was decreased in ATB patients. The expression of activation markers (HLA-DR and ICOS) and inhibitory receptors (Tim-3 and PD-1) on CD4 + CXCR5 + T cells was increased in ATB group. TB-specific antigen stimulation induced higher expression of inhibitory receptors than phytohemagglutinin stimulation in ATB group. In contrast, TB antigen stimulation did not induce a significantly increased expression of IL-21 and Ki-67 on CD4 + CXCR5 + T cells. However, blockade of inhibitory receptors Tim-3 and PD-1 not only increased the frequency of CD4 + CXCR5 + T cells, but also restored their proliferation and cytokine secretion potential. An increased expression of inhibitory receptors involves the depletion of CD4 + CXCR5 + T cells, and blockade of inhibitory receptors can restore the function of CD4 + CXCR5 + T cells in ATB patients. Copyright © 2018. Published by Elsevier Ltd.

  6. The Correlation of CD206, CD209, and Disease Severity in Behçet’s Disease with Arthritis

    Directory of Open Access Journals (Sweden)

    Bunsoon Choi

    2017-01-01

    Full Text Available The purpose of this study was to clarify the role of pattern recognition receptors in Behçet’s disease (BD. The frequencies of several pattern recognition receptors (CD11b, CD11c, CD32, CD206, CD209, and dectin-1 were analyzed in patients with BD by flow cytometry, and cytokine levels, interleukin- (IL- 18, IL-23, and IL-17A, were compared in plasma. The analysis was performed in active (n=13 and inactive (n=13 stages of BD patients. Rheumatoid arthritis patients (n=19, as a disease control, and healthy control (HC (n=19 were enrolled. The frequencies of CD11b+ and CD32+ cells were significantly increased in active BD patients compared to HC. Disease severity score was correlated to CD11c+, CD206+, and CD209+ in whole leukocytes and CD11b+, CD11c+, CD206+, CD209+, and Dectin-1+ in granulocytes. The plasma levels of IL-17A were significantly different between HC and active BD. IL-18 showed significant difference between active and inactive BD patients. From this study, we concluded the expressions of several pattern recognition receptors were correlated to the joint symptoms of BD.

  7. Taenia crassiceps Antigens Control Experimental Type 1 Diabetes by Inducing Alternatively Activated Macrophages

    Science.gov (United States)

    Espinoza-Jiménez, Arlett; De Haro, Roberto

    2017-01-01

    Type 1 diabetes (T1D) is an autoimmune disease caused by the selective destruction of the pancreatic β-cells, causing inability to produce insulin. Proinflammatory cytokines such as IL-1β, IL-6, TNF-α, IFN-γ, IL-12, IL-17, and NO can be released by CD4 and CD8+ lymphocytes as well as by classically activated macrophages (CAMϕs), which are important in the development of T1D. Helminth infections have been shown to prevent T1D, mainly through Th2-biased responses and increased recruitment of regulatory cell populations. Previously, we have shown that Taenia crassiceps infection in mice significantly reduces hyperglycemia, insulitis, and the incidence of T1D. In this study, we determined whether T. crassiceps-derived products such as soluble (TcS) or excreted/secreted (TcES) antigens might have a beneficial influence on the development of experimental T1D. Treatment with different doses before or after induction of T1D was analyzed. Mice that were pretreated with TcS were unable to develop T1D, whereas those receiving TcES early after T1D induction displayed significantly reduced insulitis and hyperglycemia along with increased recruitment of alternatively activated macrophages (AAMϕs) and myeloid-derived suppressor cells (MDSCs). Finally, we examined the modulatory role of AAMϕs on T1D by depleting macrophages with clodronate-loaded liposomes, demonstrating that AAMϕs are key cells in T1D regulation. PMID:29249872

  8. CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity.

    Science.gov (United States)

    Akane, Kazuyuki; Kojima, Seiji; Mak, Tak W; Shiku, Hiroshi; Suzuki, Haruhiko

    2016-03-01

    The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis.

  9. Cellular reactions of CD3+ CD4+ CD45RO+ T-lymphocytes on dexamethason in in normal patients and in patients with with rheumatoid arthritis in vitro

    Directory of Open Access Journals (Sweden)

    L. S. Litvinova

    2017-01-01

    Full Text Available The aim of the study was to analyze the influence of glucocorticoid (GC dexamethasone (Dex on changes in CD4+ T-cells expressing the surface molecule of activation (CD25, CD71, HLA-DR and CD95 and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes obtained from healthy donors and patients with rheumatoid arthritis in vitro.Materials and methods. The study included 50 patients and 20 healthy donors. T-cell cultures (CD3+ CD45RO+ were obtained from mononuclear leukocytes of immunomagnetic separation (MACS® technology. As an activator of T-lymphocytes, antibiotic particles with biotinylated antibodies against CD2+, CD3+, CD28+, which simulate the process of costimulation of T cells by antigen-presenting cells, were used. The following concentrations of dexamethasone (2, 8, 16, 32, 64 mg were used in the experiment. The change in the immunophenotype of T-lymphocytes was analyzed by flow cytofluoometry. The secretion of CD3+CD45RO+ T-cells of proinflammatory cytokines IL-2, IFNγ, TNFα, IL-17 and IL-21 was evaluated by enzyme-linked immunosorbent assay.Results. The general suppressor effect of Dex on CD3+CD45RO+ T-cell cultures mediated by a decrease in the number of CD4 + T cells expressing activation molecules (CD25 and proliferation (CD71, as well as inhibition of the production of inflammatory mediators: IFNγ, IL-2 and TNFα. It is shown that against the background of TCR activation Dex increases the number of CD4+CD95+HLA-DR+ cells in CD3+CD45RO+ cultures obtained from RA patients and does not change their content in the control. The correlations between the number of proinflammatory factors (IL-17, IL-21 and TNFα in CD4+CD45RO+CD95+HLA-DR+ T cells in supernatants of cell cultures in RA patients indicate the presence of a pro-inflammatory potential of this population of T cells. We assume that the resistance of CD4+CD45RO+CD95+HLA-DR+ T cells in RA patients to the suppressor effect of

  10. Reduced Plasmodium Parasite Burden Associates with CD38+ CD4+ T Cells Displaying Cytolytic Potential and Impaired IFN-γ Production

    Science.gov (United States)

    Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; Klein, Kerenaftali; McCarthy, James S.; Doolan, Denise L.

    2016-01-01

    Using a unique resource of samples from a controlled human malaria infection (CHMI) study, we identified a novel population of CD4+ T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4+ T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4+ T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-γ and other cytokines and reduced basal levels of activated STAT1. A CD38+ CD4+ T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38+ CD4+ T cells could be generated in vitro from CD38- CD4+ T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38+ CD4+ T cells with a cytotoxic phenotype and markedly impaired IFN-γ capacity in humans. The expansion of this CD38+ CD4+ T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense. Trial Registration ClinicalTrials.gov clinical trial numbers ACTRN12612000814875, ACTRN12613000565741 and ACTRN12613001040752 PMID:27662621

  11. 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

  12. 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.

  13. Establishment of HLA-DR4 transgenic mice for the identification of CD4+ T cell epitopes of tumor-associated antigens.

    Directory of Open Access Journals (Sweden)

    Junji Yatsuda

    Full Text Available Reports have shown that activation of tumor-specific CD4(+ helper T (Th cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05 transgenic mice (Tgm, since this HLA-DR allele is most frequent (13.6% in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E(d, where I-E(d α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E(d has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191

  14. Target Antigen Density Governs the Efficacy of Anti-CD20-CD28-CD3 zeta Chimeric Antigen Receptor-Modified Effector CD8(+) T Cells

    NARCIS (Netherlands)

    Watanabe, Keisuke; Terakura, Seitaro; Martens, Anton C.; van Meerten, Tom; Uchiyama, Susumu; Imai, Misa; Sakemura, Reona; Goto, Tatsunori; Hanajiri, Ryo; Imahashi, Nobuhiko; Shimada, Kazuyuki; Tomita, Akihiro; Kiyoi, Hitoshi; Nishida, Tetsuya; Naoe, Tomoki; Murata, Makoto

    2015-01-01

    The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second-and later-generation CARs simultaneously transmit costimulatory signals with CD3 zeta signals upon ligation, but may lead to severe adverse effects

  15. CD47-CAR-T Cells Effectively Kill Target Cancer Cells and Block Pancreatic Tumor Growth.

    Science.gov (United States)

    Golubovskaya, Vita; Berahovich, Robert; Zhou, Hua; Xu, Shirley; Harto, Hizkia; Li, Le; Chao, Cheng-Chi; Mao, Mike Ming; Wu, Lijun

    2017-10-21

    CD47 is a glycoprotein of the immunoglobulin superfamily that is often overexpressed in different types of hematological and solid cancer tumors and plays important role in blocking phagocytosis, increased tumor survival, metastasis and angiogenesis. In the present report, we designed CAR (chimeric antigen receptor)-T cells that bind CD47 antigen. We used ScFv (single chain variable fragment) from mouse CD47 antibody to generate CD47-CAR-T cells for targeting different cancer cell lines. CD47-CAR-T cells effectively killed ovarian, pancreatic and other cancer cells and produced high level of cytokines that correlated with expression of CD47 antigen. In addition, CD47-CAR-T cells significantly blocked BxPC3 pancreatic xenograft tumor growth after intratumoral injection into NSG mice. Moreover, we humanized mouse CD47 ScFv and showed that it effectively bound CD47 antigen. The humanized CD47-CAR-T cells also specifically killed ovarian, pancreatic, and cervical cancer cell lines and produced IL-2 that correlated with expression of CD47. Thus, CD47-CAR-T cells can be used as a novel cellular therapeutic agent for treating different types of cancer.

  16. 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.

  17. CD28-, CD45RA(null/dim) and natural killer-like CD8+ T cells are increased in peripheral blood of women with low-grade cervical lesions.

    Science.gov (United States)

    Pita-Lopez, Maria Luisa; Ortiz-Lazareno, Pablo Cesar; Navarro-Meza, Monica; Santoyo-Telles, Felipe; Peralta-Zaragoza, Oscar

    2014-01-01

    In response to antigen naive CD8+, T cells differentiate into effector cells, which express Natural killer (NK) receptors, lose CD28 expression, and die by apoptosis. However, in smaller quantities, the cells are retained for subsequent exposure to the same antigen. Knowledge is limited regarding whether the percentages of CD28-, Effector memory (EMRA(null/dim)), and the CD16+/CD56 + CD8+ T cells of women with low-grade cervical lesions are altered at a systemic level. We enrolled in this study women controls and women with Human papilloma virus infection (HPV-I) without associated cellular neoplastic changes and with Cervical Intraepithelial Neoplastic-I (CIN-I). Flow cytometry (FC) was performed for measurement of CD28-, memory subset, and NK-like CD8 + T cells, and IL-17, IFN-gamma, Tumor necrosis factor (TNF)-alpha, Interleukin (IL)-10, IL-6, IL-4, and IL-2. Finally, we genotyped the HPV. The CIN-I group increased the CD8 + CD28- and CD16+/56+ T cell percentage compared with that of HPV-I and controls (p levels among all groups. Increased levels of CD28-, EMRA(null/dim), and CD16+/CD56 + CD8+ T cells of peripheral blood in women with CIN-I may be associated with persistent HPV infection and could exert an influence on progression to cervical cancer.

  18. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

    Science.gov (United States)

    Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

    2015-02-01

    Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

  19. 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.

  20. Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

    DEFF Research Database (Denmark)

    Ladeby, Rune; Wirenfeldt, Martin; Dalmau, Ishar

    2005-01-01

    -activated microglia in the facial motor nucleus following peripheral axotomy. The results suggest lesion-reactive microglia to consist of functionally distinct subpopulations of cells; a major population of activated resident CD34(+)Mac-1(+) microglia with a high capacity for self-renewal, and a subpopulation of CD34...

  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. Global inhibition of DC priming capacity in the spleen of self-antigen vaccinated mice requires IL-10

    Directory of Open Access Journals (Sweden)

    Douglas Matthew Marvel

    2014-02-01

    Full Text Available DC in the spleen are highly activated following intravenous vaccination with a foreign antigen, promoting expansion of effector T cells, but remain phenotypically and functionally immature after vaccination with a self-antigen. Up-regulation or suppression of expression of a cohort of pancreatic enzymes 24-72 hours post-vaccination can be used as a biomarker of stimulatory versus toleragenic DC, respectively. Here we show, using MUC1 transgenic mice (MUC1.Tg and a vaccine based on the MUC1 peptide which these mice perceive as a self-antigen, that the difference in enzyme expression that predicts whether DC will promote immune response or immune tolerance, is seen as early as 4-8 hours following vaccination. We also identify early production of IL-10 as a predominant factor that both correlates with this early time point and controls DC function. Pre-treating mice with an antibody against the IL-10 receptor (IL-10R prior to vaccination results in DC that up-regulate CD40, CD80, and CD86 and promote stronger IFNγ+ T cell responses. This study suggests that transient inhibition of IL-10 prior to vaccination could improve responses to cancer vaccines that utilize self-tumor antigens.

  3. Role of CD5-negative CD8 T Cells in Adaptation to Antigenic ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of 3-oxotirucalla-7, 24-dien-21-oic acid on CD8+ T cell recovery in human immunodeficiency ..... enhances the probability of immune system recovery. ... PE: Fine tuning of TCR signaling by CD5. J Immunol.

  4. Differences between primed allogeneic T-cell responses and the primary mixed leucocyte reaction. Primed T cells become independent of the blocking effects of monoclonal antibodies against IL-1 beta and the CD5, CD11a (LFA-1), and CD11c (p 150,95) molecules

    DEFF Research Database (Denmark)

    Ødum, Niels; Hofmann, B; Morling, N

    1988-01-01

    .01) and the purified protein derivative (PPD) induced lymphocyte transformation response (42%, P less than or equal to 0.01) of peripheral blood mononuclear cells (PBMC), whereas primed allogeneic responses to PBMC and Epstein-Barr virus (EBV) cell lines were unaffected by this MoAb. In addition, preliminary data...... monoclonal antibodies (MoAb) directed against (i) adhesion molecules belonging to the CD11 cluster of leucocyte antigens (CD11a, LFA-1; CD11b, MAC1 = CR3; and CD11c, p 150,95); (ii) various T cell-related antigens (CD2, CD4, CD5 and CD8); and (iii) recombinant IL-1 beta. The CD5-, CD11a- and CD11c...

  5. Immunity to experimental Salmonella typhimurium infections in rats. Transfer of immunity with primed CD45RC+ and CD45RC- CD4 T-cell subpopulations

    DEFF Research Database (Denmark)

    Thygesen, P; Christensen, H B; Hougen, H P

    1996-01-01

    The protective effect of primed CD4 T cells against a lethal dose of Salmonella typhimurium was studied in Lewis rats. Primed CD4 T cells were obtained by inoculating Lewis rats with a non-lethal dose of S. typhimurium. Four weeks after the infection, spleen CD4 T cells were separated by antibody......-induced increase in CD45RC+ cells is most likely due to generation of antigen-specific memory T cells....

  6. 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).

  7. 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.

  8. [Regulatory B cells activated by CpG-ODN combined with anti-CD40 monoclonal antibody inhibit CD4(+)T cell proliferation].

    Science.gov (United States)

    Wang, Keng; Tao, Lei; Su, Jianbing; Zhang, Yueyang; Zou, Binhua; Wang, Yiyuan; Li, Xiaojuan

    2016-09-01

    Objective To observe the immunosuppressive function of regulatory B cells (Bregs) in vitro after activated by CpG oligodeoxynucleotide (CpG-ODN) and anti-CD40 mAb. Methods Mice splenic CD5(+)CD1d(high)B cells and CD5(-)CD1d(low)B cells were sorted by flow cytometry. These B cells were first stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours, and then co-cultured with purified CD4(+)T cells. The interleukin 10 (IL-10) expression in the activated Bregs and other B cell subset, as well as the proliferation and interferon γ (IFN-γ) expression in the CD4(+) T cells activated by anti-CD3 mAb plus anti-CD28 mAb were determined by flow cytometry. Results CD5(+)CD1d(high) B cells activated by CpG-ODN plus anti-CD40 mAb blocked the up-regulated CD4(+)T proliferation and significantly reduced the IFN-γ level. At the same time, activated CD5(-)CD1d(low)B cells showed no inhibitory effect on CD4(+)T cells. Further study revealed that IL-10 expression in the CD5(+)CD1d(high) B cells were much higher than that in the CD5(-)CD1d(low)B cells after stimulated with CpG-ODN combined with anti-CD40 mAb for 24 hours. Conclusion CD5(+)CD1d(high) B cells activated by CpG-ODN combined with anti-CD40 mAb have immune inhibitory effects on CD4(+)T cell activation in vitro , which possibly due to IL-10 secretion.

  9. HIV-1 intersection with CD4 T cell vesicle exocytosis: intercellular communication goes viral

    Directory of Open Access Journals (Sweden)

    Helena eSoares

    2014-09-01

    Full Text Available In cells of the immune system the secretion of extracellular vesicles is modulated through cellular activation. In particular, T cell activation is achieved through cell-cell contacts with antigen presenting cells and the consequent formation of a specialized signaling junction called the immunological synapse. Recent works on CD4 T cells have elucidated that cognate antigen recognition by the T cell receptor (TCR engages two distinct exocytic events. The first, involves the exocytic targeting of signaling molecules at the synaptic membrane and drives the functional architecture of the immunological synapse. The second, enlists the extracellular secretion of the TCR itself, once the functional architecture of the immunological synapse is accomplished. HIV-1, a human lymphotropic virus, has evolved sophisticated mechanisms to co-opt CD4 T cell physiology. Notably, it has become apparent that HIV-1 intersects the regulated secretory system of CD4 T cells in order to bud from the plasma membrane of the infected cell and to promote bystander cell death. Here, I review the relevance of CD4 vesicle exocytosis to immune regulation and to HIV-1 pathogenesis and discuss their potential therapeutic applications.

  10. IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells

    DEFF Research Database (Denmark)

    Skov, S; Bonyhadi, M; Odum, Niels

    2000-01-01

    The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells...... after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously...... activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels...

  11. CD4+ T regulatory cells from the colonic lamina propria of normal mice inhibit proliferation of enterobacteria-reactive, disease-inducing Th1-cells from scid mice with colitis

    DEFF Research Database (Denmark)

    Gad, M; Brimnes, J; Claesson, Mogens Helweg

    2003-01-01

    Adoptive transfer of CD4+ T cells into scid mice leads to a chronic colitis in the recipients. The transferred CD4+ T cells accumulate in the intestinal lamina propria (LP), express an activated Th1 phenotype and proliferate vigorously when exposed ex vivo to enteric bacterial antigens. As LP CD4......+ T cells from normal BALB/c mice do not respond to enteric bacterial antigens, we have investigated whether colonic LP-derived CD4+ T cells from normal mice suppress the antibacterial response of CD4+ T cells from scid mice with colitis. LP-derived CD4+ T cells cocultured with bone marrow......-derived dendritic cells effectively suppress the antibacterial proliferative response of CD4+ T cells from scid mice with colitis. The majority of these LP T-reg cells display a nonactivated phenotype and suppression is independent of antigen exposure, is partly mediated by soluble factor(s) different from IL-10...

  12. 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