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Sample records for cell antigen receptor

  1. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    Science.gov (United States)

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  2. Toxicities of chimeric antigen receptor T cells: recognition and management.

    Science.gov (United States)

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  3. Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

    Science.gov (United States)

    Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2014-12-18

    In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates.

  4. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

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

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

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

  6. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  7. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

    OpenAIRE

    Frigault, Matthew J.; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and...

  8. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter;

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  9. T Cell Receptors that Recognize the Tyrosinase Tumor Antigen | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Cancer Institute, Surgery Branch, Tumor Immunology Section, is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize T Cells Attacking Cancer: T Cell Receptors that Recognize the Tyrosinase Tumor Antigen

  10. Human antigen-specific regulatory T cells generated by T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Todd M Brusko

    Full Text Available BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff activity as determined by tumor cell growth and luciferase reporter

  11. 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.

  12. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  13. Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

    Science.gov (United States)

    Kim, Mi-Gyeong; Kim, Dongyoon; Suh, Soo-Kyung; Park, Zewon; Choi, Min Joung; Oh, Yu-Kyoung

    2016-04-01

    Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy. PMID:26895243

  14. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination.

    Science.gov (United States)

    Bonifaz, Laura C; Bonnyay, David P; Charalambous, Anna; Darguste, Dara I; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K; Moltedo, Bruno; Moran, Thomas M; Steinman, Ralph M

    2004-03-15

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic alpha-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the alphaDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell-mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models.

  15. CD28 and T cell antigen receptor signal transduction coordinately regulate interleukin 2 gene expression in response to superantigen stimulation

    OpenAIRE

    1992-01-01

    Activation of an immune response requires intercellular contact between T lymphocytes and antigen-presenting cells (APC). Interaction of the T cell antigen receptor (TCR) with antigen in the context of major histocompatibility molecules mediates signal transduction, but T cell activation appears to require the induction of a second costimulatory signal transduction pathway. Recent studies suggest that interaction of CD28 with B7 on APC might deliver such a costimulatory signal. To investigate...

  16. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination.

    Science.gov (United States)

    Qi, Qian; Cavanagh, Mary M; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E; Dekker, Cornelia L; Olshen, Richard A; Boyd, Scott D; Weyand, Cornelia M; Tian, Lu; Goronzy, Jörg J

    2016-03-30

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

  17. Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination.

    Science.gov (United States)

    Qi, Qian; Cavanagh, Mary M; Le Saux, Sabine; NamKoong, Hong; Kim, Chulwoo; Turgano, Emerson; Liu, Yi; Wang, Chen; Mackey, Sally; Swan, Gary E; Dekker, Cornelia L; Olshen, Richard A; Boyd, Scott D; Weyand, Cornelia M; Tian, Lu; Goronzy, Jörg J

    2016-03-30

    Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  18. Reconstitution of the B cell antigen receptor signaling components in COS cells.

    Science.gov (United States)

    Saouaf, S J; Kut, S A; Fargnoli, J; Rowley, R B; Bolen, J B; Mahajan, S

    1995-11-10

    To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation. PMID:7592958

  19. In Vivo Targeting of Antigens to Maturing Dendritic Cells via the DEC-205 Receptor Improves T Cell Vaccination

    Science.gov (United States)

    Bonifaz, Laura C.; Bonnyay, David P.; Charalambous, Anna; Darguste, Dara I.; Fujii, Shin-Ichiro; Soares, Helena; Brimnes, Marie K.; Moltedo, Bruno; Moran, Thomas M.; Steinman, Ralph M.

    2004-01-01

    The prevention and treatment of prevalent infectious diseases and tumors should benefit from improvements in the induction of antigen-specific T cell immunity. To assess the potential of antigen targeting to dendritic cells to improve immunity, we incorporated ovalbumin protein into a monoclonal antibody to the DEC-205 receptor, an endocytic receptor that is abundant on these cells in lymphoid tissues. Simultaneously, we injected agonistic α-CD40 antibody to mature the dendritic cells. We found that a single low dose of antibody-conjugated ovalbumin initiated immunity from the naive CD4+ and CD8+ T cell repertoire. Unexpectedly, the αDEC-205 antigen conjugates, given s.c., targeted to dendritic cells systemically and for long periods, and ovalbumin peptide was presented on MHC class I for 2 weeks. This was associated with stronger CD8+ T cell–mediated immunity relative to other forms of antigen delivery, even when the latter was given at a thousand times higher doses. In parallel, the mice showed enhanced resistance to an established rapidly growing tumor and to viral infection at a mucosal site. By better harnessing the immunizing functions of maturing dendritic cells, antibody-mediated antigen targeting via the DEC-205 receptor increases the efficiency of vaccination for T cell immunity, including systemic and mucosal resistance in disease models. PMID:15024047

  20. The T cell antigen receptor: the Swiss army knife of the immune system.

    Science.gov (United States)

    Attaf, M; Legut, M; Cole, D K; Sewell, A K

    2015-07-01

    The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These 'unconventional' T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors.

  1. Chimeric antigen receptor T cell therapy: 25years in the making.

    Science.gov (United States)

    Gill, Saar; Maus, Marcela V; Porter, David L

    2016-05-01

    Chimeric antigen receptor (CAR) T cell therapy of cancer is generating enormous enthusiasm. Twenty-five years after the concept was first proposed, major advances in molecular biology, virology, and good manufacturing practices (GMP)-grade cell production have transformed antibody-T cell chimeras from a scientific curiosity to a fact of life for academic cellular immunotherapy researchers and, increasingly, for patients. In this review, we explain the preclinical concept, outline how it has been translated to the clinic, and draw lessons from the first years of CAR T cell therapy for the practicing clinician. PMID:26574053

  2. Chimeric Antigen Receptor Therapy for B-cell Malignancies

    Directory of Open Access Journals (Sweden)

    David L Porter, Michael Kalos, Zhaohui Zheng, Bruce Levine, Carl June

    2011-01-01

    Full Text Available We presented data showing that the CART-19 cells expressing the 4-1BB signaling domain can have unprecedented and massive in-vivo expansion, traffic to tumor sites, persist long term in vivo, and induce rapid and potent anti-tumor activity in chemotherapy refractory CLL patients.

  3. Chimeric Antigen Receptor Therapy for B-cell Malignancies

    OpenAIRE

    Porter, David L.; Kalos, Michael; Zheng, Zhaohui; Levine, Bruce; June, Carl

    2011-01-01

    We presented data showing that the CART-19 cells expressing the 4-1BB signaling domain can have unprecedented and massive in-vivo expansion, traffic to tumor sites, persist long term in vivo, and induce rapid and potent anti-tumor activity in chemotherapy refractory CLL patients.

  4. Beyond the antigen receptor: editing the genome of T-cells for cancer adoptive cellular therapies

    Directory of Open Access Journals (Sweden)

    Angharad eLloyd

    2013-08-01

    Full Text Available Recent early-stage clinical trials evaluating the adoptive transfer of patient CD8+ T-cells re-directed with antigen receptors recognising tumours have shown very encouraging results. These reports provide strong support for further development of the therapeutic concept as a curative cancer treatment. In this respect combining the adoptive transfer of tumour-specific T-cells with therapies that increase their anti-tumour capacity is viewed as a promising strategy to improve treatment outcome. The ex-vivo genetic engineering step that underlies T-cell re-direction offers a unique angle to combine antigen receptor delivery with the targeting of cell intrinsic pathways that restrict T-cell effector functions. Recent progress in genome editing technologies such as protein- and RNA-guided endonucleases raise the possibility of disrupting gene expression in T-cells in order to enhance effector functions or to bypass tumour immune suppression. This approach would avoid the systemic administration of compounds that disrupt immune homeostasis, potentially avoiding autoimmune adverse effects, and could improve the efficacy of T-cell based adoptive therapies.

  5. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

    Full Text Available Natural killer (NK cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs, which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  6. Rational design of T cell receptors with enhanced sensitivity for antigen.

    Directory of Open Access Journals (Sweden)

    Rajshekhar Alli

    Full Text Available Enhancing the affinity of therapeutic T cell receptors (TCR without altering their specificity is a significant challenge for adoptive immunotherapy. Current efforts have primarily relied on empirical approaches. Here, we used structural analyses to identify a glycine-serine variation in the TCR that modulates antigen sensitivity. A G at position 107 within the CDR3β stalk is encoded within a single mouse and human TCR, TRBV13-2 and TRBV12-5 respectively. Most TCR bear a S107. The S hydroxymethyl side chain intercalates into the core of the CDR3β loop, stabilizing it. G107 TRBV possess a gap in their CDR3β where this S hydroxymethyl moiety would fit. We predicted based on modeling and molecular dynamics simulations that a G107S substitution would increase CDR3β stability and thereby augment receptor sensitivity. Experimentally, a G107S replacement led to an ∼10-1000 fold enhanced antigen sensitivity in 3 of 4 TRBV13-2(+ TCR tested. Analysis of fine specificity indicated a preserved binding orientation. These results support the feasibility of developing high affinity antigen specific TCR for therapeutic purposes through the identification and manipulation of critical framework residues. They further indicate that amino acid variations within TRBV not directly involved in ligand contact can program TCR sensitivity, and suggest a role for CDR3 stability in this programming.

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

    International Nuclear Information System (INIS)

    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)

  8. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

    Science.gov (United States)

    Levine, B L

    2015-03-01

    Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards. PMID:25675873

  9. The B cell antigen receptor and overexpression of MYC can cooperate in the genesis of B cell lymphomas.

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    Yosef Refaeli

    2008-06-01

    Full Text Available A variety of circumstantial evidence from humans has implicated the B cell antigen receptor (BCR in the genesis of B cell lymphomas. We generated mouse models designed to test this possibility directly, and we found that both the constitutive and antigen-stimulated state of a clonal BCR affected the rate and outcome of lymphomagenesis initiated by the proto-oncogene MYC. The tumors that arose in the presence of constitutive BCR differed from those initiated by MYC alone and resembled chronic B cell lymphocytic leukemia/lymphoma (B-CLL, whereas those that arose in response to antigen stimulation resembled large B-cell lymphomas, particularly Burkitt lymphoma (BL. We linked the genesis of the BL-like tumors to antigen stimulus in three ways. First, in reconstruction experiments, stimulation of B cells by an autoantigen in the presence of overexpressed MYC gave rise to BL-like tumors that were, in turn, dependent on both MYC and the antigen for survival and proliferation. Second, genetic disruption of the pathway that mediates signaling from the BCR promptly killed cells of the BL-like tumors as well as the tumors resembling B-CLL. And third, growth of the murine BL could be inhibited by any of three distinctive immunosuppressants, in accord with the dependence of the tumors on antigen-induced signaling. Together, our results provide direct evidence that antigenic stimulation can participate in lymphomagenesis, point to a potential role for the constitutive BCR as well, and sustain the view that the constitutive BCR gives rise to signals different from those elicited by antigen. The mouse models described here should be useful in exploring further the pathogenesis of lymphomas, and in preclinical testing of new therapeutics.

  10. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    Science.gov (United States)

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials.

  11. Mast cell density and the context of clinicopathological parameters and expression of p185,estrogen receptor,and proliferating cell nuclear antigen in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ying-AnJiang; You-YuanZhang; He-ShengLuo; Shou-FuXing

    2002-01-01

    AIM:To investigate the relationship between the mast cell density(MCD)and the context of clinicopathological parameters and expression of p185,estrogen receptor(ER),and proliferating cell nuclear antigen(PCNA)in gastric carcinoma.

  12. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

    Science.gov (United States)

    Mock, Ulrike; Nickolay, Lauren; Philip, Brian; Cheung, Gordon Weng-Kit; Zhan, Hong; Johnston, Ian C D; Kaiser, Andrew D; Peggs, Karl; Pule, Martin; Thrasher, Adrian J; Qasim, Waseem

    2016-08-01

    Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

  13. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam

    2009-01-01

    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  14. Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

    Science.gov (United States)

    Pinz, K; Liu, H; Golightly, M; Jares, A; Lan, F; Zieve, G W; Hagag, N; Schuster, M; Firor, A E; Jiang, X; Ma, Y

    2016-03-01

    Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

  15. Effects of dexamethasone on human natural killer cell cytotoxicity, interferon production, and interleukin-2 receptor expression induced by microbial antigens.

    OpenAIRE

    E. Piccolella; Lombardi, G.; Vismara, D; Del Gallo, F; Colizzi, V; Dolei, A; Dianzani, F

    1986-01-01

    Dexamethasone inhibits the expression of the interleukin-2 receptor, the synthesis of immune interferon, and the development of natural killer cells when added to peripheral blood mononuclear cells cultured with soluble microbial antigens (purified protein derivative and a polysaccharide extract from Candida albicans [MPPS]) or human recombinant interleukin-2.

  16. FK506 inhibits antigen receptor-mediated induction of c-rel in B and T lymphoid cells

    OpenAIRE

    1995-01-01

    Stimulation of B and T cells via the antigen receptor, by phorbol ester or by phorbol ester and ionomycin, leads to nuclear translocation of the inducible transcription factor NF-kappa B, comprising the p50 and p65 rel-related polypeptides. In this report we show that c-rel is a component of the antigen receptor-induced kappa B binding proteins in both B and T cells. Whereas NF-kappa B can be induced by phorbol ester alone, optimal induction of c-rel requires stimulation by both phorbol ester...

  17. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1.

    OpenAIRE

    Weiss, A; Koretzky, G; Schatzman, R C; Kadlecek, T

    1991-01-01

    Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nu...

  18. Adoptive Immunotherapy for Hematological Malignancies Using T Cells Gene-Modified to Express Tumor Antigen-Specific Receptors

    Directory of Open Access Journals (Sweden)

    Hiroshi Fujiwara

    2014-12-01

    Full Text Available Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs, transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

  19. Cancer T cell immunotherapy with bispecific antibodies and chimeric antigen receptors.

    Science.gov (United States)

    Lacher, Markus D; Provenzano, Maurizio

    2013-09-01

    Solid tumors contain several different types of malignant cells. This cellular heterogeneity complicates therapy for at least two reasons. First, each subpopulation may respond differently to a given treatment. Second, cancer cells are plastic, and thus may convert from a therapy-sensitive to a therapy-resistant cell type represented by another subpopulation. Therefore, successful therapies will have to target numerous malignant cell types, not just the rapidly proliferating cells as most standard treatments do. Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are particularly promising approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells. Here, we discuss several patents associated with exceptionally effective bsAbs of the tandem single-chain variable fragment (taFv) class and untangle a part of the complex network of patents directly or indirectly related to CARs. Furthermore, we speculate on the future of bsAbs and CARs for both treatment and prevention of solid tumors such as prostate cancer. PMID:23688207

  20. Chimeric Antigen Receptor-Modified T Cells for Solid Tumors: Challenges and Prospects

    Directory of Open Access Journals (Sweden)

    Yelei Guo

    2016-01-01

    Full Text Available Recent studies have highlighted the successes of chimeric antigen receptor-modified T- (CART- cell-based therapy for B-cell malignancies, and early phase clinical trials have been launched in recent years. The few published clinical studies of CART cells in solid tumors have addressed safety and feasibility, but the clinical outcome data are limited. Although antitumor effects were confirmed in vitro and in animal models, CART-cell-based therapy still faces several challenges when directed towards solid tumors, and it has been difficult to achieve the desired outcomes in clinical practice. Many studies have struggled to improve the clinical responses to and benefits of CART-cell treatment of solid tumors. In this review, the status quo of CART cells and their clinical applications for solid tumors will be summarized first. Importantly, we will suggest improvements that could increase the therapeutic effectiveness of CART cells for solid tumors and their future clinical applications. These interventions will make treatment with CART cells an effective and routine therapy for solid tumors.

  1. Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

    Directory of Open Access Journals (Sweden)

    Margaret Veselits

    Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

  2. 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.

  3. Prospects for adoptive immunotherapy of pancreatic cancer using chimeric antigen receptor-engineered T-cells.

    Science.gov (United States)

    Alrifai, Doraid; Sarker, Debashis; Maher, John

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor (CAR) engineered T-cells is emerging as a powerful new approach to cancer immunotherapy. CARs are fusion molecules that couple the antibody-like binding of a native cell surface target to the delivery of a bespoke T-cell activating signal. Recent studies undertaken by several centers have demonstrated highly compelling efficacy in patients with acute and chronic B-cell malignancies. However, comparable therapeutic activity has not been achieved in solid tumors. Modern management of pancreatic ductal adenocarcinoma (PDAC) remains ineffective, reflected in the virtual equivalence of annual incidence and mortality statistics for this tumor type. Increasing evidence indicates that these tumors are recognized by the immune system, but deploy powerful evasion strategies that limit natural immune surveillance and render efforts at immunotherapy challenging. Here, we review preclinical and clinical studies that have been initiated or completed in an effort to develop CAR-based immunotherapy for PDAC. We also consider the hurdles to the effective clinical development of this exciting new therapeutic modality.

  4. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

    Directory of Open Access Journals (Sweden)

    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. OCA-B integrates B cell antigen receptor-, CD40L- and IL 4-mediated signals for the germinal center pathway of B cell development.

    OpenAIRE

    Qin, X F; Reichlin, A; Luo, Y.; Roeder, R. G.; Nussenzweig, M.C.

    1998-01-01

    Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is re...

  7. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    OpenAIRE

    Saouaf, S J; Mahajan, S.; Rowley, R B; Kut, S A; Fargnoli, J.; Burkhardt, A L; Tsukada, S; Witte, O. N.; Bolen, J B

    1994-01-01

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity wa...

  8. A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

    Science.gov (United States)

    Lagisetty, Kiran H.; Tran, Eric; Zheng, Zhili; Gattinoni, Luca; Yu, Zhiya; Burns, William R.; Miermont, Anne M.; Teper, Yaroslav; Rudloff, Udo; Restifo, Nicholas P.; Feldman, Steven A.; Rosenberg, Steven A.; Morgan, Richard A.

    2014-01-01

    Abstract Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity. PMID:24694017

  9. The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

    Institute of Scientific and Technical Information of China (English)

    Alan DGuerrero; Judy SMoyes; Laurence JN Cooper

    2014-01-01

    The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cels to target B-cellmalignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin’s lymphoma.

  10. CD4+ T Cells and Toll-Like Receptors Recognize Salmonella Antigens Expressed in Bacterial Surface Organelles

    OpenAIRE

    Bergman, Molly A.; Cummings, Lisa A.; Barrett, Sara L. Rassoulian; Smith, Kelly D.; Lara, J. Cano; Aderem, Alan; Cookson, Brad T.

    2005-01-01

    A better understanding of immunity to infection is revealed from the characteristics of microbial ligands recognized by host immune responses. Murine infection with the intracellular bacterium Salmonella generates CD4+ T cells that specifically recognize Salmonella proteins expressed in bacterial surface organelles such as flagella and membrane vesicles. These natural Salmonella antigens are also ligands for Toll-like receptors (TLRs) or avidly associated with TLR ligands such as lipopolysacc...

  11. Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease.

    Science.gov (United States)

    Jensen, Majbrit M; Arvaniti, Maria; Mikkelsen, Jens D; Michalski, Dominik; Pinborg, Lars H; Härtig, Wolfgang; Thomsen, Morten S

    2015-04-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and modulating their function. Hence, changes in nAChR regulatory proteins such as Lynx proteins could underlie the dysregulation of nAChRs in AD. Using Western blotting, we detected bands corresponding to the Lynx proteins prostate stem cell antigen (PSCA) and Lypd6 in human cortex indicating that both proteins are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial frontal gyrus from AD patients and found significantly increased PSCA levels (approximately 70%). In contrast, no changes in Lypd6 levels were detected. In concordance with our findings in AD patients, PSCA levels were increased in the frontal cortex of triple transgenic mice with an AD-like pathology harboring human transgenes that cause both age-dependent β-amyloidosis and tauopathy, whereas Tg2576 mice, which display β-amyloidosis only, had unchanged PSCA levels compared to wild-type animals. These findings identify PSCA as a nAChR-binding protein in the human brain that is affected in AD, suggesting that PSCA-nAChR interactions may be involved in the cognitive dysfunction observed in AD. PMID:25680266

  12. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells

    OpenAIRE

    Kochenderfer, James N.; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A.

    2010-01-01

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)–expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19–CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19–CAR-tr...

  13. HLA-DR, DQ and T cell antigen receptor constant beta genes in Japanese patients with ulcerative colitis.

    Science.gov (United States)

    Kobayashi, K; Atoh, M; Konoeda, Y; Yagita, A; Inoko, H; Sekiguchi, S

    1990-01-01

    We studied the T cell antigen receptor (TcR) constant beta chain genes on HLA typed Japanese patients with ulcerative colitis (UC). A TcR constant beta EcoRI 6.0-kb fragment was present in all Japanese UC patients (n = 17) but completely absent in the controls (n = 35) (chi2 = 47.6, P less than 0.001). The frequency of HLA-DR2 antigen was significantly higher in UC patients (85% versus 28% in controls, P less than 0.001). Furthermore, HLA-DQw1 antigen was also increased in UC patients (96% versus 60% in controls, P less than 0.001). However, HLA-DR4 antigen was significantly decreased in UC patients (12% versus 37%, P = 0.02). HLA-DR1 antigen was not found in UC patients and was present in only 15% of the controls. These results suggest that TcR beta chain and HLA-DQw1 antigen may be important in the pathogenesis of Japanese UC. Images Fig. 1 PMID:1973647

  14. Protein kinase Cβ is critical for the metabolic switch to glycolysis following B-cell antigen receptor engagement.

    Science.gov (United States)

    Blair, Derek; Dufort, Fay J; Chiles, Thomas C

    2012-11-15

    Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

  15. Application of Adoptive T-Cell Therapy Using Tumor Antigen-Specific T-Cell Receptor Gene Transfer for the Treatment of Human Leukemia

    Directory of Open Access Journals (Sweden)

    Toshiki Ochi

    2010-01-01

    Full Text Available The last decade has seen great strides in the field of cancer immunotherapy, especially the treatment of melanoma. Beginning with the identification of cancer antigens, followed by the clinical application of anti-cancer peptide vaccination, it has now been proven that adoptive T-cell therapy (ACT using cancer antigen-specific T cells is the most effective option. Despite the apparent clinical efficacy of ACT, the timely preparation of a sufficient number of cancer antigen-specific T cells for each patient has been recognized as its biggest limitation. Currently, therefore, attention is being focused on ACT with engineered T cells produced using cancer antigen-specific T-cell receptor (TCR gene transfer. With regard to human leukemia, ACT using engineered T cells bearing the leukemia antigen-specific TCR gene still remains in its infancy. However, several reports have provided preclinical data on TCR gene transfer using Wilms' tumor gene product 1 (WT1, and also preclinical and clinical data on TCR gene transfer involving minor histocompatibility antigen, both of which have been suggested to provide additional clinical benefit. In this review, we examine the current status of anti-leukemia ACT with engineered T cells carrying the leukemia antigen-specific TCR gene, and discuss the existing barriers to progress in this area.

  16. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    Science.gov (United States)

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-09-27

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. PMID:7524079

  17. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

    Science.gov (United States)

    Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben; Choi, Eun Jung; Mao, Xuming; Cho, Michael Jeffrey; Di Zenzo, Giovanni; Lanzavecchia, Antonio; Seykora, John T; Cotsarelis, George; Milone, Michael C; Payne, Aimee S

    2016-07-01

    Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease. PMID:27365313

  18. Synergistic effect of chimeric antigen receptors and cytokine-induced killer cells: An innovative combination for cancer therapy

    Directory of Open Access Journals (Sweden)

    Binh Thanh Vu

    2016-06-01

    Full Text Available In recent years, the combination of gene and immunotherapy for cancer treatment has been regarded as innovative and promising; together, both therapies can help overcome limitations associated with conventional treatments. In order to augment anti-cancer efficacy and to maintain the specificity of antibody therapy, chimeric antigen receptor (CAR-modified T cells, directed toward tumor-specific antigens, have emerged as a novel and promising therapeutic platform. CARs consist of a B cell receptor (BCR-derived extracellular domain and T cell receptor (TCR-associated signaling elements. Cytokine-induced killer (CIK cells are the effector immune cells that can be activated ex vivo and possess both the anti-tumor potency of T lymphocytes and the non-major histocompatibility complex-restricted elimination of natural killer cells. With their pre-eminent ability for robust proliferation, CIK cells may overcome the main limitations of adoptive immunotherapy strategies. CIK cells have strong tumor cell killing capacity; they are effective against a wide variety of malignant tumors and have been shown to be safe in cancer patients. This review summarizes the characteristics of CARs which make them attractive for in cancer treatment strategies. In addition, the role of CIK cells and the advantages of combining CIK cells with CAR-based therapy will be discussed. Scientific evidence to support their combined therapeutic application will be highlighted, with a focus on how their innovative combination may be translated into cancer clinical trials. [Biomed Res Ther 2016; 3(6.000: 653-665

  19. It’s All About Change: The Antigen-driven Initiation of B-Cell Receptor Signaling

    Science.gov (United States)

    Liu, Wanli; Sohn, Hae Won; Tolar, Pavel; Pierce, Susan K.

    2010-01-01

    B-cell responses are initiated by the binding of foreign antigens to the clonally distributed B-cell receptors (BCRs) resulting in the triggering of signaling cascades that activate a variety of genes associated with B-cell activation. Although we now understand the molecular nature of the signaling pathways in considerable detail what remains only poorly understood are the mechanisms by which the information that antigen has bound to the BCR ectodomain is transduced across the B-cell membrane to the BCR cytoplasmic domains to trigger signaling. To a large part this gap in knowledge is because of the paucity of techniques to temporally and spatially resolve changes in the behavior of the BCR that occur within several seconds of antigen binding. With the advent of new live-cell imaging technologies we are gaining our first clear views of the events that lead up to the triggering of BCR signaling cascades. These events may provide potential new targets for therapeutic intervention in disease involving hyper or chronic activation of B cells. PMID:20591989

  20. Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T Cell Tolerance to Allogeneic Antigens.

    Science.gov (United States)

    Flies, Dallas B; Higuchi, Tomoe; Chen, Lieping

    2015-06-01

    PD-1H is a recently identified cell surface coinhibitory molecule of the B7/CD28 immune modulatory gene family. We showed previously that single injection of a PD-1H agonistic mAb protected mice from graft-versus-host disease (GVHD). In this study, we report two distinct mechanisms operate in PD-1H-induced T cell tolerance. First, signaling via PD-1H coinhibitory receptor potently arrests alloreactive donor T cells from activation and expansion in the initiation phase. Second, donor regulatory T cells are subsequently expanded to maintain long-term tolerance and GVHD suppression. Our study reveals the crucial function of PD-1H as a coinhibitory receptor on alloreactive T cells and its function in the regulation of T cell tolerance. Therefore, PD-1H may be a target for the modulation of alloreactive T cells in GVHD and transplantation. PMID:25917101

  1. 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...... responses in patients with chronic lymphocytic leukaemia (CLL) and acute lymphoblastic leukaemia (ALL). So far, eleven clinical trials including 99 CLL and ALL patients treated with CAR T cells targeting CD19 have been published, and the results from these trials are promising with impressive clinical...... responses in heavily pretreated patients. Thus, CAR T cell therapy has induced complete responses in both CLL and ALL, and surprisingly, current results indicate that patients with ALL are more prone to respond than are CLL patients. Importantly, the majority of CAR cell studies have observed severe therapy...

  2. Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin.

    OpenAIRE

    1984-01-01

    A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for pol...

  3. 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.

  4. Review of Cancer Immunotherapy: Application of Chimeric Antigen Receptor T Cells and Programmed Death 1/Programmed Death-ligand 1 Antibodies

    Directory of Open Access Journals (Sweden)

    Tengfei Zhang

    2015-01-01

    Full Text Available Cancer immunotherapy strategies based on chimeric antigen receptor (CAR transduced T cells or antibodies against immune checkpoints, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 and programmed death 1 (PD-1, achieved significant successes from bench to clinic in the past 2 years. CARs are artificial engineered receptors that can specifically target tumor cell surface antigen, activate T cell and further enhance T cell function, independent of major histocompatibility complex. CAR T cells have shown promising outcomes in cancers, especially in hematologic malignancies. CTLA-4 and PD-1 are two important immune checkpoints negatively regulating T cell activation. Clinical benefits of CTLA-4/PD-1 antibodies are significant in melanoma and other solid tumors. PD-1 is predicted to have fewer side effects and greater antitumor activity than CTLA-4. In this review, we will summarize current immunotherapies based on CAR T cells and PD-1.

  5. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    Science.gov (United States)

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  6. Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy.

    Science.gov (United States)

    Mirzaei, Hamid Reza; Mirzaei, Hamed; Lee, Sang Yun; Hadjati, Jamshid; Till, Brian G

    2016-10-01

    Excitement is growing for therapies that harness the power of patients' immune systems to combat their diseases. One approach to immunotherapy involves engineering patients' own T cells to express a chimeric antigen receptor (CAR) to treat advanced cancers, particularly those refractory to conventional therapeutic agents. Although these engineered immune cells have made remarkable strides in the treatment of patients with certain hematologic malignancies, success with solid tumors has been limited, probably due to immunosuppressive mechanisms in the tumor niche. In nearly all studies to date, T cells bearing αβ receptors have been used to generate CAR T cells. In this review, we highlight biological characteristics of γδ T cells that are distinct from those of αβ T cells, including homing to epithelial and mucosal tissues and unique functions such as direct antigen recognition, lack of alloreactivity, and ability to present antigens. We offer our perspective that these features make γδ T cells promising for use in cellular therapy against several types of solid tumors, including melanoma and gastrointestinal cancers. Engineered γδ T cells should be considered as a new platform for adoptive T cell cancer therapy for mucosal tumors. PMID:27392648

  7. Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

    Science.gov (United States)

    Globerson-Levin, Anat; Waks, Tova; Eshhar, Zelig

    2014-05-01

    Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

  8. Kinetics of human T-cell expression of LFA-1, IL-2 receptor, and ICAM-1 following antigenic stimulation in vitro

    DEFF Research Database (Denmark)

    Hviid, L; Felsing, A; Theander, T G

    1993-01-01

    in vitro is paralleled by differential kinetics in the expression of the T-cell adhesion and activation antigens leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18), interleukin-2 receptor (IL-2R; CD25), and intercellular adhesion molecule 1 (ICAM-1; CD54). Furthermore, the changes in expression...... prestimulation levels, and CD25 expression was decreasing. This indicates that T-cell expression of all the 3 surface antigens examined is reversible. While this is in agreement with previous reports of the expression kinetics of IL-2R and ICAM-1, this is the first report indicating that the regulation of T...

  9. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    Science.gov (United States)

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  10. Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

    Directory of Open Access Journals (Sweden)

    Karasuyama Hajime

    2011-04-01

    Full Text Available Abstract Background There have been few reports on the role of Fc receptors (FcRs and immunoglobulin G (IgG in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa. Methods In FcγRIIB deficient (KO and C57BL/6 (WT mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA. Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL. Results In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously. Conclusion Antigen-specific IgG ameliorates

  11. Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

    Science.gov (United States)

    Sherman, Eilon

    2016-06-01

    Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

  12. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E;

    2014-01-01

    ) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated...... by effector memory CD8+ T cells. CONCLUSION: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels......OBJECTIVES: Although CD8+ T cells play a critical role in the control of HIV-1 infection,their antiviral efficacy can be limited by antigenic variation and immune exhaustion.The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1...

  13. The Dynamics of the Human Leukocyte Antigen Head Domain Modulates Its Recognition by the T-Cell Receptor.

    Directory of Open Access Journals (Sweden)

    Estefanía García-Guerrero

    Full Text Available Generating the immune response requires the discrimination of peptides presented by the human leukocyte antigen complex (HLA through the T-cell receptor (TCR. However, how a single amino acid substitution in the antigen bonded to HLA affects the response of T cells remains uncertain. Hence, we used molecular dynamics computations to analyze the molecular interactions between peptides, HLA and TCR. We compared immunologically reactive complexes with non-reactive and weakly reactive complexes. MD trajectories were produced to simulate the behavior of isolated components of the various p-HLA-TCR complexes. Analysis of the fluctuations showed that p-HLA binding barely restrains TCR motions, and mainly affects the CDR3 loops. Conversely, inactive p-HLA complexes displayed significant drop in their dynamics when compared with its free versus ternary forms (p-HLA-TCR. In agreement, the free non-reactive p-HLA complexes showed a lower amount of salt bridges than the responsive ones. This resulted in differences between the electrostatic potentials of reactive and inactive p-HLA species and larger vibrational entropies in non-elicitor complexes. Analysis of the ternary p-HLA-TCR complexes also revealed a larger number of salt bridges in the responsive complexes. To summarize, our computations indicate that the affinity of each p-HLA complex towards TCR is intimately linked to both, the dynamics of its free species and its ability to form specific intermolecular salt-bridges in the ternary complexes. Of outstanding interest is the emerging concept of antigen reactivity involving its interplay with the HLA head sidechain dynamics by rearranging its salt-bridges.

  14. Random length assortment of human and mouse T cell receptor for antigen alpha and beta chain CDR3.

    Science.gov (United States)

    Johnson, G; Wu, T T

    1999-10-01

    In view of the recently determined three-dimensional structures of complexes formed by the T cell receptor for antigen (TCR), the processed peptide and the MHC class I molecule, it is expected that the combined configuration formed by the third complementarity determining regions (CDR3) of TCR alpha and beta chains will be very restricted in size and shape due to the limited length variations of the processed peptides. Thus, the combined TCR alpha and beta chain CDR3 lengths should have a fairly narrow distribution. This feature can be due to the selective association of long alpha chain CDR3 with short beta chain CDR3 and vice versa or due to random assortment of alpha and beta chain CDR3 of even narrower length distribution. Based on existing translated amino acid sequence data, it has been found that the latter mechanism is responsible.

  15. Aging affects B-cell antigen receptor repertoire diversity in primary and secondary lymphoid tissues.

    Science.gov (United States)

    Tabibian-Keissar, Hilla; Hazanov, Lena; Schiby, Ginette; Rosenthal, Noemie; Rakovsky, Aviya; Michaeli, Miri; Shahaf, Gitit Lavy; Pickman, Yishai; Rosenblatt, Kinneret; Melamed, Doron; Dunn-Walters, Deborah; Mehr, Ramit; Barshack, Iris

    2016-02-01

    The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms.

  16. High Throughput Sequencing of T Cell Antigen Receptors Reveals a Conserved TCR Repertoire

    Science.gov (United States)

    Hou, Xianliang; Lu, Chong; Chen, Sisi; Xie, Qian; Cui, Guangying; Chen, Jianing; Chen, Zhi; Wu, Zhongwen; Ding, Yulong; Ye, Ping; Dai, Yong; Diao, Hongyan

    2016-01-01

    Abstract The T-cell receptor (TCR) repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next-generation sequencing has become a powerful tool for deep TCR profiling. Herein, we used this technology to study the repertoire features of TCR beta chain in the blood of healthy individuals. Peripheral blood samples were collected from 10 healthy donors. T cells were isolated with anti-human CD3 magnetic beads according to the manufacturer's protocol. We then combined multiplex-PCR, Illumina sequencing, and IMGT/High V-QUEST to analyze the characteristics and polymorphisms of the TCR. Most of the individual T cell clones were present at very low frequencies, suggesting that they had not undergone clonal expansion. The usage frequencies of the TCR beta variable, beta joining, and beta diversity gene segments were similar among T cells from different individuals. Notably, the usage frequency of individual nucleotides and amino acids within complementarity-determining region (CDR3) intervals was remarkably consistent between individuals. Moreover, our data show that terminal deoxynucleotidyl transferase activity was biased toward the insertion of G (31.92%) and C (27.14%) over A (21.82%) and T (19.12%) nucleotides. Some conserved features could be observed in the composition of CDR3, which may inform future studies of human TCR gene recombination. PMID:26962778

  17. Conserved structure of amphibian T-cell antigen receptor beta chain.

    Science.gov (United States)

    Fellah, J S; Kerfourn, F; Guillet, F; Charlemagne, J

    1993-07-15

    All jawed vertebrates possess well-differentiated thymuses and elicit T-cell-like cell-mediated responses; however, no surface T-cell receptor (TCR) molecules or TCR genes have been identified in ectothermic vertebrate species. Here we describe cDNA clones from an amphibian species, Ambystoma mexicanum (the Mexican axolotl), that have sequences highly homologous to the avian and mammalian TCR beta chains. The cloned amphibian beta chain variable region (V beta) shares most of the structural characteristics with the more evolved vertebrate V beta and presents approximately 56% amino acid identities with the murine V beta 14 and human V beta 18 families. The two different cloned axolotl beta chain joining regions (J beta) were found to have conserved all the invariant mammalian J beta residues, and in addition, the presence of a conserved glycine at the V beta-J beta junction suggests the existence of diversity elements. The extracellular domains of the two axolotl beta chain constant region isotypes C beta 1 and C beta 2 show an impressively high degree of identity, thus suggesting that a very efficient mechanism of gene correction has been in operation to preserve this structure at least from the early tetrapod evolution. The transmembrane axolotl C beta domains have been less well conserved when compared to the mammalian C beta but they do maintain the lysine residue that is thought to be involved in the charged interaction between the TCR alpha beta heterodimer and the CD3 complex. PMID:8341702

  18. BRAF and MEK inhibition variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function in vitro.

    Science.gov (United States)

    Gargett, Tessa; Fraser, Cara K; Dotti, Gianpietro; Yvon, Eric S; Brown, Michael P

    2015-01-01

    Cancer immunotherapy has long been used in the treatment of metastatic melanoma, and an anti-CTLA-4 monoclonal antibody treatment has recently been approved by the US Food and Drug Administration. Targeted therapies such as small molecule kinase inhibitors targeting deregulated mitogen-activated protein kinase (MAPK) signaling have markedly improved melanoma control in up to 50% of metastatic disease patients and have likewise been recently approved. Combination therapies for melanoma have been proposed as a way to exploit the high-level but short-term responses associated with kinase inhibitor therapies and the low-level but longer-term responses associated with immunotherapy. Cancer immunotherapy now includes adoptive transfer of autologous tumor-specific chimeric antigen receptor (CAR) T cells and this mode of therapy is a candidate for combination with small molecule drugs. This paper describes CART cells that target GD2-expressing melanoma cells and investigates the effects of approved MAPK pathway-targeted therapies for melanoma [vemurafenib (Vem), dabrafenib (Dab), and trametinib (Tram)] on the viability, activation, proliferation, and cytotoxic T lymphocyte activity of these CAR T cells, as well as on normal peripheral blood mononuclear cells. We report that, although all these drugs lead to inhibition of stimulated T cells at high concentrations in vitro, only Vem inhibited T cells at concentrations equivalent to reported plasma concentrations in treated patients. Although the combination of Dab and Tram also resulted in inhibition of T-cell effector functions at some therapeutic concentrations, Dab itself had little adverse effect on CAR T-cell function. These findings may have implications for novel therapeutic combinations of adoptive CAR T-cell immunotherapy and MAPK pathway inhibitors.

  19. DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

    Science.gov (United States)

    Liu, Yang; Blanchfield, Lori; Ma, Victor Pui-Yan; Andargachew, Rakieb; Galior, Kornelia; Liu, Zheng; Evavold, Brian; Salaita, Khalid

    2016-05-17

    T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12-19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically "filtered" to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

  20. Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

    Directory of Open Access Journals (Sweden)

    Rafał Biedroń

    Full Text Available The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl, causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin and glycoproteins (human apo-transferrin, ovalbumin gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206, scavenger receptors A (CD204 and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system

  1. Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

    Institute of Scientific and Technical Information of China (English)

    杨建民; MichaelSFRIEDMAN; ChristopherMREYNOLDS; MarianneTHUBEN; LeeWILKE; JenniferFULLER; 李桥; ZeligESHHAR; JamesJMULE; KevimTMCDONAGH

    2004-01-01

    We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments,retroviral vectors expressing the N297 or N29ξ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were vitally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific immune responses. Both CD4+ and CD8+ T-cells transduced with the N297 or N29ξ chTCR demonstrated HER2-specific antigen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the feasibility of adoptive immunothempy with genetically modified T-cells expressing a chTCR specific for p185HER2.

  2. Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Yani Zhao

    Full Text Available The Duffy antigen receptor for chemokines (DARC shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated (125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. (125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression.(125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

  3. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x.

    NARCIS (Netherlands)

    Die, van I.M.; Vliet, van SJ; Nyame, AK; Cummings, RD; Bank, CM; Appelmelk, B.J.; Geijtenbeek, T.B.H.; Kooijk, van Y.

    2003-01-01

    Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies agai

  4. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    Science.gov (United States)

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  5. Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle.

    Science.gov (United States)

    Hombach, Andreas A; Abken, Hinrich

    2013-01-01

    Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1(+) CD57(+) CD7(-) CCR7(-) phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy. PMID:23761793

  6. Young T cells age during a redirected anti-tumour attack: chimeric antigen receptor (CAR-provided dual costimulation is half the battle.

    Directory of Open Access Journals (Sweden)

    Andreas A Hombach

    2013-06-01

    Full Text Available Adoptive therapy with chimeric antigen receptor (CAR-redirected T cells showed spectacular efficacy in the treatment of leukaemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG-1+ CD57+ CD7- CCR7- phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134 stimulation. We discuss the strategy with respect to prolong the anti-tumour response and to improve the over-all efficacy of adoptive cell therapy.

  7. ANALYSIS OF T CELL CLONALITY BY CDR3 SIZE OF T-CELL ANTIGEN RECEPTOR Vβ REPERTOIRE IN HCL AND c-ALL

    Institute of Scientific and Technical Information of China (English)

    LI Yang-qiu; WANG Ming-chun; Siegert W; Schmadt CA

    1999-01-01

    Objective: To analyze the distribution and clonality of TCR Vβ subfamily T cells in hairy cell leukemia (HCL) and common-acute lymphoblastic leukemia (c-ALL). Methods:Peripheral blood mononuclear cell samples from 3 cases of HCL and 1 case of c-ALL were investigated for analysis of complementarity determining region 3 (CDR3) size of T cell receptor Vβ repertoire using reverse transcriptasepolymerase chain reaction (RT-PCR). The products were further analyzed by genescan to identify T cell clonality.Results: Some Vβ subfamily PCR products from 4 patients contained monopeak (monoclone) or a dominant peak (oligoclone). In contrast, multipeak (polyclone) distributions were found in all Vβ subfamily PCR products from normal control cases. Conclusion: T cell clonal expansion may be found in HCL and c-ALL cases that may indicate a host response directed against leukemia related antigen. In addition, it may be useful to detect the minimal residual disease.

  8. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.

    Science.gov (United States)

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C; Steinman, Ralph M

    2002-12-16

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

  9. Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance

    Science.gov (United States)

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C.; Steinman, Ralph M.

    2002-01-01

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation. PMID:12486105

  10. Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease

    DEFF Research Database (Denmark)

    Jensen, Majbrit Myrup; Mikkelsen, Jens D.; Arvaniti, Maria;

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes...

  11. Cloning of T-cell antigen receptor beta chain cDNAs from Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Hordvik, I; Jacob, A L; Charlemagne, J; Endresen, C

    1996-01-01

    Atlantic salmon (Salmo salar) cDNAs encoding the T-cell antigen receptor beta chain (TCRB) were isolated from leukocyte RNA by reverse transcription - polymerase chain reaction (RT-PCR). Twenty-five distinct cDNA fragments covering the variable (V) - diversity (D) - joining (J) junction and part of the constant (C) region were characterized; the sequences of which indicate interchangeable V/D/J usage and expression in the context of one TCRBC gene. Full-length TCRBC sequence information was derived from a leukocyte cDNA library. Key residues of the salmon TCRBC region are in good agreement with those of other species. One distinct exception is the absence of the hinge region cysteine residue which is involved in covalent bonding between the alpha and beta chain in mammalian TCRs. As in amphibian and avian species, the salmon TCRBC membrane proximal region is considerably shorter than the mammalian. An octamer sequence (GGACAGGG) very similar to amphibian, avian, and mammalian D sequences could be recognized in the VDJ junctions from salmon. The pattern of VDJ variability also indicates that mechanisms like trimming and addition occur in fish as in higher vertebrates. Compared with mammals, a relatively high frequency (32%) of the VDJ junctions in salmon were out of frame. PMID:8881032

  12. A Requisite Role for Induced Regulatory T cells in Tolerance Based on Expanding Antigen Receptor Diversity

    OpenAIRE

    Haribhai, Dipica; Williams, Jason B; Jia, Shuang; Nickerson, Derek; Schmitt, Erica G.; Edwards, Brandon; Ziegelbauer, Jennifer; Yassai, Maryam; Li, Shun-Hwa; Relland, Lance M.; Wise, Petra M; Chen, Andrew; Zheng, Yu-Qian; Simpson, Pippa M.; Gorski, Jack

    2011-01-01

    Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted t...

  13. 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.

  14. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    Energy Technology Data Exchange (ETDEWEB)

    Manz, Boryana N. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Jackson, Bryan L. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Petit, Rebecca S. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Dustin, Michael L. [New York School of Medicine, New York, NY (United States); Groves, Jay [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2011-05-31

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.

  15. Assembly of the T-cell antigen receptor. Participation of the CD3 omega chain

    DEFF Research Database (Denmark)

    Neisig, A; Vangsted, A; Zeuthen, J;

    1993-01-01

    The human TCR is composed of the Ti alpha beta heterodimer in association with the CD3 chains CD3 gamma delta epsilon zeta 2. Another chain, referred to as CD3 omega, has recently been described in T cells. CD3 omega is an intracellular protein transiently associated with the CD3 complex during...... the assembly of the TCR in the endoplasmic reticulum (ER) and it is not expressed on the cell surface. The function of CD3 omega is unknown but it has been suggested that it plays an important role in the assembly of the TCR. We have studied the possible function of CD3 omega in the human leukemic T-cell line...... Jurkat and different variants of this cell line. Cells were metabolically labeled, subjected to lysis, immunoprecipitated, and analyzed by SDS-PAGE. The results indicate that: 1) CD3 omega associates primarily with the CD3 delta epsilon complex; 2) CD3 omega is not associated with single Ti alpha or Ti...

  16. Structure and diversity of the T cell antigen receptor beta-chain in a teleost fish.

    Science.gov (United States)

    Partula, S; de Guerra, A; Fellah, J S; Charlemagne, J

    1995-07-15

    Cell-mediated immunity (e.g., allograft rejection) is found in all vertebrates, and these reactions are known to depend on thymus-derived cells in amphibian, avian, and mammalian species. The participation of peripheral T cell-like lymphocytes subpopulations to fish immunity is now well documented, but the developmental origin, migration, and peripheral tissue distribution of these cells remain practically unknown. This is mainly due to the difficulty of efficiently thymectomizing fish at an early stage of development and to the lack of Ab strictly specific for thymocytes and T cell surface Ag. One strategy for analyzing T cell biology in fish would be to characterize the genes encoding polypeptides homologous to the TCR molecules. This report describes cDNA clones from the rainbow trout (Oncorhynchus mykiss) that have sequences very similar to amphibian, avian, and mammalian TCR beta-chains. Three complete trout V beta segments belonging to different families were analyzed; one of them had limited amino acid sequence similarity to the human V beta 20 family. The 10 trout beta-chain-joining segments all retain the invariant mammalian J beta residues, and comparison of 66 V beta-J beta junctions led to the identification of a D beta-like sequence (GGACAGGG) that is shorter than but very similar to the chicken D beta and mammalian D beta 1 sequences. There is considerable diversity at the V beta-D beta and D beta-J beta junctions, suggesting the presence of N-nucleotides. The trout C beta extracellular domain is shorter than mammalian C beta, and the hinge region has no cysteine residue. The transmembrane C beta domain contains a lysine residue that in mammals is thought to be involved in charged interactions with members of the CD3 complex. PMID:7608547

  17. The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

    Directory of Open Access Journals (Sweden)

    Blaise Ndjamen

    2014-03-01

    Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

  18. Antigen receptors and somatic hypermutation in B-cell chronic lymphocytic leukemia with Richter's transformation

    NARCIS (Netherlands)

    L.A. Smit; F. van Maldegem; A.W. Langerak; C.E. van der Schoot; M.J. de Wit; S. Bea; E. Campo; R.J. Bende; C.J.M. van Noesel

    2006-01-01

    Background and Objectives. Activation-induced cytidine deaminase is essential for somatic hypermutation and class switch recombination of the immunoglobulin genes in B cells. It has been proposed that aberrant targeting of the somatic hypermutation machinery is instrumental in initiation and progres

  19. A High-avidity WT1-reactive T-Cell Receptor Mediates Recognition of Peptide and Processed Antigen but not Naturally Occurring WT1-positive Tumor Cells.

    Science.gov (United States)

    Jaigirdar, Adnan; Rosenberg, Steven A; Parkhurst, Maria

    2016-04-01

    Wilms tumor gene 1 (WT1) is an attractive target antigen for cancer immunotherapy because it is overexpressed in many hematologic malignancies and solid tumors but has limited, low-level expression in normal adult tissues. Multiple HLA class I and class II restricted epitopes have been identified in WT1, and multiple investigators are pursuing the treatment of cancer patients with WT1-based vaccines and adoptively transferred WT1-reactive T cells. Here we isolated an HLA-A*0201-restricted WT1-reactive T-cell receptor (TCR) by stimulating peripheral blood lymphocytes of healthy donors with the peptide WT1:126-134 in vitro. This TCR mediated peptide recognition down to a concentration of ∼0.1 ng/mL when pulsed onto T2 cells as well as recognition of HLA-A*0201 target cells transfected with full-length WT1 cDNA. However, it did not mediate consistent recognition of many HLA-A*0201 tumor cell lines or freshly isolated leukemia cells that endogeneously expressed WT1. We dissected this pattern of recognition further and observed that WT1:126-134 was more efficiently processed by immunoproteasomes compared with standard proteasomes. However, pretreatment of WT1 tumor cell lines with interferon gamma did not appreciably enhance recognition by our TCR. In addition, we highly overexpressed WT1 in several leukemia cell lines by electroporation with full-length WT1 cDNA. Some of these lines were still not recognized by our TCR suggesting possible antigen processing defects in some leukemias. These results suggest WT1:126-134 may not be a suitable target for T-cell based tumor immunotherapies. PMID:26938944

  20. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

    Science.gov (United States)

    Suarez, Eloah Rabello; Chang, De-Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J.; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A.

    2016-01-01

    Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen. PMID:27145284

  1. Chimeric antigen receptor-engineered cytokine-induced killer cells overcome treatment resistance of pre-B-cell acute lymphoblastic leukemia and enhance survival.

    Science.gov (United States)

    Oelsner, Sarah; Wagner, Juliane; Friede, Miriam E; Pfirrmann, Verena; Genßler, Sabrina; Rettinger, Eva; Buchholz, Christian J; Pfeifer, Heike; Schubert, Ralf; Ottmann, Oliver G; Ullrich, Evelyn; Bader, Peter; Wels, Winfried S

    2016-10-15

    Pre-emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease. CIK cells are a heterogeneous effector cell population including T cells (CD3(+) CD56(-) ), natural killer (NK) cells (CD3(-) CD56(+) ) and natural killer T (T-NK) cells (CD3(+) CD56(+) ) that exhibit non-major histocompatibility complex (MHC)-restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)-γ, anti-CD3 antibody, interleukin-2 (IL-2) and interleukin-15 (IL-15). To facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19-targeted CIK/63.28.z cells against otherwise CIK-resistant cancer cell lines and primary B-ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre-B-ALL. Our results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL. PMID:27253354

  2. The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

    Directory of Open Access Journals (Sweden)

    M.I. Hiyane

    2006-03-01

    Full Text Available Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

  3. Antigen and transforming growth factor beta receptors contribute to long term functional and phenotypic heterogeneity of memory CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Yinghong eHu

    2013-08-01

    Full Text Available Pathogen-specific CD8 T cells provide a mechanism for selectively eliminating host cells that are harboring intracellular pathogens. The pathogens are killed when lytic molecules are injected into the cytoplasm of the infected cells and begin an apoptotic cascade. Activated CD8 T cells also release large quantities of proinflammatory cytokines that stimulate other immune cells in the local vicinity. As the alveoli are extraordinarily sensitive to cytokine induced damage, multiple layers of immune regulation limit the activities of immune cells that enter the lungs. These mechanisms include receptor-mediated signaling pathways in CD8 T cells that respond to peptide antigens and transforming growth factor-beta. Both pathways influence the functional and phenotypic properties of long-lived CD8 T cells populations in peripheral and lymphoid tissues.

  4. Blockade of the interleukin-2 receptor by anti-Tac antibody inhibits the generation of antigen-nonspecific suppressor T cells in vitro.

    OpenAIRE

    Oh-Ishi, T; Goldman, C K; Misiti, J; Waldmann, T. A.

    1988-01-01

    The role of interleukin 2 (IL-2) in the activation of suppressor T cells was investigated by using the monoclonal antibody anti-Tac, which blocks the binding of IL-2 to the 55-kDa peptide of the high-affinity IL-2 receptor. Anti-Tac was added to an antigen-nonspecific suppressor system in which Con A-induced suppressor T cells were generated during a preculture period, and their effects on immunoglobulin production were assessed in second, indicator cultures containing pokeweed mitogen and pe...

  5. Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.

    Science.gov (United States)

    Krenciute, Giedre; Krebs, Simone; Torres, David; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Li, Xiao-Nan; Lesniak, Maciej S; Balyasnikova, Irina V; Gottschalk, Stephen

    2016-02-01

    Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies. PMID:26514825

  6. Mechanistic Assessment of PD-1H Coinhibitory Receptor-Induced T-Cell Tolerance to Allogeneic Antigens1

    Science.gov (United States)

    Flies, Dallas B.; Higuchi, Tomoe; Chen, Lieping

    2015-01-01

    PD-1H is a recently identified cell surface co-inhibitory molecule of the B7/CD28 immune modulatory gene family. We showed previously that single injection of a PD-1H agonistic monoclonal antibody (mAb) protected mice from graft versus host disease (GVHD). We report here two distinct mechanisms operate in PD-1H-induced T cell tolerance. First, signaling via PD-1H co-inhibitory receptor potently arrests allo-reactive donor T cells from activation and expansion in the initiation phase. Second, donor regulatory T cells are subsequently expanded to maintain long-term tolerance and GVHD suppression. Our study reveals the crucial function of PD-1H as a co-inhibitory receptor on allo-reactive T cells and its function in the regulation of T cell tolerance. Therefore, PD-1H may be a target for the modulation of allo-reactive T cells in GVHD and transplantation. PMID:25917101

  7. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    Science.gov (United States)

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  8. Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

    Directory of Open Access Journals (Sweden)

    Lihua E Budde

    Full Text Available Modification of T cells with chimeric antigen receptors (CAR has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9, and a truncated CD19 selectable marker. Rapid expansion (2000 fold of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.

  9. Identification of prostate-specific G-protein coupled receptor as a tumor antigen recognized by CD8(+ T cells for cancer immunotherapy.

    Directory of Open Access Journals (Sweden)

    Satoko Matsueda

    Full Text Available BACKGROUND: Prostate cancer is the most common cancer among elderly men in the US, and immunotherapy has been shown to be a promising strategy to treat patients with metastatic castration-resistant prostate cancer. Efforts to identify novel prostate specific tumor antigens will facilitate the development of effective cancer vaccines against prostate cancer. Prostate-specific G-protein coupled receptor (PSGR is a novel antigen that has been shown to be specifically over-expressed in human prostate cancer tissues. In this study, we describe the identification of PSGR-derived peptide epitopes recognized by CD8(+ T cells in an HLA-A2 dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-one PSGR-derived peptides were predicted by an immuno-informatics approach based on the HLA-A2 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs obtained from either HLA-A2(+ healthy donors or HLA-A2(+ prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Among the 21 PSGR-derived peptides, three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients. Importantly, these peptide-specific T cells recognized and killed LNCaP prostate cancer cells in an HLA class I-restricted manner. CONCLUSIONS/SIGNIFICANCE: We have identified three novel HLA-A2-restricted PSGR-derived peptides recognized by CD8(+ T cells, which, in turn, recognize HLA-A2(+ and PSGR(+ tumor cells. The PSGR-derived peptides identified may be used as diagnostic markers as well as immune targets for development of anticancer vaccines.

  10. The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor-β receptor on dendritic cells potentiates tumour antigen-specific CD8(+) T cell immunity.

    Science.gov (United States)

    Ahn, Y-H; Hong, S-O; Kim, J H; Noh, K H; Song, K-H; Lee, Y-H; Jeon, J-H; Kim, D-W; Seo, J H; Kim, T W

    2015-07-01

    Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune-priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)-10 and transforming growth factor (TGF)-β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL-10 receptor alpha (siIL-10RA) initiated the strongest antigen-specific CD8(+) T cell immune responses. The potency of siIL-10RA was enhanced further by combining it with siRNA targeting TGF-β receptor (siTGF-βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl-2-like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL-10RA and siTGF-βR generated the strongest antigen-specific CD8(+) T cell immunity. Concordantly, the knock-down of both IL-10RA and TGF-βR in DCs induced the strongest anti-tumour effects in the TC-1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)-16 E7 antigen, and even in the immune-resistant TC-1 (P3) tumour model that secretes more IL-10 and TGF-β than the parental tumour cells (TC-1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail-mediated strategy by co-targeting immunosuppressive molecules to enhance the potency of DC-based vaccines. PMID:25753156

  11. Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

    Science.gov (United States)

    Peralta-Zaragoza, O; Martínez-Valdez, H; Madrid-Marina, V

    1996-01-01

    The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization. PMID:8854386

  12. Antigen Specificity of Clonally Expanded and Receptor Edited Cerebrospinal Fluid B cells from Patients with Relapsing Remitting MS

    OpenAIRE

    Lambracht-Washington, Doris; O’Connor, Kevin C.; Cameron, Elizabeth; Jowdry, Andrea; Ward, E. Sally; Frohman, Elliot; Racke, Michael K.; Monson, Nancy L.

    2007-01-01

    We re-engineered the immunoglobulin rearrangements from clonally expanded CSF B cells of three Multiple Sclerosis patients as Fab fragments, and used three methods to test for their Ag-specificity. Nine out of ten Fab fragments were reactive to Myelin Basic Protein (MBP). The one Fab that did not react to MBP was a product of receptor editing. Two of the nine MBP-reactive Fabs were also reactive to GFAP and CNPase, indicating that these clones were polyreactive. Targeting the mechanisms that ...

  13. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

    Directory of Open Access Journals (Sweden)

    Ana C. Parente-Pereira

    2014-07-01

    Full Text Available Chimeric antigen receptors (CARs are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation.

  14. Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group

    DEFF Research Database (Denmark)

    Jensen, T; Nielsen, M; Gad, Monika;

    2001-01-01

    A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O...

  15. Immunotherapy of non-Hodgkin's 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

    2016-09-01

    CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion 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's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The 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 single-chain variable fragment component of the CAR, which limited CAR-T cell expansion 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 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 lymphodepletion that improved disease response and overall and progression-free survival. PMID:27605551

  16. Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

    Science.gov (United States)

    Rast, J P; Haire, R N; Litman, R T; Pross, S; Litman, G W

    1995-01-01

    Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene superfamily in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems. PMID:7642232

  17. Distinct domains of the CD3-gamma chain are involved in surface expression and function of the T cell antigen receptor

    DEFF Research Database (Denmark)

    Wegener, A M; Hou, X; Dietrich, J;

    1995-01-01

    these two subunits. To identify CD3-gamma-specific domains that are required for assembly of the complete TcR and for surface expression and function of the TcR, chimeric CD3-gamma/CD3-delta molecules were constructed and expressed in T cells devoid of endogenous CD3-gamma. Substitution of the extracellular...... that the extracellular domain of CD3-gamma plays a unique role in TcR assembly. Functional analyses of the transfectants demonstrated that the intracellular domain of CD3-gamma is required for protein kinase C-mediated down-regulation of TcR but is dispensable for the pattern of tyrosine phosphorylation observed......The T cell antigen receptor (TcR) is a multisubunit complex that consists of at least six different polypeptides. We have recently demonstrated that the CD3-delta subunit cannot substitute for the CD3-gamma subunit in TcR cell surface expression, in spite of significant amino acid homology between...

  18. 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.

  19. Relationship between serum carcinoembryonic antigen level and epidermal growth factor receptor mutations with the influence on the prognosis of non-small-cell lung cancer patients

    Directory of Open Access Journals (Sweden)

    Cai ZX

    2016-06-01

    Full Text Available Zuxun Cai Department of Thoracic Surgery, Henan Provincial Chest Hospital, Zhengzhou City, People’s Republic of China Objective: To investigate the relationship between serum carcinoembryonic antigen (CEA level and epidermal growth factor receptor (EGFR gene mutations in non-small-cell lung cancer (NSCLC patients and to analyze the influence of CEA level on postoperative survival time in lung cancer patients. Methods: A total of 296 patients who were treated in Thoracic Surgery Department of Henan Provincial Chest Hospital from September 2011 to September 2013 were recruited. The level of tumor markers, such as CEA, was determined before the surgery, and EGFR gene mutations were detected after surgery. Thereby, the relationship between tumor makers, including CEA, and EGFR mutation and its influence on prognosis could be investigated. Results: Among 296 patients, the positive rate of EGFR gene mutation was 37.84% (112/296; the mutation occurred more frequently in nonsmokers, adenocarcinoma patients, women, and patients aged <60 years (P<0.05. Both tumor markers and chemosensitivity indicators were related to the profile of EGFR mutations. Elevated squamous cell carcinoma and Cyfra21-1 as well as positively expressed ERCC1 were more common in patients with wild-type EGFR (P<0.05, whereas increased CEA level was observed more frequently in patients with EGFR gene mutation (P=0.012. The positive rate of EGFR gene mutations was higher as the serum CEA level increased, that is, the positive rate in patients with serum CEA level <5, 5–20, and >20 µg/L was 39.81%, 45.32%, and 65.47%, respectively (P=0.004. Logistic regression analysis showed that CEA level was an independent factor in predicting EGFR gene mutations, and serum CEA level was also an independent factor in affecting the prognosis of NSCLC patients, as the overall 2-year survival rate was 73.86% in elevated CEA group and 86.43% in normal group (P<0.01. Conclusion: The prognosis of

  20. Antigen-Specific CD4+ T Cells Recognize Epitopes of Protective Antigen following Vaccination with an Anthrax Vaccine

    OpenAIRE

    Laughlin, Elsa M.; Miller, Joseph D.; James, Eddie; Fillos, Dimitri; Ibegbu, Chris C.; Mittler, Robert S.; Akondy, Rama; Kwok, William; Ahmed, Rafi; Nepom, Gerald,

    2007-01-01

    Detection of antigen-specific CD4+ T cells is facilitated by the use of fluorescently labeled soluble peptide-major histocompatibility complex (MHC) multimers which mirror the antigen specificity of T-cell receptor recognition. We have used soluble peptide-MHC class II tetramers containing peptides from the protective antigen (PA) of Bacillus anthracis to detect circulating T cells in peripheral blood of subjects vaccinated with an anthrax vaccine. PA-specific HLA class II-restricted T lympho...

  1. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Monica Casucci, Attilio Bondanza

    2011-01-01

    Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

  2. The proliferating cell nuclear antigen regulates retinoic acid receptor transcriptional activity through direct protein–protein interaction

    OpenAIRE

    Martin, Perrine J; Lardeux, Virginie; Lefebvre, Philippe

    2005-01-01

    Retinoic acid receptors (RARs) interact, in a ligand-dependent fashion, with many coregulators that participate in a wide spectrum of biological responses, ranging from embryonic development to cellular growth control. The transactivating function of these ligand-inducible transcription factors reside mainly, but not exclusively, in their ligand-binding domain (AF2), which recruits or dismiss coregulators in a ligand-dependent fashion. However, little is known about AF2-independent function(s...

  3. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    Science.gov (United States)

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  4. Schistosoma mansoni Soluble Egg Antigens Induce Expression of the Negative Regulators SOCS1 and SHP1 in Human Dendritic Cells via Interaction with the Mannose Receptor.

    Directory of Open Access Journals (Sweden)

    Elsenoor J Klaver

    Full Text Available Schistosomiasis is a common debilitating human parasitic disease in (subtropical areas, however, schistosome infections can also protect against a variety of inflammatory diseases. This has raised broad interest in the mechanisms by which Schistosoma modulate the immune system into an anti-inflammatory and regulatory state. Human dendritic cells (DCs show many phenotypic changes upon contact with Schistosoma mansoni soluble egg antigens (SEA. We here show that oxidation of SEA glycans, but not heat-denaturation, abrogates the capacity of SEA to suppress both LPS-induced cytokine secretion and DC proliferation, indicating an important role of SEA glycans in these processes. Remarkably, interaction of SEA glycans with DCs results in a strongly increased expression of Suppressor Of Cytokine Signalling1 (SOCS1 and SH2-containing protein tyrosine Phosphatase-1 (SHP1, important negative regulators of TLR4 signalling. In addition, SEA induces the secretion of transforming growth factor β (TGF-β, and the surface expression of the costimulatory molecules Programmed Death Ligand-1 (PD-L1 and OX40 ligand (OX40L, which are known phenotypic markers for the capacity of DCs to polarize naïve T cells into Th2/Treg cell subsets. Inhibition of mannose receptor (MR-mediated internalization of SEA into DCs by blocking with allyl α-D-mannoside or anti-MR antibodies, significantly reduced SOCS1 and SHP1 expression. In conclusion, we demonstrate that SEA glycans are essential for induction of enhanced SOCS1 and SHP1 levels in DCs via the MR. Our data provide novel mechanistic evidence for the potential of S. mansoni SEA glycans to modulate human DCs, which may contribute to the capacity of SEA to down-regulate inflammatory responses.

  5. Schistosoma mansoni Soluble Egg Antigens Induce Expression of the Negative Regulators SOCS1 and SHP1 in Human Dendritic Cells via Interaction with the Mannose Receptor.

    Science.gov (United States)

    Klaver, Elsenoor J; Kuijk, Loes M; Lindhorst, Thisbe K; Cummings, Richard D; van Die, Irma

    2015-01-01

    Schistosomiasis is a common debilitating human parasitic disease in (sub)tropical areas, however, schistosome infections can also protect against a variety of inflammatory diseases. This has raised broad interest in the mechanisms by which Schistosoma modulate the immune system into an anti-inflammatory and regulatory state. Human dendritic cells (DCs) show many phenotypic changes upon contact with Schistosoma mansoni soluble egg antigens (SEA). We here show that oxidation of SEA glycans, but not heat-denaturation, abrogates the capacity of SEA to suppress both LPS-induced cytokine secretion and DC proliferation, indicating an important role of SEA glycans in these processes. Remarkably, interaction of SEA glycans with DCs results in a strongly increased expression of Suppressor Of Cytokine Signalling1 (SOCS1) and SH2-containing protein tyrosine Phosphatase-1 (SHP1), important negative regulators of TLR4 signalling. In addition, SEA induces the secretion of transforming growth factor β (TGF-β), and the surface expression of the costimulatory molecules Programmed Death Ligand-1 (PD-L1) and OX40 ligand (OX40L), which are known phenotypic markers for the capacity of DCs to polarize naïve T cells into Th2/Treg cell subsets. Inhibition of mannose receptor (MR)-mediated internalization of SEA into DCs by blocking with allyl α-D-mannoside or anti-MR antibodies, significantly reduced SOCS1 and SHP1 expression. In conclusion, we demonstrate that SEA glycans are essential for induction of enhanced SOCS1 and SHP1 levels in DCs via the MR. Our data provide novel mechanistic evidence for the potential of S. mansoni SEA glycans to modulate human DCs, which may contribute to the capacity of SEA to down-regulate inflammatory responses. PMID:25897665

  6. Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals.

    OpenAIRE

    Oyaizu, N; Chirmule, N; Pahwa, S.

    1992-01-01

    The CD4 molecule plays an essential role in antigen-induced activation of T helper (Th) cells, but its contribution to signal transduction events resulting in physiologic T cell function is ill defined. By utilizing anti-CD4 monoclonal antibodies (MAbs) that recognize distinct epitopes of CD4, we have investigated the role of CD4 molecule in antigen-induced interleukin 2 (IL-2) and IL-2 receptor (IL-2R) alpha chain expression in class II major histocompatibility complex-restricted antigen-spe...

  7. Coexpressed Catalase Protects Chimeric Antigen Receptor-Redirected T Cells as well as Bystander Cells from Oxidative Stress-Induced Loss of Antitumor Activity.

    Science.gov (United States)

    Ligtenberg, Maarten A; Mougiakakos, Dimitrios; Mukhopadhyay, Madhura; Witt, Kristina; Lladser, Alvaro; Chmielewski, Markus; Riet, Tobias; Abken, Hinrich; Kiessling, Rolf

    2016-01-15

    Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.

  8. Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen

    OpenAIRE

    1995-01-01

    The cutaneous lymphocyte-associated antigen (CLA) is the major T cell ligand for the vascular adhesion molecule E-selectin, and it has been proposed to be involved in the selective targeting of memory T cells reactive with skin-associated Ag to cutaneous inflammatory sites. To further investigate the relation of CLA and cutaneous T cell responses, we analyzed the CLA phenotype of circulating memory T cells in patients with allergic contact dermatitis and atopic dermatitis (AD) alone vs in pat...

  9. Prolonged androgen deprivation leads to downregulation of androgen receptor and prostate-specific membrane antigen in prostate cancer cells

    OpenAIRE

    Liu, Tiancheng; Wu, Lisa Y.; Fulton, Melody D.; JOHNSON, JACQUELINE M.; Berkman, Clifford E.

    2012-01-01

    Emergence of androgen-independent cancer cells during androgen deprivation therapy presents a significant challenge to successful treatment outcomes in prostate cancer. Elucidating the role of androgen deprivation in the transition from an androgen-dependent to an androgen-independent state may enable the development of more effective therapeutic strategies against prostate cancer. Herein, we describe an in vitro model for assessing the effects of continuous androgen-deprivation on prostate c...

  10. Structural and antigenic types of cell wall polysaccharides from viridans group streptococci with receptors for oral actinomyces and streptococcal lectins.

    OpenAIRE

    Cisar, J O; Sandberg, A L; Reddy, G P; Abeygunawardana, C; Bush, C A

    1997-01-01

    Lectin-mediated interactions between oral viridans group streptococci and actinomyces may play an important role in microbial colonization of the tooth surface. The presence of two host-like motifs, either GalNAc beta1-->3Gal (Gn) or Gal beta1-->3GalNAc (G), in the cell wall polysaccharides of five streptococcal strains accounts for the lactose-sensitive coaggregations of these bacteria with Actinomyces naeslundii. Three streptococcal strains which have Gn-containing polysaccharides also part...

  11. Control of T cell antigen reactivity via programmed TCR downregulation.

    Science.gov (United States)

    Gallegos, Alena M; Xiong, Huizhong; Leiner, Ingrid M; Sušac, Bože; Glickman, Michael S; Pamer, Eric G; van Heijst, Jeroen W J

    2016-04-01

    The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage. PMID:26901151

  12. 嵌合抗原受体T细胞在B细胞淋巴瘤中的研究进展%Research progress of chimeric antigen receptor-modified T cell in B cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    应志涛

    2014-01-01

    B细胞淋巴瘤的预后在利妥昔单抗出现之后有了明显改善,但仍有一部分患者会复发进展.嵌合抗原受体T细胞(CAR T细胞)是通过基因修饰的方法获得的针对肿瘤细胞表面特定抗原的特异性T细胞,在复发难治B细胞淋巴瘤的治疗中取得了很好疗效,目前研究最多的是针对B细胞淋巴瘤表面的CD19抗原.文章综述了抗CD19 CAR T细胞在复发难治B细胞淋巴瘤中的疗效、不良反应及目前存在的问题.%The prognosis of B cell lymphoma has been significantly improved in the rituximab era,but there are also patients who are refractory or relapsed after rituximab-containing treatment.Chimeric antigen receptor T (CAR T) cell is a kind of T cell which is genetically modified to target specific antigen expressed on the lymphoma cell surface.The response rate of CAR T cell in relapsed or refractory B cell lymphoma is inspiring.Anti-CD19 CAR T cell is the most popular cell being tested in clinical studies.This article discussed the present situation of CAR T cell in B cell lymphoma,including the efficacy,toxicity,side effects and open questions existing in this treatment.

  13. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves' Disease.

    Science.gov (United States)

    Inaba, Hidefumi; De Groot, Leslie J; Akamizu, Takashi

    2016-01-01

    Graves' disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  14. 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.

    Science.gov (United States)

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    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.

  15. 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.

    Science.gov (United States)

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    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. PMID:27548616

  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

    Science.gov (United States)

    Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E.; Laskowski, Tamara; McNamara, George; Cooper, Laurence J. N.

    2016-01-01

    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. PMID:27548616

  17. Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

    Science.gov (United States)

    Vela Ramirez, J E; Roychoudhury, R; Habte, H H; Cho, M W; Pohl, N L B; Narasimhan, B

    2014-01-01

    The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1. PMID:25068589

  18. Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Expression of Proliferating Cell Nuclear Antigen and Vascular Endothelial Growth Factor in Primary Culture Cells Derived from Human Ovarian Mucinous Cystadenocarcino

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The prlmary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups (P<0.05 or P< 0.01), while decreased significantly in antisense ODN groups (P<0. 05 or P<0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly (P<0.01). It was suggested that FSH might promotethe development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.

  19. The inducible caspase-9 suicide gene system as a ‘safety switch’ to limit on-target, off-tumor toxicities of chimeric antigen receptor T-cells.

    Directory of Open Access Journals (Sweden)

    Tessa eGargett

    2014-10-01

    Full Text Available Immune modulation has become a central element in many cancer treatments, and T cells genetically engineered to express chimeric antigen receptors (CAR may provide a new approach to cancer immunotherapy. Autologous CAR T cells that have been re-directed towards tumor-associated antigens (TAA have shown promising results in phase 1 clinical trials, with some patients undergoing complete tumor regression. However this T-cell therapy must carefully balance effective T-cell activation, to ensure antitumor activity, with the potential for uncontrolled activation that may produce immunopathology. An inducible Caspase 9 (iCasp9 ‘safety switch’ offers a solution that allows for the removal of inappropriately activated CAR T cells. The induction of iCasp9 depends on the administration of the small molecule dimerizer drug AP1903 and dimerization results in rapid induction of apoptosis in transduced cells, preferentially killing activated cells expressing high levels of transgene. The iCasp9 gene has been incorporated into vectors for use in preclinical studies and demonstrates effective and reliable suicide gene activity in phase 1 clinical trials. A third-generation CAR incorporating iCasp9 re-directs T cells towards the GD2 TAA. GD2 is over-expressed in melanoma and other malignancies of neural crest origin and the safety and activity of these GD2-iCAR T cells will be investigated in CARPETS and other actively recruiting phase 1 trials.

  20. Germinal center B cells recognize antigen through a specialized immune synapse architecture.

    Science.gov (United States)

    Nowosad, Carla R; Spillane, Katelyn M; Tolar, Pavel

    2016-07-01

    B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we found that, in contrast with naive and memory B cells, which gathered antigen toward the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β-NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T cell-dependent selection of high-affinity B cells in GCs. PMID:27183103

  1. CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin’s Lymphomas

    Science.gov (United States)

    Onea, Alexandra S; Jazirehi, Ali R

    2016-01-01

    Recovery rates for B-cell Non-Hodgkin’s Lymphoma (NHL) are up to 70% with current standard-of-care treatments including rituximab (chimeric anti-CD20 monoclonal antibody) in combination with chemotherapy (R-CHOP). However, patients who do not respond to first-line treatment or develop resistance have a very poor prognosis. This signifies the need for the development of an optimal treatment approach for relapsed/refractory B-NHL. Novel CD19- chimeric antigen receptor (CAR) T-cell redirected immunotherapy is an attractive option for this subset of patients. Anti-CD19 CAR T-cell therapy has already had remarkable efficacy in various leukemias as well as encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. In going forward with additional clinical trials, complementary treatments that may circumvent potential resistance mechanisms should be used alongside anti-CD19 T-cells in order to prevent relapse with resistant strains of disease. Some such supplementary tactics include conditioning with lymphodepletion agents, sensitizing with kinase inhibitors and Bcl-2 inhibitors, enhancing function with multispecific CAR T-cells and CD40 ligand-expressing CAR T-cells, and safeguarding with lymphoma stem cell-targeted treatments. A therapy regimen involving anti-CD19 CAR T-cells and one or more auxiliary treatments could dramatically improve prognoses for patients with relapsed/refractory B-cell NHL. This approach has the potential to revolutionize B-NHL salvage therapy in much the same way rituximab did for first-line treatments. PMID:27186412

  2. Expressions of Proliferating Cell Nuclear Antigen and Wheat Germ Agglutinin Receptor in Human Bladder Carcinoma%膀胱癌增殖细胞核抗原与麦胚凝集素受体的相关关系

    Institute of Scientific and Technical Information of China (English)

    张士文; 葛根; 金伯涛

    2001-01-01

    [Purpose]To probe the relation of proliferating cell nuclear antigen (PCNA) and wheat germ agglutinin (WGA) receptors expressed in human bladder transitional cell carcinoma (TCC).[Methods]PCNA and WGA receptors were detected by immunohistochemical method (ABC method) in 63 specimens of TCC.[Results]We found that the distributions of PCNA and WGA receptors were increased with increase of histopathological grade in TCC (P<0.01).There was a higher expression in invasive tumors than that in superficial tumors (P<0.005),and there was a positive relation between PCNA and WGA receptors also.[Conclusion]It is shown that PCNA and WGA can be used as tumor markers for bladder cancer.%[目的 ]探讨增殖细胞核抗原 (proliferating cell nuclear antigen,PCNA)和麦胚凝集素 (wheat germ agglutinin,WGA)在膀胱移行细胞癌 (TCC)中表达的相关关系。 [方法 ]采用免疫组织化学 ABC法对 63例 TCC标本进行 PCNA和 WGA受体检测。 [结果 ]PCNA与 WGA的强阳性表达随着肿瘤的病理分级升高而增高;浸润性肿瘤中的 WGA受体的强阳性表达显著高于浅表性肿瘤 (P<0.05); PCNA与 WGA受体表达一致性良好,呈显著性相关 (P<0.005)。 [结论 ]我们认为 PCNA和 WGA受体均可作为 TCC的肿瘤标记物,证明了 TCC细胞的增殖活性增强将改变其细胞膜的抗原性。

  3. Research progress of chimeric antigen receptor-transduced-T cells in B-cell malignancies%嵌合抗原受体T细胞在B细胞性血液肿瘤中的研究进展

    Institute of Scientific and Technical Information of China (English)

    徐丽丽; 王扬

    2014-01-01

    Chimeric antigen receptor-transduced-T (CAR-T) cells shows strong cytotoxicity to the tumor with specific antigen by CTL effects and cytokines releasing without MHC-restriction.As multiple clinical trials from cancer centers around the West countries reported recent years,anti-CD19-CAR T cells can not only induce complete remission of B-cell malignancies resistant,but also eliminate the minimal residual disease (MRD) at the molecular level.It is believed that the obstacles from bench to clinic will be cleared and CAR will become one of the main cancer therapies with breakthroughs in comprehensive treatment of tumors.%嵌合抗原受体(CAR)转染的T细胞通过释放细胞因子及细胞毒性T淋巴细胞(CTL)效应可高效杀伤靶抗原阳性的肿瘤细胞,且不受相容性复合体(MHC)的限制.近年欧美多个癌症中心的临床试验表明,CD19靶向的CAR-T细胞输注不仅可使复发状态的B细胞急性淋巴细胞白血病(B-ALL)患者达完全缓解,且分子水平的微小残留病(MRD)也可转阴.其在慢性淋巴细胞白血病、B细胞性恶性淋巴瘤患者中也取得了显著疗效,相信不久的将来该技术必将成为肿瘤综合治疗的重要手段.

  4. Reassessing target antigens for adoptive T cell therapy

    Science.gov (United States)

    Hinrichs, Christian S.; Restifo, Nicholas P.

    2014-01-01

    Adoptive T cell therapy can target and kill widespread malignant cells thereby inducing durable clinical responses in melanoma and selected other malignances. However, many commonly targeted tumor antigens are also expressed by healthy tissues, and T cells do not distinguish between benign and malignant tissues if both express the target antigen. As such, autoimmune toxicity from T-cell-mediated destruction of normal tissue has limited the development and adoption of this otherwise promising type of cancer therapy. A review of the unique biology of T-cell therapy and of recent clinical experience compels a reassessment of target antigens that traditionally have been viewed from the perspective of weaker immunotherapeutic modalities. In selecting target antigens for adoptive T-cell therapy, expression by tumors and not by essential healthy tissues is of paramount importance. The risk of autoimmune adverse events can be further mitigated by generating antigen receptors using strategies that reduce the chance of cross-reactivity against epitopes in unintended targets. In general, a circumspect approach to target selection and thoughtful preclinical and clinical studies are pivotal to the ongoing advancement of these promising treatments. PMID:24142051

  5. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

    OpenAIRE

    Zheng Zhili; Chinnasamy Nachimuthu; Morgan Richard A

    2012-01-01

    Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymp...

  6. Antigen Export Reduces Antigen Presentation and Limits T Cell Control of M. tuberculosis.

    Science.gov (United States)

    Srivastava, Smita; Grace, Patricia S; Ernst, Joel D

    2016-01-13

    Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion.

  7. 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.

  8. Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

    International Nuclear Information System (INIS)

    The frequency of mutant T lymphocytes defective in T-cell receptor gene (α or β) expression was measured using the two-color flow cytometric technique. Results for a total of 203 atomic bomb survivors, 78 of whom were proximally exposed (DS86 doses of ≥ 1.5 Gy) and 125 of whom were distally exposed (DS86 doses of 228Th formerly used for radiodiagnosis. In addition, thyroid disease patients treated with 131I showed a dose-related increase of mutant frequency. It was suggested that the present T-cell receptor mutation assay has a unique characteristic as a biological dosimeter for the measurement of recent exposures to genotoxic agents. (author)

  9. Hepatitis B virus antigens impair NK cell function.

    Science.gov (United States)

    Yang, Yinli; Han, Qiuju; Zhang, Cai; Xiao, Min; Zhang, Jian

    2016-09-01

    An inadequate immune response of the host is thought to be a critical factor causing chronic hepatitis B virus (CHB) infection. Natural killer (NK) cells, as one of the key players in the eradication and control of viral infections, were functionally impaired in CHB patients, which might contribute to viral persistence. Here, we reported that HBV antigens HBsAg and HBeAg directly inhibited NK cell function. HBsAg and/or HBeAg blocked NK cell activation, cytokine production and cytotoxic granule release in human NK cell-line NK-92 cells, which might be related to the downregulation of activating receptors and upregulation of inhibitory receptor. Furthermore, the underlying mechanisms likely involved the suppression of STAT1, NF-κB and p38 MAPK pathways. These findings implicated that HBV antigen-mediated inhibition of NK cells might be an efficient strategy for HBV evasion, targeting the early antiviral responses mediated by NK cells and resulting in the establishment of chronic virus infection. Therefore, this study revealed the relationship between viral antigens and human immune function, especially a potential important interaction between HBV and innate immune responses. PMID:27341035

  10. Interleukin-21-dependent modulation of T cell antigen receptor reactivity towards low affinity peptide ligands in autoreactive CD8(+) T lymphocytes.

    Science.gov (United States)

    Bobbala, Diwakar; Orkhis, Sakina; Kandhi, Rajani; Ramanathan, Sheela; Ilangumaran, Subburaj

    2016-09-01

    IL-21 promotes autoimmune type-1 diabetes (T1D) in NOD mice by facilitating CD4(+) T cell help to CD8(+) T cells. IL-21 also enables autoreactive CD8(+) T cells to respond to weak TCR ligands and induce T1D. Here, we assessed whether IL-21 is essential for T1D induction in a mouse model where the disease can occur independently of CD4 help. In this model, which expresses lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) antigen under the rat insulin promoter (RIP-GP), LCMV infection activates CD8(+) T cells reactive to the GP-derived GP33 peptide that attack pancreatic islets and cause T1D. We show that IL-21 deficiency in RIP-GP mice did not impair T1D induction by LCMV expressing the wildtype GP33 peptide. Surprisingly, LCMV-L6F, expressing a weak peptide mimic of GP33, induced T1D more efficiently in Il21(-/-)RIP-GP mice than in controls. However, LCMV-C4Y expressing a very weak peptide mimic of GP33 did not induce T1D in Il21(-/-) mice, but T cells from the infected mice caused disease in lymphopenic RIP-GP mice upon adoptive transfer. Using Nur77(GFP) reporter mice, we show that CD8(+) T cells from Il21(-/-) mice expressing the GP33-specific transgenic P14 TCR showed increased reactivity towards low affinity TCR ligands. Collectively, our findings show that IL-21 is not always required for T1D induction by autoreactive CD8(+) T cells, and suggest that IL-21 may play an important role in regulating CD8(+) T cell reactivity towards low affinity TCR ligands. PMID:27300756

  11. The effect of Chinese herbal medicine"heche assisted pregnancy recipe"on endometrial estrogen and progesterone receptor, proliferating cell nuclear antigen and vascular endothelial growth factor in the patients with infertility

    Institute of Scientific and Technical Information of China (English)

    刘效群; 阚国英; 彭玉梅; 樊瑞琴; 齐惠敏; 焦妹芬; 李忠; 石彬; 尹桂然; 董锡月

    2003-01-01

    Objectives:To investigate the effect of Chinese herbal medicine"heche assisted preg-nancy recipe (HCAPR)" on estrogen receptor(ER), progesterone receptor (PR), pro-lifierating cell nuclear antigen(PCNA) and vascular endothelial growth factor (VEGF)in endometrium of infertile women.Methods: The S-P immunohistochemical assay was used to observe expression ofER, PR , PCNA and VEGF in late proliferative phase before and after the HCAPR treat-ment.Results: After the treatment, the expression of ER,PR,PCNA and VEGF in nucleiof glandular epithelium and stromal cells was significantly stronger (all P<0. 001) re-spectively than that before treatment , especially the expression of PCNA and VEGF.Conclusions: These results suggest that traditional Chinese medicine HCAPR oftonifying kidney and regulating menstruation increased the synthesis of ER,PR, PCNAand VEGF, which may promote normal growth and development of the endometrium ,improve the micro-environment of the endometrium, and enhance uterine receptivity.The evidence may provide theoretical basis for therapy infertility with Chinese herbalmedicine.

  12. Profile of killer cell immunoglobulin-like receptor and its human leucocyte antigen ligands in dengue-infected patients from Western India.

    Science.gov (United States)

    Alagarasu, K; Bachal, R V; Shah, P S; Cecilia, D

    2015-12-01

    Killer cell immunoglobulin-like receptors (KIRs) regulate the activation of natural killer cells (NKs). Qualitative and quantitative differences in the type and the number of KIRs expressed on NK cells affect its activation which would influence the outcome of the disease. In this study, 114 hospitalized cases of dengue [82 dengue fever (DF) and 32 dengue haemorrhagic fever (DHF) cases] and 104 healthy controls (HC) without no known history of hospitalization for dengue-like illness were investigated for their KIR gene profile to find out the association of KIR genes with dengue disease severity. KIR gene profile was investigated using duplex sequence-specific priming polymerase chain reaction-based typing system. The results revealed a higher frequency of KIR3DL1 gene [P = 0.0225; odds ratio (OR) 4.1 95% confidence interval (CI) 1.1-14.8] and lower frequency of KIR3DS1/3DS1 genotype [P = 0.0225; OR 0.24 95% CI (0.068-0.88)] in DF cases compared to HC. Immunoglobulin-like receptor gene frequencies were not different between DHF and DF or HC. The results suggest that KIR3DL1/KIR3DS1 locus might be associated with the risk of developing DF. PMID:26385514

  13. Virus-Like Display of a Neo-Self Antigen Reverses B Cell Anergy in a B Cell Receptor Transgenic Mouse Model1

    Science.gov (United States)

    Chackerian, Bryce; Durfee, Marisa R.; Schiller, John T.

    2012-01-01

    The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4+ Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance. PMID:18424700

  14. Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata

    OpenAIRE

    Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I.; Eichhorn, M; Williams, R O; Ahmed, J. S.; Baldwin, C L; Clevers, J.C.

    1990-01-01

    The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested...

  15. Antigen dynamics of follicular dendritic cells

    NARCIS (Netherlands)

    Heesters, B.A.

    2015-01-01

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

  16. Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

    Directory of Open Access Journals (Sweden)

    Judith Abelha

    1981-06-01

    Full Text Available Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e não-imunes.Todos os soros humanos usados tinham títulos elevados de anticorpos das classes IgM ou IgG. Aglutinação e entumescimento dos parasitos eram observados apos encubação a 37ºC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a três dias. A quantidade de soro imune capaz de lisar um máximo de 10 x 10 [elevado a 6] hemácias sensibilizadas não foi capaz de lisar 4 x 10 [elevado a 3] tripomastigotas. Tipicamente, os parasitos apresentavam alterações cíclicas com formação de

  17. Mast Cell and Immune Inhibitory Receptors

    Institute of Scientific and Technical Information of China (English)

    Lixin Li; Zhengbin Yao

    2004-01-01

    Modulation by balancing activating and inhibitory receptors constitutes an important mechanism for regulating immune responses. Cells that are activated following ligation of receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs) can be negatively regulated by other receptors bearing immunoreceptor tyrosine-based inhibition motifs (ITIMs). Human mast cells (MCs) are the major effector cells of type I hypersensitivity and important participants in a number of disease processes. Antigen-mediated aggregation of IgE bound to its high-affinity receptor on MCs initiates a complex series of biochemical events leading to MC activation. With great detailed description and analysis of several inhibitory receptors on human MCs, a central paradigm of negative regulation of human MC activation by these receptors has emerged. Cellular & Molecular Immunology. 2004;1(6):408-415.

  18. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

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

  19. Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

    Science.gov (United States)

    Argüello, Rafael J; Reverendo, Marisa; Gatti, Evelina; Pierre, Philippe

    2016-07-01

    Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

  20. Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

    OpenAIRE

    Kloog, Yoel; Mor, Adam

    2014-01-01

    T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed th...

  1. Adoptive immunotherapy for acute leukemia:New insights in chimeric antigen receptors

    Institute of Scientific and Technical Information of China (English)

    Ma?l; Heiblig; Mohamed; Elhamri; Mauricette; Michallet; Xavier; Thomas

    2015-01-01

    Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells(LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despitethe introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90’s, chimeric antigen receptors(CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term antitumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia.

  2. An Overview of B-1 Cells as Antigen-Presenting Cells

    Science.gov (United States)

    Popi, Ana F.; Longo-Maugéri, Ieda M.; Mariano, Mario

    2016-01-01

    The role of B cells as antigen-presenting cells (APCs) has been extensively studied, mainly in relation to the activation of memory T cells. Considering the B cell subtypes, the role of B-1 cells as APCs is beginning to be explored. Initially, it was described that B-1 cells are activated preferentially by T-independent antigens. However, some reports demonstrated that these cells are also involved in a T-dependent response. The aim of this review is to summarize information about the ability of B-1 cells to play a role as APCs and to briefly discuss the role of the BCR and toll-like receptor signals in this process. Furthermore, some characteristics of B-1 cells, such as natural IgM production and phagocytic ability, could interfere in the participation of these cells in the onset of an adaptive response. PMID:27148259

  3. The structure, rearrangement, and ontogenic expression of DB and JB gene segments of the Mexican axolotl T-cell antigen receptor beta chain (TCRB).

    Science.gov (United States)

    Kerfourn, F; Charlemagne, J; Fellah, J S

    1996-01-01

    We sequenced a total of 189 independent rearrangements in which the VB7.1 element is associated with CB1 (99 clones) or CB2 (90 clones) isotypes of the T-cell receptor (TCR) beta chain in the Mexican axolotl. Three stages of development were analyzed: 2.5 months, 10 months, and 25 months. Three JB1 segments were associated with the VB-CB1 rearrangements and six JB2 segments with VB-CB2. As in other vertebrates, some amino acid positions were conserved in all Jbetas (e. g., Phe-108, Gly-109, Gly-111, Thr-112, and Val-116). Two 11 nucleotides DB-like sequences, differed by one (A or T) central residue and could be productively read in the three putative reading frames. Most of the DB1 and JB1 segments were in the VB-CB1 clones, and most of the DB2 and JB2 segments were in the VB-CB2 clones, suggesting that the TCRB locus is organized into independent DB-JB-CB clusters that used the same collection of VB segments. About 40% of the beta-chain VDJ junctions in 2.5-month-old larvae had N nucleotides, compared with about 73% in 10 - 25-month old animals. The beta-chain VDJ junctions had about 30% of defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs, and the large genome in this urodele. Many of the axolotl CDRbeta3 sequences deduced for in frame VDJ rearrangements are the same in animals of different origins. Such redundancy could be a statistical effect due to the small number of thymocytes in the developing axolotl, rather than to some bias due to junctional preferences.

  4. The structure, rearrangement, and ontogenic expression of DB and JB gene segments of the Mexican axolotl T-cell antigen receptor beta chain (TCRB).

    Science.gov (United States)

    Kerfourn, F; Charlemagne, J; Fellah, J S

    1996-01-01

    We sequenced a total of 189 independent rearrangements in which the VB7.1 element is associated with CB1 (99 clones) or CB2 (90 clones) isotypes of the T-cell receptor (TCR) beta chain in the Mexican axolotl. Three stages of development were analyzed: 2.5 months, 10 months, and 25 months. Three JB1 segments were associated with the VB-CB1 rearrangements and six JB2 segments with VB-CB2. As in other vertebrates, some amino acid positions were conserved in all Jbetas (e. g., Phe-108, Gly-109, Gly-111, Thr-112, and Val-116). Two 11 nucleotides DB-like sequences, differed by one (A or T) central residue and could be productively read in the three putative reading frames. Most of the DB1 and JB1 segments were in the VB-CB1 clones, and most of the DB2 and JB2 segments were in the VB-CB2 clones, suggesting that the TCRB locus is organized into independent DB-JB-CB clusters that used the same collection of VB segments. About 40% of the beta-chain VDJ junctions in 2.5-month-old larvae had N nucleotides, compared with about 73% in 10 - 25-month old animals. The beta-chain VDJ junctions had about 30% of defective rearrangements at all stages of development, which could be due to the slow rate of cell division in the axolotl lymphoid organs, and the large genome in this urodele. Many of the axolotl CDRbeta3 sequences deduced for in frame VDJ rearrangements are the same in animals of different origins. Such redundancy could be a statistical effect due to the small number of thymocytes in the developing axolotl, rather than to some bias due to junctional preferences. PMID:8753858

  5. Combination of Human Leukocyte Antigen and Killer Cell Immunoglobulin-Like Receptor Genetic Background Influences the Onset Age of Hepatocellular Carcinoma in Male Patients with Hepatitis B Virus Infection

    Directory of Open Access Journals (Sweden)

    Ning Pan

    2013-01-01

    Full Text Available To investigate whether killer cell immunoglobulin-like receptor (KIR and human leukocyte antigen (HLA genetic background could influence the onset age of hepatocellular carcinoma (HCC in patients with hepatitis B virus (HBV infection, one hundred and seventy-one males with HBV-related HCC were enrolled. The presence of 12 loci of KIR was detected individually. HLA-A, -B, and -C loci were genotyped with high resolution by a routine sequence-based typing method. The effect of each KIR locus, HLA ligand, and HLA-KIR combination was examined individually by Kaplan-Meier (KM analysis. Multivariate Cox hazard regression model was also applied. We identified C1C1-KIR2DS2/2DL2 as an independent risk factor for earlier onset age of HCC (median onset age was 44 for C1C1-KIR2DS2/2DL2 positive patients compared to 50 for negative patients, P=0.04 for KM analysis; HR = 1.70, P=0.004 for multivariate Cox model. We conclude that KIR and HLA genetic background can influence the onset age of HCC in male patients with HBV infection. This study may be useful to improve the current HCC surveillance program in HBV-infected patients. Our findings also suggest an important role of natural killer cells (or other KIR-expressing cells in the progress of HBV-related HCC development.

  6. Chagas' disease and Duffy antigen/receptor for chemokine (DARC: a mini-review

    Directory of Open Access Journals (Sweden)

    AP Oliveira

    2011-01-01

    Full Text Available Duffy gene (FY codifies the transmembrane glycoprotein Duffy (gp-Fy of 35 to 43 kDa which is moderately immunogenic. This glycoprotein is polymorphic, and constitutes the antigens of the Duffy histo-blood system which were designated receptors for chemokines and denominated DARC (Duffy antigen/receptor for chemokine. This receptor has an important role in the regulation of chemokine levels in the circulation, as it binds and adsorbs them on the surface of red cells as a reservoir. It plays a "sink" role, which can contribute to homeostasis by removing inflammatory chemokines from circulation as well as maintaining them in plasmatic levels. Chronic Chagas' cardiopathy (CCC is the most frequent form of the disease. It is an inflammatory disease, in which infiltrated inflammatory cells play an important role in the development and progress of the infection. High chemokine levels in the plasma have been associated with the disease severity in patients with heart failure. In this context, the profile of DARC expression could play an important function as a receptor for chemokines in Chagas' disease, in patients with CCC, as it can modulate damage from this inflammatory disease.

  7. Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

    DEFF Research Database (Denmark)

    Andersen, Rikke Sick; Kvistborg, Pia; Frøsig, Thomas Mørch;

    2012-01-01

    Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specif......(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h....

  8. 嵌合型抗原受体T细胞治疗技术在B细胞肿瘤中的研究进展:第56届美国血液学会年会报道%Progress of chimeric antigen receptor T cell immunotherapy in B cell hematologic malignancies:reports from the 56th American Society of Hematology annual meeting

    Institute of Scientific and Technical Information of China (English)

    邓碧萍; 刘红星; 童春容

    2015-01-01

    Chimeric antigen receptor T cell (CAR-T) therapy has shown promising perspective in clinical trails of B cell hematologic malignancies.Meanwhile,this therapy still need to be further improved in the following aspects:design of CAR-T,cancer antigens selection,T cells origin,and clinical application strategy.CAR-T immunotherapy is one of hot topics in the 56th American Society of Hematology (ASH) annual meeting.Some breakthroughs have been reported in both basic research and clinical trails.%嵌合型抗原受体T细胞(chimeric antigen receptorT cell,CAR-T)在B细胞肿瘤临床试验中已经显示了良好的前景,但是CAR-T的设计、肿瘤抗原的选择、T细胞的来源、CAR-T的临床应用策略等还有待进一步开发完善.CAR-T免疫细胞治疗技术是2014年第56届美国血液学会(ASH)年会的热点话题之一,其基础研究及临床试验均有突破性进展.

  9. [Mucose associated lymphoid tissue. Antigen presenting cells].

    Science.gov (United States)

    Luzardo-Baptista, Mario J; Luzardo, José Rafael

    2013-12-01

    We studied samples of normal and abnormal human mucosae, including oral tissue and uterine cervix, using electron microscopy. Special attention was given to the functions and mechanisms of defense carried out by the epithelial (EC) and dendritic cells (DC). Activated epithelial cells posses the capacity to uptake and process antigens, in order to present them, subsequently, to the dendritic cells. The structures and elements of the cells intervening on this function are: micropinocytic vesicles, multivesicular bodies, lysosomes, phagosomes, clathrin-covered vesicles, dense granules covered by a unit membrane, granules with onion likes leaves, microbodies, and dense granules with acid phosphatase activity. When they first arrive within the epithelial layers, the DC are clear with long cytoplasmic projections, which later become short, and the density of their cytoplasm increases. They possess mycropinocytic vesicles, some clathrine-covered vesicles, lysososmes and Birbeck granules. At this moment, they are known as Langerhans cells. EC and DC present many surface folds rich in micropynocytic vesicles. Between EC and DC there are many contacts (close junctions or tight junctions), through which antigens, phagocitized and processed by the EC, are given to the DC. These cells join them to major histocompatibility complex molecules or to other molecules with similar functions (CD1). Then the Langerhans cells travel to the lymphatic node to activate T cells and continue the immunologic task. So, in this way, both the EC and the DC are a link between the natural and the acquired immunological mechanisms. PMID:24502183

  10. Biochemical basis of synergy between antigen and T-helper (Th) cell-mediated activation of resting human B cells.

    OpenAIRE

    Chartash, E K; Crow, M K; Friedman, S M

    1989-01-01

    We have utilized CD23 expression as a marker for B cell activation in order to investigate the biochemical basis for synergy between antigen and T helper (Th) cells in the activation of resting human B cells. Our results confirm that while ligation of surface immunoglobulin (sIg) receptors by antigen analogues (e.g., F(ab')2 goat anti-human IgM) does not lead to CD23 expression, this stimulus markedly enhances CD23 expression induced during antigen specific Th-B cell interaction or by rIL-4. ...

  11. The progression of chimeric antigen receptor modified T cells in malignant tumor%嵌合抗原受体修饰T细胞在恶性肿瘤中的研究进展

    Institute of Scientific and Technical Information of China (English)

    张少华; 毕经旺

    2014-01-01

    近年来在肿瘤免疫治疗领域,嵌合抗原受体(CAR)修饰T细胞在基础研究和临床试验中取得了巨大进展。CAR由特异性单克隆抗体的可变区和CD3ζ结构域组成。CAR修饰T细胞的胞外区直接识别肿瘤相关抗原(TAA)。T细胞和靶抗原结合后可直接介导细胞毒性,并释放一些细胞因子如穿孔素、颗粒酶、干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α),最终导致肿瘤细胞的坏死。尽管CAR-T细胞具有显著的抗肿瘤效应,一些基础研究和临床实验中观察到的安全性问题值得关注。%Recentyearshavewitnessedmuchprogressinbothbasicresearchandclinicaltrialsregar-ding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells.CAR combine the varia-ble regions of a specific monoclonal antibody (scFv)with the CD3ζendodomain.The extracellular domain of CAR-engineered T cells directly dock to the tumor-associated antigen (TAA).When T cells bind to target anti-gens,they mediated redirected cytotoxicity and secrete a series of cytokines such as Perforin,Granzyme,Inter-feron-γ(IFN-γ)and Tumor necrosis factor-α(TNF-α),which would eventually lead to the necrosis of tumor cells.Although the antitumor response of the CAR-engineered T cells is considered as successful and surpri-sing,it should be noted that some safety issues have been observed in other several basic researches and clinical trials.This overview focuses upon the utility and safety of the CAR-engineered T cells.

  12. Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping.

    Science.gov (United States)

    Prodinger, W M

    1999-01-01

    Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed. PMID:10741859

  13. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen specific Interleukin 17 response

    Science.gov (United States)

    Zeng, Xun; Wei, Yu-ling; Huang, Jun; Newell, Evan W.; Yu, Hongxiang; Kidd, Brian A.; Kuhns, Michael S.; Waters, Ray W.; Davis, Mark M.; Weaver, Casey T.; Chien, Yueh-hsiu

    2012-01-01

    Summary γδ T cells contribute uniquely to host immune defense. However, how they function remains an enigma. Although it is unclear what most γδ T cells recognize, common dogma asserts that they recognize self-antigens. While they are the major initial Interleukin-17 (IL-17) producers in infections, it is unclear what is required to trigger these cells to act. Here, we report that a noted B cell antigen, the algae protein-phycoerythrin (PE) is an antigen for murine and human γδ T cells. PE also stained specific bovine γδ T cells. Employing this specificity, we demonstrated that antigen recognition, but not extensive clonal expansion, was required to activate naïve γδ T cells to make IL-17. In this activated state, γδ T cells gained the ability to respond to cytokine signals that perpetuated the IL-17 production. These results underscore the adaptability of lymphocyte antigen receptors and suggest a previously unrecognized antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

  14. γδ T cells recognize a microbial encoded B cell antigen to initiate a rapid antigen-specific interleukin-17 response.

    Science.gov (United States)

    Zeng, Xun; Wei, Yu-Ling; Huang, Jun; Newell, Evan W; Yu, Hongxiang; Kidd, Brian A; Kuhns, Michael S; Waters, Ray W; Davis, Mark M; Weaver, Casey T; Chien, Yueh-hsiu

    2012-09-21

    γδ T cells contribute uniquely to immune competence. Nevertheless, how they function remains an enigma. It is unclear what most γδ T cells recognize, what is required for them to mount an immune response, and how the γδ T cell response is integrated into host immune defense. Here, we report that a noted B cell antigen, the algae protein phycoerythrin (PE), is a murine and human γδ T cell antigen. Employing this specificity, we demonstrated that antigen recognition activated naive γδ T cells to make interleukin-17 and respond to cytokine signals that perpetuate the response. High frequencies of antigen-specific γδ T cells in naive animals and their ability to mount effector response without extensive clonal expansion allow γδ T cells to initiate a swift, substantial response. These results underscore the adaptability of lymphocyte antigen receptors and suggest an antigen-driven rapid response in protective immunity prior to the maturation of classical adaptive immunity. PMID:22960222

  15. Research progress of application of immunotherapy by chimeric antigen receptor-engineered T cells%嵌合抗原受体修饰的T细胞免疫治疗研究进展

    Institute of Scientific and Technical Information of China (English)

    李伟; 于慧杰

    2015-01-01

    The technique of chimeric antigen receptor-engineered T cells (CAR-T) is combined single chain antibody and T-cell activation modification and has specific and durable killing effect on tumors.It had achieved stunning results on clinical trials of B lymphocytic leukemias,B lymphomas and solid tumors.Meanwhile it also had potential risk on the off target effects,cytokine release syndrome,graft versus host disease and so on.This paper briefly reviews the principal of CAR-T cellular mechanism,advantages and clinical applications.%嵌合抗原受体修饰的T细胞(CAR-T细胞)是将单链抗体技术和T细胞活化基序结合应用的一种新的免疫治疗技术,具有特异、持久的肿瘤杀伤效应,在B淋巴细胞白血病、B淋巴瘤及实体瘤的临床试验中取得了令人震惊的效果,但也存在着脱靶效应、细胞因子释放综合征、移植物抗宿主病等潜在的风险.文章就CAR-T细胞原理、优势、临床应用等方面进行简要综述.

  16. New strategy of tumor immunotherapy based on chimeric antigen receptor engineered T cells%基于嵌合抗原受体修饰T细胞的肿瘤免疫治疗新策略

    Institute of Scientific and Technical Information of China (English)

    王艺; 赵颖颖; 韩双印

    2013-01-01

    Chimeric antigen receptor (CAR)-engineered T cell is a newly developed strategy of adoptive immunotherapy.Its unique theoretical superiority and attractive application prospects open up a promising arena for anticancer therapy.CAR combines single chain variable fragment (scFv) antibody recognizing tumor-associated antigen with T cell activation motif,which endows T cells with tumor-orientated targeting ability,stronger killing activity,and prolonged survival by genetic modification.Since first proposed by Dr.Eshhar in 1989,CAR has been developed from the first generation to the second and the third generations containing costimulatory molecular.The clinical trials in leukemia,lymphoma,and melanoma have obtained exciting results.However,the off-target effect,cytokine strom,and graft-versus-host disease are potential challenges for clinical use.Future research will focus on designing safer CAR of the fourth generation,selecting good therapeutic T cell subsets,optimizing clinical scheme of administration,and improving pre-clinical models.It is believed that the obstacles from bench to clinic will be cleared and that CAR will become one of the main cancer therapies with breakthroughs in immunology,gene therapy and cell engineering.%嵌合抗原受体(chimeric antigen receptor,CAR)修饰T细胞是近年来迅速发展的肿瘤过继免疫治疗新手段,其独特的作用机制和诱人的应用前景为肿瘤生物治疗开辟了一个崭新的舞台.CAR将识别肿瘤相关抗原的单链抗体和T细胞的活化基序相结合,通过基因转导赋予T细胞肿瘤靶向性、更强的杀伤活性和持久的生命力.自1989年Eshhar等首次提出CAR以来,CAR已从第一代发展至含有共刺激分子的第二、三代,CAR的Ⅰ/Ⅱ期临床试验在白血病、淋巴瘤、黑素瘤等恶性肿瘤中取得了可喜的成果,但是也面临脱靶效应、细胞因子风暴、移植物抗宿主病等潜在的安全性问题,未来研究将集中于设计更安全

  17. Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hai-Jie Yang; Ning-Shao Xia; Min Chen; Tong Cheng; Shui-Zhen He; Shao-Wei Li; Bao-Quan Guan; Zi-Heng Zhu; Ying Gu; Jun Zhang

    2005-01-01

    AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier.METHODS: One to 6 tandem copies of HBV preS1 (21-47)fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E. coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric partides was detected with immuno-capture PCR.RESULTS: Recombinant antigens CⅠ, CⅡ, CⅢ carrying 1-3 copies of HBV preS1 (21-47) individually could form viruslike particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CⅠ, CⅡ, CⅢ could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CⅠ, CⅡ and CⅢ) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CⅠ, CⅡ and CⅢ were able to capture HBV virions in immuno-capture PCR assay in vitro.CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

  18. TIM-4, expressed by medullary macrophages, regulates respiratory tolerance by mediating phagocytosis of antigen-specific T cells

    OpenAIRE

    Albacker, Lee A; Yu, Sanhong; Bedoret, Denis; Lee, Wan-Ling; Umetsu, Sarah E.; Monahan, Sheena; Freeman, Gordon J.; Umetsu, Dale T.; DeKruyff, Rosemarie H.

    2012-01-01

    Respiratory exposure to antigen induces T cell tolerance via several overlapping mechanisms that limit the immune response. While the mechanisms involved in the development of Treg cells have received much attention, those that result in T cell deletion are largely unknown. Herein, we show that F4/80+ lymph node medullary macrophages expressing TIM-4, a phosphatidylserine receptor, remove antigen-specific T cells during respiratory tolerance, thereby reducing secondary T cell responses. Block...

  19. A Stochastic Model of the Germinal Center Integrating Local Antigen Competition, Individualistic T-B Interactions, and B Cell Receptor Signaling.

    Science.gov (United States)

    Wang, Peng; Shih, Chang-Ming; Qi, Hai; Lan, Yue-Heng

    2016-08-15

    The germinal center (GC) reaction underlies productive humoral immunity by orchestrating competition-based affinity maturation to produce plasma cells and memory B cells. T cells are limiting in this process. How B cells integrate signals from T cells and BCRs to make fate decisions while subjected to a cyclic selection process is not clear. In this article, we present a spatiotemporally resolved stochastic model that describes cell behaviors as rate-limited stochastic reactions. We hypothesize a signal integrator protein integrates follicular helper T (Tfh)- and Ag-derived signals to drive different B cell fates in a probabilistic manner and a dedicated module of Tfh interaction promoting factors control the efficiency of contact-dependent Tfh help delivery to B cells. Without assuming deterministic affinity-based decisions or temporal event sequence, this model recapitulates GC characteristics, highlights the importance of efficient T cell help delivery during individual contacts with B cells and intercellular positive feedback for affinity maturation, reveals the possibility that antagonism between BCR signaling and T cell help accelerates affinity maturation, and suggests that the dichotomy between affinity and magnitude of GC reaction can be avoided by tuning the efficiency of contact-dependent help delivery during reiterative T-B interactions. PMID:27421481

  20. Strategies for B-cell receptor repertoire analysis in Primary Immunodeficiencies:From severe combined immunodeficiency to common variable immunodeficiency

    Directory of Open Access Journals (Sweden)

    Hanna eIJspeert

    2015-04-01

    Full Text Available The antigen receptor repertoires of B and T cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective we describe strategies and considerations for analysis of the naive and antigen selected B-cell repertoires in primary immunodeficiency (PID patients with a focus on severe combined immunodeficiency (SCID and common variable immunodeficiency (CVID.

  1. Antigen selection in B-cell lymphomas--tracing the evidence.

    Science.gov (United States)

    Sutton, Lesley-Ann; Agathangelidis, Andreas; Belessi, Chrysoula; Darzentas, Nikos; Davi, Frederic; Ghia, Paolo; Rosenquist, Richard; Stamatopoulos, Kostas

    2013-12-01

    While signaling through the B cell receptor (BcR) facilitates B cell development and maintenance, it also carries intertwined risks for the development of lymphomas since malignant B cells can exploit these pathways in order to trigger and fuel clonal expansion. This corruption of the normal B cell response to antigens, leading to sustained BcR signaling, has given great impulse to investigate in detail the role of antigen in lymphomas. Suffice it to conclude from such studies, largely immunogenetics based, that the evidence implicating antigens (exogenous or self) in lymphoma development is substantial and that lymphomagenesis is functionally driven and dynamic, rather than a simple stochastic process. As the paradigm of antigen-driven lymphoma evolves, further investigation will be paramount to the identification of the inciting agent(s) that may be responsible for immunoproliferative neoplasms and also for the development of therapeutic agents targeting effectors of the BcR signaling pathway.

  2. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    Science.gov (United States)

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  3. Strategies for B-cell receptor repertoire analysis in Primary Immunodeficiencies:From severe combined immunodeficiency to common variable immunodeficiency

    OpenAIRE

    Hanna eIJspeert; Marjolein eWentink; David evan Zessen; Driessen, Gertjan J.; Virgil A.S.H. Dalm; Martin P van Hagen; Ingrid ePico-Knijnenburg; Erik J Simons; van Dongen, Jacques J.M.; Stubbs, Andrew P; Mirjam eVan Der Burg

    2015-01-01

    The antigen receptor repertoires of B and T cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many ...

  4. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation.

    Science.gov (United States)

    Alam, S; Rangaswamy, D; Prakash, S; Sharma, R K; Khan, M I; Sonawane, A; Agrawal, S

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-07-01

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

  6. Targeting self- and foreign antigens to dendritic cells via DC-ASGPR generates IL-10-producing suppressive CD4+ T cells.

    Science.gov (United States)

    Li, Dapeng; Romain, Gabrielle; Flamar, Anne-Laure; Duluc, Dorothée; Dullaers, Melissa; Li, Xiao-Hua; Zurawski, Sandra; Bosquet, Nathalie; Palucka, Anna Karolina; Le Grand, Roger; O'Garra, Anne; Zurawski, Gerard; Banchereau, Jacques; Oh, Sangkon

    2012-01-16

    Dendritic cells (DCs) can initiate and shape host immune responses toward either immunity or tolerance by their effects on antigen-specific CD4(+) T cells. DC-asialoglycoprotein receptor (DC-ASGPR), a lectinlike receptor, is a known scavenger receptor. Here, we report that targeting antigens to human DCs via DC-ASGPR, but not lectin-like oxidized-LDL receptor, Dectin-1, or DC-specific ICAM-3-grabbing nonintegrin favors the generation of antigen-specific suppressive CD4(+) T cells that produce interleukin 10 (IL-10). These findings apply to both self- and foreign antigens, as well as memory and naive CD4(+) T cells. The generation of such IL-10-producing CD4(+) T cells requires p38/extracellular signal-regulated kinase phosphorylation and IL-10 induction in DCs. We further demonstrate that immunization of nonhuman primates with antigens fused to anti-DC-ASGPR monoclonal antibody generates antigen-specific CD4(+) T cells that produce IL-10 in vivo. This study provides a new strategy for the establishment of antigen-specific IL-10-producing suppressive T cells in vivo by targeting whole protein antigens to DCs via DC-ASGPR.

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

    DEFF Research Database (Denmark)

    Owens, T; Fazekas de St Groth, B

    1987-01-01

    The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4...... the formation of TCR complexes and so prevent activation. However, by increasing the epitope density of the activating ligand, the avidity of the T cell/ligand interaction can be increased sufficiently to prevent this disruption.(ABSTRACT TRUNCATED AT 400 WORDS)...

  8. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

    OpenAIRE

    Wu, Chia-Yung; Kole T Roybal; Puchner, Elias M.; Onuffer, James; Lim, Wendell A.

    2015-01-01

    There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen spec...

  9. The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31).

    Science.gov (United States)

    Sachs, Ulrich J H; Andrei-Selmer, Cornelia L; Maniar, Amudhan; Weiss, Timo; Paddock, Cathy; Orlova, Valeria V; Choi, Eun Young; Newman, Peter J; Preissner, Klaus T; Chavakis, Triantafyllos; Santoso, Sentot

    2007-08-10

    Human neutrophil-specific CD177 (NB1 and PRV-1) has been reported to be up-regulated in a number of inflammatory settings, including bacterial infection and granulocyte-colony-stimulating factor application. Little is known about its function. By flow cytometry and immunoprecipitation studies, we identified platelet endothelial cell adhesion molecule-1 (PECAM-1) as a binding partner of CD177. Real-time protein-protein analysis using surface plasmon resonance confirmed a cation-dependent, specific interaction between CD177 and the heterophilic domains of PECAM-1. Monoclonal antibodies against CD177 and against PECAM-1 domain 6 inhibited adhesion of U937 cells stably expressing CD177 to immobilized PECAM-1. Transendothelial migration of human neutrophils was also inhibited by these antibodies. Our findings provide direct evidence that neutrophil-specific CD177 is a heterophilic binding partner of PECAM-1. This interaction may constitute a new pathway that participates in neutrophil transmigration. PMID:17580308

  10. Expression and Purification of the Bacillus anthracis Protective Antigen Receptor-binding Domain

    Institute of Scientific and Technical Information of China (English)

    葛猛; 徐俊杰; 李冰; 董大勇; 宋小红; 郭强; 赵剑; 陈薇

    2004-01-01

    The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E. coli. Signal sequence of the outer membrane protein A (OmpA) of E. coli was attached to the 5' end of the gene encoding protective antigen receptor-binding domain (the 4th domain of PA, PALM). The plasmid carrying the fusion gene was then transformed into E. coli and induced to express recombinant PAlM by IFFG. The recombinant protein was purified by chromatography and then identified by N-terrainal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ionexchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E. coli. The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.

  11. Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells

    DEFF Research Database (Denmark)

    Sette, A; Buus, S; Colon, S;

    1987-01-01

    A detailed analysis of the residues within an immunogenic peptide that endow it with the capacity to interact with Ia and to be recognized by T cells is presented. Ia interacts with only a few of the peptide residues and overall exhibits a very broad specificity. Some residues appear to interact...... both with Ia and with T cells, leading to a model in which a peptide antigen is 'sandwiched' between Ia and the T-cell receptor....

  12. Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

    OpenAIRE

    Kawahara, Masahiro; Ueda, Hiroshi; Morita, Sumiyo; Tsumoto, Kouhei; Kumagai, Izumi; Nagamune, Teruyuki

    2003-01-01

    While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin recepto...

  13. Proliferating cell nuclear antigen in neutrophil fate.

    Science.gov (United States)

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  14. T-Cell Tumor Elimination as a Result of T-Cell Receptor-Mediated Activation

    Science.gov (United States)

    Ashwell, Jonathan D.; Longo, Dan L.; Bridges, Sandra H.

    1987-07-01

    It has recently been shown that activation of murine T-cell hybridomas with antigen inhibits their growth in vitro. The ``suicide'' of these neoplastic T cells upon stimulation with antigen suggested the possibility that activation via the antigen-specific receptor could also inhibit the growth of neoplastic T cells in vivo. To test this, mice were subcutaneously inoculated with antigen-specific T-cell hybridomas and then treated intraperitoneally with antigen. Administration of the appropriate antigen immediately after inoculation with the T-cell hybridoma abrogated tumor formation; antigen administered after tumors had become established decreased the tumor burden and, in a substantial fraction of animals, led to long-term survival. The efficacy of antigen therapy was due to both a direct inhibitory effect on tumor growth and the induction of host immunity. These studies demonstrate the utility of cellular activation as a means of inhibiting neoplastic T-cell growth in vivo and provide a rationale for studying the use of less selective reagents that can mimic the activating properties of antigen, such as monoclonal antibodies, in the treatment of T-cell neoplasms of unknown antigen specificity.

  15. Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

    NARCIS (Netherlands)

    Tacken, P.J.; Vries, I.J.M. de; Gijzen, K.; Joosten, B.H.G.M.; Wu, D.; Rother, R.P.; Faas, S.J.; Punt, C.J.A.; Torensma, R.; Adema, G.J.; Figdor, C.G.

    2005-01-01

    Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized ant

  16. Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

    Directory of Open Access Journals (Sweden)

    Brian D Hondowicz

    Full Text Available The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

  17. Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred T cells in glioblastoma.

    Science.gov (United States)

    Hegde, Meenakshi; Corder, Amanda; Chow, Kevin K H; Mukherjee, Malini; Ashoori, Aidin; Kew, Yvonne; Zhang, Yi Jonathan; Baskin, David S; Merchant, Fatima A; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Wu, Meng Fen; Liu, Hao; Heslop, Helen E; Gottschalk, Stephen; Gottachalk, Stephen; Yvon, Eric; Ahmed, Nabil

    2013-11-01

    Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

  18. In situ Delivery of Tumor Antigen- and Adjuvant-Loaded Liposomes Boosts Antigen-Specific T-Cell Responses by Human Dermal Dendritic Cells.

    Science.gov (United States)

    Boks, Martine A; Bruijns, Sven C M; Ambrosini, Martino; Kalay, Hakan; van Bloois, Louis; Storm, Gert; de Gruijl, Tanja; van Kooyk, Yvette

    2015-11-01

    Dendritic cells (DCs) have an important role in tumor control via the induction of tumor-specific T-cell responses and are therefore an ideal target for immunotherapy. The human skin is an attractive site for tumor vaccination as it contains various DC subsets. The simultaneous delivery of tumor antigen with an adjuvant is beneficial for cross-presentation and the induction of tumor-specific T-cell responses. We therefore developed liposomes that contain the melanoma-associated antigen glycoprotein 100280-288 peptide and Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) as adjuvant. These liposomes are efficiently taken up by monocyte-derived DCs, and antigen presentation to CD8(+) T cells was significantly higher with MPLA-modified liposomes as compared with non-modified liposomes or the co-administration of soluble MPLA. We used a human skin explant model to evaluate the efficiency of intradermal delivery of liposomes. Liposomes were efficiently taken up by CD1a(+) and especially CD14(+) dermal DCs. Induction of CD8(+) T-cell responses by emigrated dermal DCs was significantly higher when MPLA was incorporated into the liposomes as compared with non-modified liposomes or co-administration of soluble MPLA. Thus, the modification of antigen-carrying liposomes with TLR ligand MPLA significantly enhances tumor-specific T-cell responses by dermal DCs and is an attractive vaccination strategy in human skin. PMID:26083554

  19. Engineering antigen-specific T cells from genetically modified human hematopoietic stem cells in immunodeficient mice.

    Directory of Open Access Journals (Sweden)

    Scott G Kitchen

    Full Text Available There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR. Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.

  20. Prostate specific antigen gene expression in androgen insensitive prostate carcinoma subculture cell line.

    Science.gov (United States)

    Tsui, Ke-Hung; Feng, Tsui-Hsia; Chung, Li-Chuan; Chao, Chun-Hsiang; Chang, Phei-Lang; Juang, Horng-Heng

    2008-01-01

    A novel prostate cancer cell line (PC-J) was isolated from an androgen independent non-prostate specific antigen (non-PSA) producing carcinoma cell line. The homologous correlation between PC-J and PC-3 was determined by short tandem repeat analysis. The PSA promoter activity was detected by transient expression assay in the PC-J and LNCaP cells but not in androgen insensitive PC-3 cells. When the PC-J cells were cotransfected with androgen receptor, androgen receptor coactivators and PSA reporter vector cells, the reporter assays indicated that nuclear receptor coactivator 4 (NCOA4) but not androgen receptor activator 24 (ARA24) increased the sensitivity and maximum stimulation of dihydrotestosterone (DHT)-inducing PSA promoter activity. The RT-PCR assays revealed that the expression of several tumor markers, including interleukin-6, prostate stem cell antigen (PSCA), prostate epithelium-specific Ets transcription factor (PDEF) and matriptase, was lower in the PC-J cells than in the PC-3 cells. This cell model elucidated the regulation of PSA expression and enabled comparison of the gene profile at different stages of metastasis in prostatic carcinoma.

  1. Cytotoxicity of tumor antigen specific human T cells is unimpaired by arginine depletion.

    Directory of Open Access Journals (Sweden)

    Markus Munder

    Full Text Available Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8(+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i CD8(+ T cells with specificity against the MART-1aa26-35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii clonal CMV pp65aa495-503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495-503 specific T cell receptor were analyzed. Our data demonstrate that human CD8(+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency.

  2. Tracking antigen-specific T-cells during clinical tolerance induction in humans.

    Directory of Open Access Journals (Sweden)

    Aamir Aslam

    Full Text Available Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen

  3. Fibroblasts as Efficient Antigen-Presenting Cells in Lymphoid Organs

    Science.gov (United States)

    Kundig, Thomas M.; Bachmann, Martin F.; Dipaolo, Claudio; Simard, John J. L.; Battegay, Manuel; Lother, Heinz; Gessner, Andre; Kuhlcke, Klaus; Ohashi, Pamela S.; Hengartner, Hans; Zinkernagel, Rolf M.

    1995-06-01

    Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.

  4. Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice.

    Directory of Open Access Journals (Sweden)

    Annalisa Ciabattini

    Full Text Available Priming of T cells is a key event in vaccination, since it bears a decisive influence on the type and magnitude of the immune response. T-cell priming after mucosal immunization via the nasal route was studied by investigating the distribution of antigen-loaded antigen presenting cells (APCs and primed antigen-specific T cells. Nasal immunization studies were conducted using the model protein antigen ovalbumin (OVA plus CpG oligodeoxynucleotide adjuvant. Trafficking of antigen-specific primed T cells was analyzed in vivo after adoptive transfer of OVA-specific transgenic T cells in the presence or absence of fingolimod, a drug that causes lymphocytes sequestration within lymph nodes. Antigen-loaded APCs were observed in mediastinal lymph nodes, draining the respiratory tract, but not in distal lymph nodes. Antigen-specific proliferating T cells were first observed within draining lymph nodes, and later in distal iliac and mesenteric lymph nodes and in the spleen. The presence at distal sites was due to migration of locally primed T cells as shown by fingolimod treatment that caused a drastic reduction of proliferated T cells in non-draining lymph nodes and an accumulation of extensively divided T cells within draining lymph nodes. Homing of nasally primed T cells in distal iliac lymph nodes was CD62L-dependent, while entry into mesenteric lymph nodes depended on both CD62L and α4β7, as shown by in vivo antibody-mediated inhibition of T-cell trafficking. These data, elucidating the trafficking of antigen-specific primed T cells to non-draining peripheral and mucosa-associated lymph nodes following nasal immunization, provide relevant insights for the design of vaccination strategies based on mucosal priming.

  5. Delayed type hypersensitivity to allogeneic mouse epidermal cell antigens, 2

    International Nuclear Information System (INIS)

    A low dose of ultraviolet B radiation impairs the effectiveness of epidermal cell antigens. We studied the effect of ultraviolet B radiation on the delayed type hypersensitivity induced by allogeneic epidermal cell antigen. The delayed type hypersensitivity response was assayed by footpad swelling in mice. When epidermal cells were exposed to ultraviolet B radiation (660 J/m2), their ability to induce T cells of delayed type hypersensitivity activation was markedly inhibited in any combination of recipient mice and allogeneic epidermal cells. The effect of ultraviolet B radiation on epidermal cells was observed before immunization and challenge. Ultraviolet B treated epidermal cells did not induce suppressor T cells in mice. These results indicate that ultraviolet B radiation destroys the antigenicity of epidermal cells. (author)

  6. Antigen loading on dendritic cells affects the lell function in stimulating T cells.

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To study the effect of antigen loading on dendritic cells (DC). Methods: DCs collected from peripheral blood monocytes were loaded with a tumor antigen from XG-7 cell line. These DCs were then co-cultured with allogeneic T cells and were compared with those DCs without antigen exposure.

  7. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity.

    Science.gov (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha

    2011-01-15

    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  8. MHC class I is functionally associated with antigen receptors in human T and B lymphomas

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Jacoby, B F; Skov, S;

    1996-01-01

    We have studied the antibody-induced effect of cross-linking the major histocompatibility complex class I (MHC-I) in human T leukemic cells (Jurkat) and human B lymphoma cells (Solubo, Burkitts lymphoma) on intracellular [Ca2+]i levels. The increase in [Ca2+]i after MHC-I cross-linking in Jurkat...... lines the increase in [Ca2+]i after MHC-I cross-linking caused upregulation of CD69, an early marker of activation. When studying the effect of MHC-I cross-linking on the TCR- and B cell antigen receptor (BCR)- mediated increase in [Ca2+]i, respectively, we observed that MHC-I had a costimulatory effect......, respectively, were positively correlated with the level of MHC-I expressed on the cell surface. These observations suggest two different roles in signal transduction for the MHC-I molecules in the T and B cells studied. First, by themselves MHC-I complexes are able to induce activation of intracellular second...

  9. Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

    Science.gov (United States)

    Yang, Lili; Baltimore, David

    2005-03-01

    A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

  10. Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation.

    Science.gov (United States)

    Koh, Sarene; Shimasaki, Noriko; Bertoletti, Antonio

    2016-01-01

    Autologous T lymphocytes genetically modified to express T cell receptors or chimeric antigen receptors have shown great promise in the treatment of several cancers, including melanoma and leukemia. In addition to tumor-associated antigens and tumor-specific neoantigens, tumors expressing viral peptides can also be recognized by specific T cells and are attractive targets for cell therapy. Hepatocellular carcinoma cells often have hepatitis B virus DNA integration and can be targeted by hepatitis B virus-specific T cells. Here, we describe a method to engineer hepatitis B virus-specific T cell receptors in primary human T lymphocytes based on electroporation of hepatitis B virus T cell receptor messenger RNA. This method can be extended to a large scale therapeutic T cell production following current good manufacturing practice compliance and is applicable to the redirection of T lymphocytes with T cell receptors of other virus specificities such as Epstein-Barr virus, cytomegalovirus, and chimeric receptors specific for other antigens expressed on cancer cells. PMID:27236807

  11. Survival and signaling changes in antigen presenting cell subsets after radiation

    Science.gov (United States)

    Parker, Jennifer Janell

    Radiation therapy is a widely used cancer treatment that has the potential to influence anti-tumor immune responses. Both myeloablative and non-myeloablative radiation are often used as part of preparatory regimens for hematopoetic stem cell transplantation, in combination with other chemotherapy or immuno-modulatory (e.g. Anti-thymocyte globulin (ATG)) therapies for both cytotoxic and immune modulatory purposes. However, the mechanisms responsible for the effect of radiation on antigen presenting cell (APC) responsiveness and radioresistance are poorly understood. The first studies described in this thesis were designed to identify and characterize early radiation-induced signaling changes in antigen presenting cells and to determine the effects of these signaling changes on APC receptor expression and function. The NFkappaB pathway in antigen presenting cells was chosen for study because it is activated by radiation in a wide range of other cell types and plays a vital role in the maintenance and regulation of the immune system. The effects of therapeutically relevant doses radiation (2 and 20 Gy) were compared at various timepoints in the human monocytic cell line (U937) using phospho-flow cytometry staining methods and cytometric analysis. These studies demonstrated that radiation-induced changes in the phosphorylation state of NFkappaB family members that were p53 independent. However, these changes were dependent upon activation of ATM in response to single or double-stranded breaks in DNA, as shown in experiments using an inhibitor of ATM and ATM siRNA knockdown U937 cells. In addition, studies examining the effect of radiation on co-stimulatory receptors with and without inhibition of the NFkappaB pathway via phospho-flow cytometry revealed that radiation-induced phosphorylation of NEMO promoted the activation and functional maturation of U937 cells. Furthermore, functional studies using both phospho-flow cytometry and/or mixed lymphocyte reactions to

  12. Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.

    Science.gov (United States)

    Anastassiadis, Konstantinos; Rostovskaya, Maria; Lubitz, Sandra; Weidlich, Stefanie; Stewart, A Francis

    2010-04-01

    Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of the androgen receptor (irtTA-ABD) or of a mutated glucocorticoid receptor (irtTA-GBD*). Induction of T-antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA-ABD or dexamethasone for irtTA-GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T-antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T-antigen. PMID:20146354

  13. Molecular cloning of a cDNA for the chicken progesterone receptor B antigen.

    OpenAIRE

    Zarucki-Schulz, T; Kulomaa, M S; Headon, D R; N.L. Weigel; Baez, M; Edwards, D.P.; McGuire, W L; Schrader, W T; O'Malley, B W

    1984-01-01

    A cDNA for the chicken progesterone receptor B subunit antigen (Mr, 108,000) has been isolated from a cDNA library prepared from size-selected chicken oviduct poly(A)+RNA. A specific monoclonal antibody raised against hen progesterone receptor B subunit (alpha PR-B) was used to screen the library. Recombinant clones reacting with the antibody by virtue of antigen expression were used in hybrid-selected translation. A single clone, pPRB-1, hybridized specifically to a mRNA that yielded a Mr 10...

  14. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    Science.gov (United States)

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed. PMID:2973431

  15. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    Science.gov (United States)

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

  16. Dual effect of CD85/leukocyte Ig-like receptor-1/Ig-like transcript 2 and CD152 (CTLA-4) on cytokine production by antigen-stimulated human T cells.

    Science.gov (United States)

    Saverino, Daniele; Merlo, Andrea; Bruno, Silvia; Pistoia, Vito; Grossi, Carlo E; Ciccone, Ermanno

    2002-01-01

    The functional outcome of a T cell response to Ag is the result of a balance between coactivation and inhibitory signals. In this study we have investigated the effects of the CD85/leukocyte Ig-like receptor (LIR)-1/Ig-like transcript (ILT) 2 and of CD152 (CTLA-4) inhibitory receptors on the modulation of cell-mediated immune responses to specific Ags, both at the effector and at the resting/memory cell level. Proliferation and cytokine production of CD4+ T lymphocytes stimulated by recall Ags have been evaluated. Cross-linking of CD85/LIR-1/ILT2 or CD152 molecules on cultured T cells using specific mAb and goat anti-mouse antiserum inhibits Ag-specific T cell proliferation. This inhibition is always paralleled by increased production of cytokines that down-regulate immune responses, e.g., IL-10 and TGF-beta. In contrast, the production of cytokines that support T cell expansion and function (e.g., IL-2, IFN-gamma, and IL-13) is significantly decreased. A long-term effect of CD85/LIR-1/ILT2 and of CD152 occurs during Ag-specific T cell activation and expansion. T cells, primed in the presence of anti-CD85/LIR-1/ILT2 and anti-CD152 blocking mAb (but in the absence of cross-linking), proliferate at higher rates and produce higher amounts of IL-2, IFN-gamma, and IL-13, in comparison with T cells stimulated with the Ag alone. We also show that the inhibitory receptors exert a similar effect during Ag activation of specific CD4+ effector T cells. Ag-specific polyclonal CD4+ T cell lines exhibit increased proliferation and IL-2, IFN-gamma, and IL-13 production when the CD85/LIR-1/ILT2 receptor is blocked by specific mAb. In contrast, cross-linking of this receptor down-regulates Ag-specific CD4+ T cell proliferation and increases IL-10 and TGF-beta production. PMID:11751964

  17. Monoclonal T-cell receptors: new reagents for cancer therapy.

    Science.gov (United States)

    Stauss, Hans J; Cesco-Gaspere, Michela; Thomas, Sharyn; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; Wright, Graham; Perro, Mario; Little, Ann-Margaret; Pospori, Constantina; King, Judy; Morris, Emma C

    2007-10-01

    Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate "undifferentiated" T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells. PMID:17637721

  18. Structure-Based, Rational Design of T Cell Receptors

    OpenAIRE

    Zoete, V; Irving, M.; Ferber, M.; Cuendet, M. A.; Michielin, O

    2013-01-01

    Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding...

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

    DEFF Research Database (Denmark)

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

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic ...

  20. CD8+ T cell priming by dendritic cell vaccines requires antigen transfer to endogenous antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Alice W Yewdall

    Full Text Available Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.

  1. Staphylococcus aureus Targets the Duffy Antigen Receptor for Chemokines (DARC) to Lyse Erythrocytes

    NARCIS (Netherlands)

    Spaan, András N.; Reyes-Robles, Tamara; Badiou, Cédric; Cochet, Sylvie; Boguslawski, Kristina M.; Yoong, Pauline; Day, Christopher J.; Gosselaar-de Haas, Carla J C; van Kessel, Kok P M; Vandenesch, François; Jennings, Michael P.; Le Van Kim, Caroline; Colin, Yves; Van Strijp, Jos A G; Henry, Thomas; Torres, Victor J.

    2015-01-01

    In order for Staphylococcus aureus to thrive inside the mammalian host, the bacterium has to overcome iron scarcity. S. aureus is thought to produce toxins that lyse erythrocytes, releasing hemoglobin, the most abundant iron source in mammals. Here we identify the Duffy antigen receptor for chemokin

  2. Typing of murine cell-surface antigens by cellular radioimmunoassay

    International Nuclear Information System (INIS)

    A cellular radioimmunoassay utilizing 125I-labelled Protein A was used for detecting antigen-antibody complexes on gultaraldehyde fixed cells attached to microtiter plates. This method is rapid, sensitive and specific for revealing H-2 private and public specificities as well as Ia and Lyt antigens. As plates may be kept for months, several reactivities can be tested in one step on a large panel rendering a regular supply of animals unnecessary. (Auth.)

  3. An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv

    International Nuclear Information System (INIS)

    Selection and production of specific antibodies are limiting the development of high-throughput immunoassays such as antibody chips. In this study, we propose an antigen-mediated selection of antibody producers (ASAP) system in mammalian cells. As a model system, transgenes encoding anti-fluorescein ScFv fused to cytokine receptors were introduced to IL-3-dependent cell lines. Addition of fluorescein-conjugated BSA induced growth signal through the ScFv/receptor chimeras, leading to selective expansion of the transduced cells. Cre recombinase was then used to excise the receptor gene flanked by two loxP recognition sites in the introns, resulting in secretion of his-myc-tagged ScFv to the culture medium. When the first loxP site was used in the exon as a linker between ScFv and receptor, enhanced antigen-mediated cell proliferation and production of unexpectedly glycosylated ScFv were achieved. ASAP is the first mammalian selection/production system of recombinant human ScFvs, without need for subcloning and with the advantage of glycosylated product

  4. Requirements for Peptide-induced T Cell Receptor Downregulation on Naive CD8+ T Cells

    OpenAIRE

    Cai, Zeling; Kishimoto, Hidehiro; Brunmark, Anders; Jackson, Michael R.; Peterson, Per A.; Sprent, Jonathan

    1997-01-01

    The requirements for inducing downregulation of α/β T cell receptor (TCR) molecules on naive major histocompatibility complex class I–restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent re...

  5. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

    Directory of Open Access Journals (Sweden)

    Zheng Zhili

    2012-02-01

    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  6. Antigen-induced and non-antigen-induced histamine release from rat mast cells sensitized with mouse antiserum.

    Directory of Open Access Journals (Sweden)

    Kurose,Masao

    1981-10-01

    Full Text Available Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS, although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s, which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.

  7. Clinical application of active B-cell antigen receptor signaling as novel therapeutic target in B-cell non-Hodgkin lymphoma%靶向B细胞抗原受体信号转导通路小分子抑制剂在B细胞非霍奇金淋巴瘤中的临床应用

    Institute of Scientific and Technical Information of China (English)

    丁宁

    2013-01-01

    The chronic B-cell antigen receptor (BCR) pathway plays an important role in malignant proliferation of B-cell lymphoma.Several tyrosine kinase inhibitors have shown impressive anti-tumor activity in relapsed and refractory B-cell non-Hodgkin lymphoma.This review discussed essential tyrosine kinases in BCR signaling pathway and present data from clinical usage of these related novel target agents.%B细胞抗原受体(BCR)信号转导通路对于B细胞非霍奇金淋巴瘤(B-NHL)恶性增殖的调控作用日益受到关注.针对BCR信号转导通路关键分子所研发的酪氨酸激酶抑制剂,已经在临床研究中初步显现出对B细胞淋巴瘤的良好疗效.文章就BCR信号转导通路中的关键酪氨酸激酶及相应靶向药物的临床应用状况作一综述.

  8. Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8+ T Cells Ex Vivo

    Institute of Scientific and Technical Information of China (English)

    Weijuan Gong; Mingchun Ji; Zhengfeng Cao; Liheng Wang; Yayun Qian; Maozhi Hu; Li Qian; Xingyuan Pan

    2008-01-01

    Atificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of CO. stimulating receptors of CD28 and 4-1BB. respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32, CD86/4-IBBL cell with OKT3 monoclonal antibody against CD3.named K32/CD86/4-lBBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8+ T cell activation. promoting CD8+ T cell proliferation, division, and long-term growth, inhibiting CD8+ T cell apoptosis, and enhancing CD8+ T cell secretion of IFN-Y and perforin. Furthermore, antigen. secific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 day This approach was robust, simple, reproducible and economical for expansion and activation of CD8+ T cells and may have important therapeutic implications for adoptive immunotherapy. Cellular & Molecular Immunology.2007;5(1):47-53.

  9. Polymeric Structure and Host Toll-like Receptor 4 Dictate Immunogenicity of NY-ESO-1 Antigen in Vivo*

    Science.gov (United States)

    Liu, Yanan; Tian, Xiaoli; Leitner, Wolfgang W.; Aldridge, Michael E.; Zheng, Junying; Yu, Zhiya; Restifo, Nicholas P.; Weiss, Richard; Scheiblhofer, Sandra; Xie, Chong; Sun, Ren; Cheng, Genhong; Zeng, Gang

    2011-01-01

    In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern. PMID:21900253

  10. Polymeric structure and host Toll-like receptor 4 dictate immunogenicity of NY-ESO-1 antigen in vivo.

    Science.gov (United States)

    Liu, Yanan; Tian, Xiaoli; Leitner, Wolfgang W; Aldridge, Michael E; Zheng, Junying; Yu, Zhiya; Restifo, Nicholas P; Weiss, Richard; Scheiblhofer, Sandra; Xie, Chong; Sun, Ren; Cheng, Genhong; Zeng, Gang

    2011-10-28

    In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.

  11. Nematode-Derived Proteins Suppress Proliferation and Cytokine Production of Antigen-Specific T Cells via Induction of Cell Death

    Science.gov (United States)

    Hartmann, Wiebke; Brenz, Yannick; Kingsley, Manchang Tanyi; Ajonina-Ekoti, Irene; Brattig, Norbert W.; Liebau, Eva; Breloer, Minka

    2013-01-01

    In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro. PMID:23861729

  12. Nematode-derived proteins suppress proliferation and cytokine production of antigen-specific T cells via induction of cell death.

    Directory of Open Access Journals (Sweden)

    Wiebke Hartmann

    Full Text Available In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host's immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocercavolvulus-derived excreted/secreted proteins. Addition of two recombinant O. volvulus proteins, abundant larval transcript-2 (OvALT-2 and novel larval transcript-1 (OvNLT-1 to cell cultures of T cell receptor transgenic CD4(+ and CD8(+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4(+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by (3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8(+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O. volvulus protein, microfilariae surface-associated antigen (Ov103, did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O. volvulus, which interfere with the function of antigen-specific T cells in vitro.

  13. HIV-1 Trans Infection of CD4+ T Cells by Professional Antigen Presenting Cells

    Directory of Open Access Journals (Sweden)

    Charles R. Rinaldo

    2013-01-01

    Full Text Available Since the 1990s we have known of the fascinating ability of a complex set of professional antigen presenting cells (APCs; dendritic cells, monocytes/macrophages, and B lymphocytes to mediate HIV-1 trans infection of CD4+ T cells. This results in a burst of virus replication in the T cells that is much greater than that resulting from direct, cis infection of either APC or T cells, or trans infection between T cells. Such APC-to-T cell trans infection first involves a complex set of virus subtype, attachment, entry, and replication patterns that have many similarities among APC, as well as distinct differences related to virus receptors, intracellular trafficking, and productive and nonproductive replication pathways. The end result is that HIV-1 can sequester within the APC for several days and be transmitted via membrane extensions intracellularly and extracellularly to T cells across the virologic synapse. Virus replication requires activated T cells that can develop concurrently with the events of virus transmission. Further research is essential to fill the many gaps in our understanding of these trans infection processes and their role in natural HIV-1 infection.

  14. Antigenicity and immunogenicity of an extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

    OpenAIRE

    Gómez, A M; Rhodes, J C; Deepe, G S

    1991-01-01

    In order to identify T-cell antigens from Histoplasma capsulatum yeast cells, we prepared a detergent extract of the cell wall and cell membrane of yeast-phase H. capsulatum G217B and analyzed its antigenicity and immunogenicity. Mice injected with viable H. capsulatum yeast cells or with 500 or 1,000 micrograms of the extract mounted a delayed-type hypersensitivity response to solubilized cell wall and cell membrane. Vaccination with this antigenic preparation conferred a protective immune r...

  15. Development of antibodies to human embryonic stem cell antigens

    OpenAIRE

    Stanley Marisa; Rao Mahendra S; Olson Judith M; Cai Jingli; Taylor Eva; Ni Hsiao-Tzu

    2005-01-01

    Abstract Background Using antibodies to specific protein antigens is the method of choice to assign and identify cell lineage through simultaneous analysis of surface molecules and intracellular markers. Embryonic stem cell research can be benefited from using antibodies specific to transcriptional factors/markers that contribute to the "stemness" phenotype or critical for cell lineage. Results In this report, we have developed and validated antibodies (either monoclonal or polyclonal) specif...

  16. Cross-presentation of cell-associated antigens by MHC class I in dendritic cell subsets

    Directory of Open Access Journals (Sweden)

    Enric eGutiérrez-Martínez

    2015-07-01

    Full Text Available Dendritic cells have the unique ability to pick up dead cells carrying antigens in tissue and migrate to the lymph nodes where they can cross-present cell-associated antigens by MHC class I to CD8+ T cells. There is strong in vivo evidence that the mouse XCR1+ dendritic cells subset acts as a key player in this process. The intracellular processes underlying cross-presentation remain controversial and several pathways have been proposed. Indeed, a wide number of studies have addressed the cellular process of cross-presentation in vitro using a variety of sources of antigen and antigen presenting cells. Here we review the in vivo and in vitro evidence supporting the current mechanistic models and disscuss their physiological relevance to the cross-presentation of cell-associated antigens by dendritic cells subsets

  17. Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

    DEFF Research Database (Denmark)

    Sørensen, Anja Elaine; Johnsen, Claus R; Dalgaard, Louise Torp;

    2013-01-01

    of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4......). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated...

  18. Efficacy improvement of chimeric antigen receptor T-cell immunotherapy: reports from the 57th American Society of Hematology annual meeting%提高嵌合抗原受体T细胞疗效的研究进展:第57届美国血液学会年会报道

    Institute of Scientific and Technical Information of China (English)

    李扬秋; 曾成武

    2016-01-01

    How to improve the efficacy of chimeric antigen receptor T-cell (CAR-T) is one of the key points for manufacture and application of CAR-T. In this review, the studies from the 57th American Society of Hematology (ASH) annual meeting regarding to the strategies for optimal CAR-T activity were summarized, including pathway inhibitors, enhancement antigen expression of target cells, optimization of conditioning chemotherapy, as well as the novel technology for CAR-T generation.%如何增强嵌合抗原受体T细胞(CAR-T)效应是CAR-T研制和应用的一个关键问题.文章主要总结第57届美国血液学会(ASH)年会中针对这一问题的研究成果,包括一些信号通路的抑制、联合表达刺激分子、提高靶细胞的抗原性、优化CAR-T治疗前处理方案以及新的CAR-T制作策略等.

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

    Directory of Open Access Journals (Sweden)

    Juliette Faraco

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

  20. Human leukocyte antigen-DO regulates surface presentation of human leukocyte antigen class II-restricted antigens on B cell malignancies

    NARCIS (Netherlands)

    Kremer, A.N.; Meijden, E.D. van der; Honders, M.W.; Pont, M.J.; Goeman, J.J.; Falkenburg, J.H.F.; Griffioen, M.

    2014-01-01

    Hematological malignancies often express surface HLA class II, making them attractive targets for CD4+ T cell therapy. We previously demonstrated that HLA class II ligands can be divided into DM-resistant and DM-sensitive antigens. In contrast to presentation of DM-resistant antigens, presentation o

  1. Molecular structure and biological function of proliferating cell nuclear antigen

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the core component of replication complex in eukaryote.As a processive factor of DNA polymerase delta, PCNA coordinates the replication process by interacting with various replication proteins. PCNA appears to play an essential role in many cell events, such as DNA damage repair, cell cycle regulation, and apoptosis, through the coordination or organization of different partners. PCNA is an essential factor in cell proliferation, and has clinical significance in tumor research. In this article we review the functional structure of PCNA, which acts as a function switch in different cell events.

  2. Adsorption of multimeric T cell antigens on carbon nanotubes

    DEFF Research Database (Denmark)

    Fadel, Tarek R; Li, Nan; Shah, Smith;

    2013-01-01

    Antigen-specific activation of cytotoxic T cells can be enhanced up to three-fold more than soluble controls when using functionalized bundled carbon nanotube substrates ((b) CNTs). To overcome the denaturing effects of direct adsorption on (b) CNTs, a simple but robust method is demonstrated...... to stabilize the T cell stimulus on carbon nanotube substrates through non-covalent attachment of the linker neutravidin....

  3. Antigen presenting cells in the skin of a patient with hair loss and systemic lupus erythematosus

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2009-01-01

    Full Text Available Context: Hair loss is one of the most striking clinical features of active systemic lupus erythematosus (SLE, however, very few studies have investigated the immunological features of this process. Case report: We describe a 33 years old female who presented with scalp hair loss and arthralgias. Physical examination revealed erythematous plaques on the nose and scalp, with bitemporal hair loss. Scalp biopsies revealed epidermal hyperkeratosis, with a mild interface infiltrate of lymphocytes and histiocytes and a superficial and deep, perivascular and periadnexal infiltrate of mostly CD4 positive cells. Antibodies to HAM 56, CD68, CD1a, S-100, mast cell tryptase and c-kit/CD117 were strongly positive around the hair follicles, and in the adjacent sebaceous glands. Conclusion : We present the first report showing a significant presence of several antigen presenting cells around the hair follicular units in a patient with alopecia in active SLE. Today, antigen presenting cells and dendritic cells (DC are modeled as the master regulators of human immunity. One aspect that has become clearly appreciated is the great diversity of DC subtypes, each with considerable functional differences. Thus, we suggest that APC and DCs are equipped with Pattern Recognition Receptors (PRRs to some hair follicular unit antigens; that these innate sensors recognize conserved molecular patterns on self- tissue, and play a significant role in the pathophysiology of alopecia in SLE patients

  4. Antigen transfer from exosomes to dendritic cells as an explanation for the immune enhancement seen by IgE immune complexes.

    Directory of Open Access Journals (Sweden)

    Rebecca K Martin

    Full Text Available IgE antigen complexes induce increased specific T cell proliferation and increased specific IgG production. Immediately after immunization, CD23(+ B cells capture IgE antigen complexes, transport them to the spleen where, via unknown mechanisms, dendritic cells capture the antigen and present it to T cells. CD23, the low affinity IgE receptor, binds IgE antigen complexes and internalizes them. In this study, we show that these complexes are processed onto B-cell derived exosomes (bexosomes in a CD23 dependent manner. The bexosomes carry CD23, IgE and MHC II and stimulate antigen specific T-cell proliferation in vitro. When IgE antigen complex stimulated bexosomes are incubated with dendritic cells, dendritic cells induce specific T-cell proliferation in vivo, similar to IgE antigen complexes. This suggests that bexosomes can provide the essential transfer mechanism for IgE antigen complexes from B cells to dendritic cells.

  5. Chromatin architecture, CTCF and V(D)J recombination: managing long-distance relationships at antigen receptor loci1

    OpenAIRE

    Shih, Han-Yu; Krangel, Michael S.

    2013-01-01

    The rearrangement of T and B lymphocyte antigen receptor loci occurs within a highly complex chromosomal environment and is orchestrated through complex mechanisms. Over the past decade, a large body of literature has highlighted the significance of chromatin architecture at antigen receptor loci in supporting the genomic assembly process: in preparation for recombination, these loci tend to contract and form multiple loops that shorten the distances between gene segments and facilitate recom...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma-as...... from different fragments of resected melanoma lesions. In summary, our findings provide an initial definition of T-cell populations contributing to tumor recognition in TILs although the specificity of many tumor-reactive TILs remains undefined.......Tumor-infiltrating lymphocytes (TIL) isolated from melanoma patients and expanded in vitro by interleukin (IL)-2 treatment can elicit therapeutic response after adoptive transfer, but the antigen specificities of the T cells transferred have not been determined. By compiling all known melanoma......-associated antigens and applying a novel technology for high-throughput analysis of T-cell responses, we dissected the composition of melanoma-restricted T-cell responses in 63 TIL cultures. T-cell reactivity screens against 175 melanoma-associated epitopes detected 90 responses against 18 different epitopes...

  7. Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad A;

    2013-01-01

    Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking...

  8. Single-cell analysis of glandular T cell receptors in Sjögren’s syndrome

    Science.gov (United States)

    Joachims, Michelle L.; Leehan, Kerry M.; Lawrence, Christina; Pelikan, Richard C.; Moore, Jacen S.; Pan, Zijian; Rasmussen, Astrid; Radfar, Lida; Lewis, David M.; Grundahl, Kiely M.; Kelly, Jennifer A.; Wiley, Graham B.; Shugay, Mikhail; Chudakov, Dmitriy M.; Lessard, Christopher J.; Stone, Donald U.; Scofield, R. Hal; Montgomery, Courtney G.; Sivils, Kathy L.; Thompson, Linda F.; Farris, A. Darise

    2016-01-01

    CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren’s syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA− T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies. PMID:27358913

  9. Archaeosomes varying in lipid composition differ in receptor-mediated endocytosis and differentially adjuvant immune responses to entrapped antigen

    Directory of Open Access Journals (Sweden)

    G. Dennis Sprott

    2003-01-01

    Full Text Available Archaeosomes prepared from total polar lipids extracted from six archaeal species with divergent lipid compositions had the capacity to deliver antigen for presentation via both MHC class I and class II pathways. Lipid extracts from Halobacterium halobium and from Halococcus morrhuae strains 14039 and 16008 contained archaetidylglycerol methylphosphate and sulfated glycolipids rich in mannose residues, and lacked archaetidylserine, whereas the opposite was found in Methanobrevibacter smithii, Methanosarcina mazei and Methanococcus jannaschii. Annexin V labeling revealed a surface orientation of phosphoserine head groups in M. smithii, M. mazei and M. jannaschii archaeosomes. Uptake of rhodamine-labeled M. smithii or M. jannaschii archaeosomes by murine peritoneal macrophages was inhibited by unlabeled liposomes containing phosphatidylserine, by the sulfhydryl inhibitor N-ethylmaleimide, and by ATP depletion using azide plus fluoride, but not by H. halobium archaeosomes. In contrast, N-ethylmaleimide failed to inhibit uptake of the four other rhodamine-labeled archaeosome types, and azide plus fluoride did not inhibit uptake of H. halobium or H. morrhuae archaeosomes. These results suggest endocytosis of archaeosomes rich in surface-exposed phosphoserine head groups via a phosphatidylserine receptor, and energy-independent surface adsorption of certain other archaeosome composition classes. Lipid composition affected not only the endocytic mechanism, but also served to differentially modulate the activation of dendritic cells. The induction of IL-12 secretion from dendritic cells exposed to H. morrhuae 14039 archaeosomes was striking compared with cells exposed to archaeosomes from 16008. Thus, archaeosome types uniquely modulate antigen delivery and dendritic cell activation.

  10. Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Reichert Torsten E

    2009-04-01

    Full Text Available Abstract Background The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. Methods The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B and compared with those of an adult keratinocyte cell line (NHEK. Results All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. Conclusion MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.

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

    International Nuclear Information System (INIS)

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

  12. Participation of CD45, NKR-P1A and ANK61 antigen in rat hepatic NK cell (pit cell)-mediated target cell cytotoxicity

    Institute of Scientific and Technical Information of China (English)

    Dian Zhong Luo; David Vermijlen; B lent Ahishali; Vasilis Triantis; Eddie Wisse; Karin Vanderkerken; Peter J.K. Kuppen

    2000-01-01

    AIM Several triggering receptors have been described to be involved in natural killer (NK) cellmediated target cytotoxicity. In these studies, NK cells derived from blood or spleen were used. Pit cells are liver-specific NK cells that possess a higher level of natural cytotoxicity and a different morphology when compared to blood NK cells. The aim of this study was to characterize the role of the NK-triggering molecules NKR-P1A, ANK61 antigen, and CD45 in pit cell-mediated killing of target cells. METHODS 51 Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. RESULTS Flow cytometric analysis showed that pit cells expressed CD45, NKR-P1A, and ANK61 antigen. Treatment of pit cells with monoclonal antibody ( mAb ) to CD45 ( ANK74 ) not only inhibited CC531s or YAC-1 target lysis but also apoptosis induced by pit cells. The mAbs to NKRP1A (3.2.3) and ANK61 antigen (ANK61) had no effect on pit cell-mediated CC531s or YAC-1 target cytolysis or apoptosis, while they did increase the Fcγ receptor positive (FcγR+) P815 cytolysis and apoptosis. This enhanced cytotoxicity could he inhibited by 3,4-dichloroisocoumarin, an inhibitor of granzymes. CONCLUSION These results indicate that CD45 participates in pit cell-mediated CC531s and YAC-1 target cytolysis and apoptosis. NKR-P1A and ANK61 antigen on pit cells function as activation structures against FcγR+ P815 cells, which was mediated by the perforin/granzyme pathway.

  13. 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.

  14. Specificity for the tumor-associated self-antigen WT1 drives the development of fully functional memory T cells in the absence of vaccination.

    Science.gov (United States)

    Pospori, Constandina; Xue, Shao-An; Holler, Angelika; Voisine, Cecile; Perro, Mario; King, Judith; Fallah-Arani, Farnaz; Flutter, Barry; Chakraverty, Ronjon; Stauss, Hans J; Morris, Emma C

    2011-06-23

    Recently, vaccines against the Wilms Tumor antigen 1 (WT1) have been tested in cancer patients. However, it is currently not known whether physiologic levels of WT1 expression in stem and progenitor cells of normal tissue result in the deletion or tolerance induction of WT1-specific T cells. Here, we used an human leukocyte antigen-transgenic murine model to study the fate of human leukocyte antigen class-I restricted, WT1-specific T cells in the thymus and in the periphery. Thymocytes expressing a WT1-specific T-cell receptor derived from high avidity human CD8 T cells were positively selected into the single-positive CD8 population. In the periphery, T cells specific for the WT1 antigen differentiated into CD44-high memory phenotype cells, whereas T cells specific for a non-self-viral antigen retained a CD44(low) naive phenotype. Only the WT1-specific T cells, but not the virus-specific T cells, displayed rapid antigen-specific effector function without prior vaccination. Despite long-term persistence of WT1-specific memory T cells, the animals did not develop autoimmunity, and the function of hematopoietic stem and progenitor cells was unimpaired. This is the first demonstration that specificity for a tumor-associated self-antigen may drive differentiation of functionally competent memory T cells. PMID:21447831

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

    DEFF Research Database (Denmark)

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

    1998-01-01

    We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (r....... tuberculosis, MT-CF and M. bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T-cell specificities induced by natural infection is directed towards several unrelated culture filtrate...... as well as somatic-derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M. tuberculosis and that some antigens, such as ESAT-6, are recognized by a high number of individuals...

  16. Recombinant T-cell receptors : An immunologic link to cancer therapy

    NARCIS (Netherlands)

    Calogero, A; de Leij, YFMH; Mulder, NH; Hospers, GAP

    2000-01-01

    Cytotoxic T cells can specifically kill target cells that express antigens recognized by the T-cell receptor. These are membrane-bound proteins that are not ubiquitous and thus are difficult to purify and study at the protein level. The advent of recombinant DNA technology has facilitated these obje

  17. Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.

    OpenAIRE

    Strasser, A.; Harris, A W; von Boehmer, H; Cory, S

    1994-01-01

    To explore the role of bcl-2 in T-cell development, a bcl-2 transgene was introduced into mice expressing a T-cell receptor (TCR) transgene encoding reactivity for the mouse male antigen HY presented by the H-2Db class I antigen of the major histocompatibility complex (MHC). Normal thymic development is contingent on the ability of immature thymocytes to interact with self-MHC molecules presented by thymic stroma (positive selection). Thus, thymocyte numbers are low in femal...

  18. Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

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    Stewart Nuttall

    2013-01-01

    Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

  19. Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies

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    Jennifer D. Stone

    2013-08-01

    Full Text Available Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional alpha-beta T cell receptor (TCR against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR consisting of a single-chain antibody as an Fv fragment (scFv linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the alpha-beta TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly.

  20. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration

    Science.gov (United States)

    Lopes Pinheiro, Melissa A; Kamermans, Alwin; Garcia-Vallejo, Juan J; van het Hof, Bert; Wierts, Laura; O'Toole, Tom; Boeve, Daniël; Verstege, Marleen; van der Pol, Susanne MA; van Kooyk, Yvette; de Vries, Helga E; Unger, Wendy WJ

    2016-01-01

    Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment. DOI: http://dx.doi.org/10.7554/eLife.13149.001 PMID:27336724

  1. Interferon-gamma-like molecule induces Ia antigens on cultured mast cell progenitors.

    OpenAIRE

    Wong, G H; Clark-lewis, I.; McKimm-Breschkin, J L; Schrader, J W

    1982-01-01

    Persisting (P) cells (murine cells that resemble mast cells and grow continuously in vitro for prolonged periods in the presence of a specific growth factor) did not express detectable levels of Ia antigens (murine class II major histocompatibility antigens) when their growth was supported by partially purified P cell-stimulating factor. However, when these Ia-negative P cells were transferred to medium conditioned by concanavalin A-stimulated spleen cells, Ia antigens appeared within 24 hr. ...

  2. Distribution features of lymphocytes with peanut agglutinin positive receptors in gums epithelium of rats in norm and after intrauterine antigenic action

    Directory of Open Access Journals (Sweden)

    Burega Yu.

    2015-09-01

    Full Text Available Background. According to the conception “Lymphocyte is the main factor of morphogenesis” changes in lymphocyte receptor repertory, induced by antigenic action in the fetal period of development, influence on organs and tissues development after birth. Functional activity of immunological immature PNA+ lymphocytes inducing the change in functioning, imbalance in formation cells of microenvironment, synthesis of intracellular substance and the fibers of extracellular matrix leads to violation of morphological and functional condition of organs. Objective. Determine the features of distribution of lymphocytes with receptors to peanut agglutinin in gingival epithelium of rats in norm and after intrauterine antigenic action. Methods. The object of the research was: 224 jaws of 112 white laboratory rats. The rats divided into three groups. First group – intact rats. Second group –rats, which were introduced 0,05 ml solution of antigen in the amniotic fluid on the 18th day of pregnancy by the method of N. Voloshyn, the third group – control, the animals were introduced intrauterine 0,05 ml of physiological solution on the 18th day of pregnancy. The antigen was split vaccine Vaxigrip 2009. Results and conclusion. In newborn animals, after intrauterine antigen action it was determined significantly increased content of PNA+ lymphocytes in the epithelium of gingival mucous, compared with control group, where PNA+ lymphocytes number gradually decreases. On the 11 th day of life, in animals of second group, quantity of intraepithelial PNA+ lymphocytes remains higher. On 45th day of postnatal formation its share does not significantly differ from similar indicators in all groups and decreases compared with neonatal period. Citation: Burega Yu. Distribution features of lymphocytes with peanut agglutinin positive receptors in gums epithelium of rats in norm and after intrauterine antigenic action. Morphologia. 2015;9(3:8-11.

  3. Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae of Brugia pahangi

    Science.gov (United States)

    O'Connor, Richard A.; Devaney, Eileen

    2002-01-01

    Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematode Brugia pahangi results in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4hi). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4hi cells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4hi cells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-γR−/−) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4+ T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4+ T cells from IFN-γR−/− mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO. PMID:12379675

  4. Cell wall anchoring of the Campylobacter antigens to Lactococcus lactis

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    Patrycja Anna Kobierecka

    2016-02-01

    Full Text Available Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered

  5. Original encounter with antigen determines antigen-presenting cell imprinting of the quality of the immune response in mice.

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    Valérie Abadie

    Full Text Available BACKGROUND: Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed in vivo mechanisms triggered following intradermal (i.d. and intramuscular (i.m. Modified Vaccinia virus Ankara (MVA administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MPhis, myeloid dendritic cells (DCs, and neutrophils (PMNs. MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MPhis, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA. CONCLUSIONS/SIGNIFICANCE: This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  7. Lipopolysaccharide O-antigen prevents phagocytosis of Vibrio anguillarum by rainbow trout (Oncorhynchus mykiss skin epithelial cells.

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    Kristoffer Lindell

    Full Text Available Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.

  8. Circadian control of antigen-specific T cell responses

    Directory of Open Access Journals (Sweden)

    Nobis CC

    2016-09-01

    Full Text Available Chloé C Nobis,1–3 Nathalie Labrecque,2–4 Nicolas Cermakian1,5–8 1Douglas Mental Health University Institute, 2Maisonneuve-Rosemont Hospital Research Centre, 3Department of Microbiology, Infectious Diseases and Immunology, 4Department of Medicine, University of Montreal, 5Department of Psychiatry, 6Department of Microbiology and Immunology, 7Department of Neurology and Neurosurgery, 8Department of Physiology, McGill University, Montreal, QC, Canada Abstract: The immune system is composed of two arms, the innate and the adaptive immunity. While the innate response constitutes the first line of defense and is not specific for a particular pathogen, the adaptive response is highly specific and allows for long-term memory of the pathogen encounter. T lymphocytes (or T cells are central players in the adaptive immune response. Various aspects of T cell functions vary according to the time of day. Circadian clocks located in most tissues and cell types generate 24-hour rhythms of various physiological processes. These clocks are based on a set of clock genes, and this timing mechanism controls rhythmically the expression of numerous other genes. Clock genes are expressed in cells of the immune system, including T cells. In this review, we provide an overview of the circadian control of the adaptive immune response, with emphasis on T cells, including their development, trafficking, response to antigen, and effector functions. Keywords: circadian clock, adaptive immune response, T lymphocyte, antigen, cytokine, proliferation

  9. Vaccination with Antigen Combined with αβ-ATP as a Vaccine Adjuvant Enhances Antigen-Specific Antibody Production via Dendritic Cell Activation.

    Science.gov (United States)

    Matsuo, Kazuhiko; Nishiuma, Satoshi; Hasegawa, Yuta; Kawabata, Fumika; Kitahata, Kosuke; Nakayama, Takashi

    2016-01-01

    Adjuvants are required to enhance antigen-specific immune responses by vaccines. Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X and P2Y receptors and triggers the activation of dendritic cells (DCs). Here we investigated the in vivo adjuvant efficacy of α,β-methylene-ATP (αβ-ATP), a non-hydrolysable form of ATP. We found that intradermal injection of ovalbumin (OVA), as a model antigen, combined with αβ-ATP, as the adjuvant, enhanced OVA-specific immune responses more than OVA alone. Additionally, DCs in the skin of mice injected with OVA and αβ-ATP had increased expression of major histocompatibility complex class II and co-stimulator molecules, CD40, CD80, and CD86, suggesting that αβ-ATP activated DC. These findings indicate that αβ-ATP functions as a potent vaccine adjuvant. PMID:27251512

  10. Immune complexes that contain HIV antigens activate peripheral blood T cells.

    Science.gov (United States)

    Korolevskaya, L B; Shmagel, K V; Saidakova, E V; Shmagel, N G; Chereshnev, V A

    2016-07-01

    Uninfected donor T cells were treated in vitro by model immune complexes that contained either HIV or hepatitis C virus (HCV) antigens. Unlike HCV antigen-containing complexes, the immune complexes that contained HIV antigens have been shown to activate peripheral blood T cells of uninfected donors under in vitro conditions. Both the antiviral antibodies and HIV antigen were involved in the activation process. The unique properties of the immune complexes formed by HIV antigens and antiviral antibodies are believed to result from the virus-specific antibody properties and molecular conformation of the antigen-antibody complex. PMID:27595830

  11. Phase variable O antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1.

    Directory of Open Access Journals (Sweden)

    Kimberley D Seed

    2012-09-01

    Full Text Available The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.

  12. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  13. T-cell recognition of a cross-reactive antigen(s) in erythrocyte stages of Plasmodium falciparum and Plasmodium yoelii: inhibition of parasitemia by this antigen(s).

    OpenAIRE

    Lucas, B.; Engels, A; Camus, D; Haque, A.

    1993-01-01

    In the current study, we investigated the presence of a cross-reactive antigen(s) in the erythrocyte stage from Plasmodium yoelii (265 BY strain) and Plasmodium falciparum through recognition by T cells primed in vivo with antigens from each of these parasites. BALB/c mice are naturally resistant to P. falciparum but are susceptible to P. yoelii infection. Mice that had recovered from P. yoelii primary infection became resistant to a second infection. A higher in vitro proliferative response ...

  14. Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers.

    Science.gov (United States)

    Frenz, Theresa; Grabski, Elena; Durán, Verónica; Hozsa, Constantin; Stępczyńska, Anna; Furch, Marcus; Gieseler, Robert K; Kalinke, Ulrich

    2015-09-01

    Targeted drug delivery systems hold promise for selective provision of active compounds to distinct tissues or cell subsets. Thus, locally enhanced drug concentrations are obtained that would confer improved efficacy. As a consequence adverse effects should be diminished, as innocent bystander cells are less affected. Currently, several controlled drug delivery systems based on diverse materials are being developed. Some systems exhibit material-associated toxic effects and/or show low drug loading capacity. In contrast, liposomal nanocarriers are particularly favorable because they are well tolerated, poorly immunogenic, can be produced in defined sizes, and offer a reasonable payload capacity. Compared with other immune cells, professional antigen-presenting cells (APCs) demonstrate enhanced liposome uptake mediated by macropinocytosis, phagocytosis and presumably also by clathrin- and caveolae-mediated endocytosis. In order to further enhance the targeting efficacy toward APCs, receptor-mediated uptake appears advisable. Since APC subsets generally do not express single linage-specific receptors, members of the C-type lectin receptor (CLR) family are compelling targets. Examples of CLR expressed by APCs include DEC-205 (CD205) expressed by myeloid dendritic cells (DC) and monocytes, the mannose receptor C type 1 (MR, CD206) expressed by DC, monocytes and macrophages, DC-SIGN (CD209) expressed by DC, and several others. These receptors bind glycans, which are typically displayed by pathogens and thus support pathogen uptake and endocytosis. Further research will elucidate whether glycan-decorated liposomes will not only enhance APCs targeting but also enable preferential delivery of their payload to discrete subcellular compartments. PMID:25701806

  15. New advances in leukaemia immunotherapy by the use of Chimeric Artificial Antigen Receptors (CARs: state of the art and perspectives for the near future

    Directory of Open Access Journals (Sweden)

    Cribioli Elisabetta

    2011-09-01

    Full Text Available Abstract Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs, which are artificial molecules containing antibody-derived fragments (to bind the specific target, joined with potent signalling T-Cell Receptor (TCR-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future.

  16. Tumor-derived death receptor 6 modulates dendritic cell development.

    Science.gov (United States)

    DeRosa, David C; Ryan, Paul J; Okragly, Angela; Witcher, Derrick R; Benschop, Robert J

    2008-06-01

    Studies in murine models of cancer as well as in cancer patients have demonstrated that the immune response to cancer is often compromised. This paradigm is viewed as one of the major mechanisms of tumor escape. Many therapies focus on employing the professional antigen presenting dendritic cells (DC) as a strategy to overcome immune inhibition in cancer patients. Death receptor 6 (DR6) is an orphan member of the tumor necrosis factor receptor superfamily (TNFRSF21). It is overexpressed on many tumor cells and DR6(-/-) mice display altered immunity. We investigated whether DR6 plays a role in tumorigenesis by negatively affecting the generation of anti-tumor activity. We show that DR6 is uniquely cleaved from the cell surface of tumor cell lines by the membrane-associated matrix metalloproteinase (MMP)-14, which is often overexpressed on tumor cells and is associated with malignancy. We also demonstrate that >50% of monocytes differentiating into DC die when the extracellular domain of DR6 is present. In addition, DR6 affects the cell surface phenotype of the resulting immature DC and changes their cytokine production upon stimulation with LPS/IFN-gamma. The effects of DR6 are mostly amended when these immature DC are matured with IL-1beta/TNF-alpha, as measured by cell surface phenotype and their ability to present antigen. These results implicate MMP-14 and DR6 as a mechanism tumor cells can employ to actively escape detection by the immune system by affecting the generation of antigen presenting cells.

  17. Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells?

    Science.gov (United States)

    Desai, S V; Love, R M; Rich, A M; Seymour, G J

    2011-02-01

    Bacteria are the prime cause of periapical diseases and root canal microbiology is a well-researched area of endodontics. Antigen-presenting cells (APCs) are present in periapical lesions of endodontic origin and play a substantial role in recognizing, processing and presenting pathogenic antigens to the adaptive immune system such as an effective and long-lasting immune response is generated against the specific pathogens. Toll-like receptors (TLRs) are germ-line encoded pathogen recognition receptors (PRR) expressed by various APCs which induce their maturation, lead to gene transcription in the nucleus and the production of several pro- and anti-inflammatory cytokines. Thirteen TLRs have been discovered, 10 of which have been identified in humans so far. Preliminary studies of dental pulp tissue have demonstrated various cell types expressing different TLRs in response to commonly encountered microorganisms. However, there is little information available regarding the expression and function of the various TLRs in human periapical lesions. This review discusses the interactions of various APCs in periapical lesions and the possible roles of different TLRs and APCs in pulp/periapical pathogen recognition and presentation to the adaptive immune system in the initiation and sustaining of periapical diseases.

  18. Erythrocyte Duffy antigen receptor for chemokines (DARC):diagnostic and therapeutic implications in atherosclerotic Cardiovascular disease

    Institute of Scientific and Technical Information of China (English)

    Stavros APOSTOLAKIS; Georgios K CHALIKIAS; Dimitrios N TZIAKAS; Stavros KONSTANTINIDES

    2011-01-01

    Atherosclerosis is an inflammatory disease.The last three decades efforts have been made to elucidate the biochemical pathways that are implicated in the process of atherogenesis and plaque development.Chemokines are crucial mediators in every step of this process.Additionally.cellular components of the peripheral blood have been proved important mediators in the formation and progression of atherosclerotic lesions.However,until recently data were mostly focusing on leukocytes and platelets.Erythrocytes were considered unreceptive bystanders and limited data supported their importance in the progression and destabilization of the atherosclerotic plaque.Recently erythrocytes, through their Duffy antigen receptor for chemokines(DARC),have been proposed as appealing regulators of chemokine-induced pathways.Dissimilar to every other chemokine receptor DARC possesses high affinity for severalligands from both CC and CXC chemokine sub-families.Moreover,DARC is not coupled to a G-protein or any other intracellular signalling system;thus it is incapable of generating second messages.The exact biochemical role of erythrocyte DARC remains to be determined.It is however challenging the fact that DARC is a regulator of almost every CC and CXC chemokine ligand and therefore DARC antagonism could efiectively block the complex pre-inflammatory chemokine network.In the present review we intent to provid recent evidence supporting the role of erythrocytes in atherosclerosis focusing on the erythrocyte-chemokine interaction through the Duffy antigen system.

  19. Stable isotope labeling of oligosaccharide cell surface antigens

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A. [and others

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  20. In situ Delivery of Antigen to DC-SIGN(+)CD14(+) Dermal Dendritic Cells Results in Enhanced CD8(+) T-Cell Responses.

    Science.gov (United States)

    Fehres, Cynthia M; van Beelen, Astrid J; Bruijns, Sven C M; Ambrosini, Martino; Kalay, Hakan; van Bloois, Louis; Unger, Wendy W J; Garcia-Vallejo, Juan J; Storm, Gert; de Gruijl, Tanja D; van Kooyk, Yvette

    2015-09-01

    CD14(+) dendritic cells (DCs) present in the dermis of human skin represent a large subset of dermal DCs (dDCs) that are considered macrophage-like cells with poor antigen (cross)-presenting capacity and limited migratory potential to the lymph nodes. CD14(+) dDC highly express DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), a receptor containing potent endocytic capacity, facilitating intracellular routing of antigens to major histocompatibility complex I and II (MHC-I andII) loading compartments for the presentation to antigen-specific CD8(+) and CD4(+) T cells. Here we show using a human skin explant model that the in situ targeting of antigens to DC-SIGN using glycan-modified liposomes enhances the antigen-presenting capacity of CD14(+) dDCs. Intradermal vaccination of liposomes modified with the DC-SIGN-targeting glycan Lewis(X), containing melanoma antigens (MART-1 or Gp100), accumulated in CD14(+) dDCs and resulted in enhanced Gp100- or MART-1-specific CD8(+) T-cell responses. Simultaneous intradermal injection of the cytokines GM-CSF and IL-4 as adjuvant enhanced the migration of the skin DCs and increased the expression of DC-SIGN on the CD14(+) and CD1a(+) dDCs. These data demonstrate that human CD14(+) dDCs exhibit potent cross-presenting capacity when targeted in situ through DC-SIGN. PMID:25885805

  1. TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

    Science.gov (United States)

    Takada, Kensuke; Van Laethem, Francois; Xing, Yan; Akane, Kazuyuki; Suzuki, Haruhiko; Murata, Shigeo; Tanaka, Keiji; Jameson, Stephen C; Singer, Alfred; Takahama, Yousuke

    2015-10-01

    In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

  2. Density distribution of receptors for peannut lectin in the structure of wall rats’ pharynx in the postnatal period after antenatal antigen influence

    Directory of Open Access Journals (Sweden)

    Voloshin M.A.

    2012-01-01

    Full Text Available It was determined that in animals with antenatal antigen influence density peanut lectin receptors on the membrane and in the cytoplasm of epithelial cells mucus layer of the pharynx′ nasal part on the first day of life less in comparison intact animals group. Starting from the third day of life, the intensity of deposition benzydyn mark in epithelial cells cytoplasm of the upper and lower walls of the pharynx’ nasal part, and the secret of goblet cells in experimental animals is higher than in intact rats. Content carbohydrate residues β-D-galactose in the intercellular substance of submucous connective tissue of the pharynx above in experimental animals compared with intact during all periods of observation. Antenatal antigen influence leads to an increase in PNA+-lymphocytes in the mucous layer of the pharynx.

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

    DEFF Research Database (Denmark)

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

    1984-01-01

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

  4. Effect on Retinoic Acid Receptor and Proliferating Cell Nuclear Antigen of Retinoic Acid in Colorectal Carcinoma%肠癌组织维甲酸受体的测定和维甲酸对其表达的影响

    Institute of Scientific and Technical Information of China (English)

    樊卫; 卫洪波; 韩晓燕

    2001-01-01

    Objective To investigate the effect on retinoic acid receptor (RAR) of retinoic acid (RA) in colorectal carcinoma. Methods 160 cases of health male Wistar rats were divided into 4 groups, and each group was of 40 cases.80 rats in group 1 and 2 induced by dimethylhydrazine (DMH) (20mg/kg,once a week,injected subdermally) for 7~13 weeks;after that,rats in group 2 and 3 were treated with RA (50mg/kg,every day,orally) for 8 weeks;others were was control. In 7th\14th\21th week, 8 rats were killed in each group. The others were killed in 28th week.Colorectal tumors in mucosa were examined. The RAR concentration was studied. Results The incidence of colorectal carcinoma induced by DMH between group 1and 2 was different significantly (p<0.05),and higher than group 3 and 4.The content of RAR in cancer groups was lower than normal one (p<0.05).RA may increase RAR concentration of cancer tissue progressively (p<0.05).Conclusions RA could decrease the incidence of colorectal carcinoma induced by DMH. Colorectal cancer tissue existed abnormal expression of RAR,RA could regulate RAR concentrations of intestine cells.%目的用放射配体结合分析法测定肠癌组织细胞内维甲酸受体 (retinoic acid receptor, RAR)含量,探讨维甲酸 (Retinoic acid, RA)对肠癌组织内RAR表达的影响.方法取Wistar大鼠160只,随机分4组,每组40只,其中第1、2组二甲基肼诱癌,第3、4组用生理盐水注射;于第7周开始,第2、3组按50mg/kg体重灌服维甲酸,每日1次,共8周.于7,14, 21周每组处死8只,于28周全部处死大鼠.测定RAR的含量.结果第1组肠癌发生率100%,第2组20%,差异有显著性(p<0.01);结肠组织细胞核内存在丰富的RAR,而肠癌组织RAR含量明显减少,并主要表现为数量的减少; RA对正常组织、肠癌组织内的RAR表达有显著性影响,差别有统计学意义(p<0.05).结论 RA可以减少二甲基肼诱发肠癌的发生;肠癌组织

  5. Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Danilo Santana Alessio Franceschi

    2011-01-01

    Full Text Available Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

  6. Serological analysis of cell surface antigens of null cell acute lymphocytic leukemia by mouse monoclonal antibodies.

    OpenAIRE

    Ueda, R; Tanimoto, M; Takahashi, T.; Ogata, S; Nishida, K; Namikawa, R.; Nishizuka, Y; Ota, K.

    1982-01-01

    Nine antigens systems were defined. Two were related to HLA-A,B,C and to Ia-like antigens; the others could be grouped into three categories. (i) NL-22, NL-1: NL-22 antibody reacted with leukemia cells from 12 to 16 cases of null cell acute lymphocytic leukemia (null-ALL) but not with any other type of leukemia tested or with lymphoid cells of various origins. Among cultured cell lines tested, one (NALM-6) of three null-ALL cell lines was positive, the others were negative. Absorption analysi...

  7. Mast cells modulate transport of CD23/IgE/antigen complex across human intestinal epithelial barrier

    Directory of Open Access Journals (Sweden)

    Ping-Chang Yang

    2009-06-01

    Full Text Available Background: Food allergy and chronic intestinal inflammation are common in western countries. The complex of antigen/IgE is taken up into the body from the gut lumen with the aid of epithelial cell-derived CD23 (low affinity IgE receptor II that plays an important role in the pathogenesis of intestinal allergy. This study aimed to elucidate the role of mast cell on modulation of antigen/IgE complex transport across intestinal epithelial barrier. Methods: Human intestinal epithelial cell line HT29 cell monolayer was used as a study platform. Transepithelial electric resistance (TER and permeability to ovalbumin (OVA were used as the markers of intestinal epithelial barrier function that were recorded in response to the stimulation of mast cell-derived chemical mediators. Results: Conditioned media from naïve mast cell line HMC-1 cells or monocyte cell line THP-1 cells significantly upregulated the expression of CD23 and increased the antigen transport across the epithelium. Treatment with stem cell factor (SCF, nerve growth factor (NGF, retinoic acid (RA or dimethyl sulphoxide (DMSO enhanced CD23 expression in HT29 cells. Conditioned media from SCF, NGF or RA-treated HMC-1 cells, and SCF, NGF, DMSO or RA-treated THP-1 cells enhanced immune complex transport via enhancing the expression of the CD23 in HT29 cells and the release of inflammatory mediator TNF-α. Nuclear factor kappa B inhibitor, tryptase and TNF-α inhibited the increase in CD23 in HT29 cells and prevents the enhancement of epithelial barrier permeability. Conclusions: Mast cells play an important role in modulating the intestinal CD23 expression and the transport of antigen/IgE/CD23 complex across epithelial barrier.

  8. Antigen Processing by Autoreactive B Cells Promotes Determinant Spreading

    Institute of Scientific and Technical Information of China (English)

    Yang D.Dai; George Carayanniotis; Eli Sercarz

    2005-01-01

    Acute primary immune responses tend to focus on few immunodominant determinants using a very limited number of T cell clones for expansion, whereas chronic inflammatory responses generally recruit a large number of different T cell clones to attack a broader range of determinants of the invading pathogens or the inflamed tissues.In T cell-mediated organ-specific autoimmune disease, a transition from the acute to the chronic phase contributes to pathogenesis, and the broadening process is called determinant spreading. The cellular components catalyzing the spreading reaction are not identified. It has been suggested that autoreactive B cells may play a central role in diversifying autoreactive T cell responses, possibly through affecting antigen processing and presentation. The clonal identity and diversity of the B cells and antibodies seem critical in regulating T cell activity and subsequent tissue damage or repair. Here, we use two autoimmune animal models, experimental autoimmune thyroiditis (EAT)and type 1 diabetes (T1D), to discuss how autoreactive B cells or antibodies alter the processing and presentation of autoantigens to regulate specific T cell response.

  9. Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

    NARCIS (Netherlands)

    Rodriguez, A; Regnault, A; Kleijmeer, M; Ricciardi-Castagnoli, P; Amigorena, S

    1999-01-01

    In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune r

  10. Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells

    NARCIS (Netherlands)

    J.J. García-Vallejo; M. Ambrosini; A. Overbeek; W.E. van Riel; K. Bloem; W.W.J. Unger; F. Chiodo; J.G. Bolscher; K. Nazmi; H. Kalay; Y. van Kooyk

    2013-01-01

    Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells

  11. Simple solid-phase radioimmunoassay for human leukemia-associated cell membrane antigens

    International Nuclear Information System (INIS)

    In the present study, a simple solid-phase radioimmunoassay was developed to determine detergent-extracted human leukemia-associated cell membrane antigens. In the assay, 96-well microtiter plates are coated with human leukemia cell membrane antigens containing a T cell leukemia or a non-T cell leukemia antigen in the presence of a detergent, and treated with 1.6% bovine serum albumin solution. The coated antigens were reacted with an appropriate murine monoclonal antibody (mAb). The bound mAb is determined by a second reaction with 125I-labeled F(ab')2 of goat anti-mouse Ig. The best antigen dose-dependent antibody binding results were obtained using the plates coated with antigens in the presence of taurocholate. In addition, the usefulness of the present assay with taurocholate during the purification of the antigens was demonstrated. (Auth.)

  12. Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

    Directory of Open Access Journals (Sweden)

    Athanasios Niarchos

    Full Text Available We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR in lepidopteran insect cells (i-α1-ECD and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG. Compared to the previously expressed protein in P. pastoris (y-α1-ECD, i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.

  13. Different-Sized Gold Nanoparticle Activator/Antigen Increases Dendritic Cells Accumulation in Liver-Draining Lymph Nodes and CD8+ T Cell Responses.

    Science.gov (United States)

    Zhou, Qianqian; Zhang, Yulong; Du, Juan; Li, Yuan; Zhou, Yong; Fu, Qiuxia; Zhang, Jingang; Wang, Xiaohui; Zhan, Linsheng

    2016-02-23

    The lack of efficient antigen and activator delivery systems, as well as the restricted migration of dendritic cells (DCs) to secondary lymph organs, dramatically limits DC-based adoptive immunotherapy. We selected two spherical gold nanoparticle (AuNP)-based vehicles of optimal size for activator and antigen delivery. Their combination (termed the NanoAu-Cocktail) was associated with the dual targeting of CpG oligonucleotides (CpG-ODNs) and an OVA peptide (OVAp) to DC subcellular compartments, inducing enhanced antigen cross-presentation, upregulated expression of costimulatory molecules and elevated secretion of T helper1 cytokines. We demonstrated that the intravenously transfused NanoAu-Cocktail pulsed DCs showed dramatically improved in vivo homing ability to lymphoid tissues and were settled in T cell area. Especially, by tissue-distribution analysis, we found that more than 60% of lymphoid tissues-homing DCs accumulated in liver-draining lymph nodes (LLNs). The improved homing ability of NanoAu-Cocktail pulsed DCs was associated with the high expression of chemokine receptor 7 (CCR7) and rearrangement of the cytoskeletons. In addition, by antigen-specific tetramers detection, NanoAu-Cocktail pulsed DCs were proved able to elicit strong antigen-specific CD8+ T cell responses, which provided enhanced protection from viral invasions. This study highlights the importance of codelivering antigen/adjuvant using different sized gold nanoparticles to improve DC homing and therapy. PMID:26771692

  14. MONOCLONAL-ANTIBODIES TO HUMAN EMBRYONAL CARCINOMA-CELLS - ANTIGENIC RELATIONSHIPS OF GERM-CELL TUMORS

    NARCIS (Netherlands)

    DEWIT, TFR; WILSON, L; VANDENELSEN, PJ; THIELEN, F; BREKHOFF, D; OOSTERHUIS, JW; PERA, MF; STERN, PL

    1991-01-01

    Fifteen monoclonal antibodies (mAb) that show specificity for human embryonal carcinoma cells are described. C57BL/6 mice were immunized with Tera-2 embryonal carcinoma cells, and hybridomas were isolated and tested versus a set of human developmental tumor cell lines. The antigens exhibit relativel

  15. Computational Modeling of T Cell Receptor Complexes.

    Science.gov (United States)

    Riley, Timothy P; Singh, Nishant K; Pierce, Brian G; Weng, Zhiping; Baker, Brian M

    2016-01-01

    T-cell receptor (TCR) binding to peptide/MHC determines specificity and initiates signaling in antigen-specific cellular immune responses. Structures of TCR-pMHC complexes have provided enormous insight to cellular immune functions, permitted a rational understanding of processes such as pathogen escape, and led to the development of novel approaches for the design of vaccines and other therapeutics. As production, crystallization, and structure determination of TCR-pMHC complexes can be challenging, there is considerable interest in modeling new complexes. Here we describe a rapid approach to TCR-pMHC modeling that takes advantage of structural features conserved in known complexes, such as the restricted TCR binding site and the generally conserved diagonal docking mode. The approach relies on the powerful Rosetta suite and is implemented using the PyRosetta scripting environment. We show how the approach can recapitulate changes in TCR binding angles and other structural details, and highlight areas where careful evaluation of parameters is needed and alternative choices might be made. As TCRs are highly sensitive to subtle structural perturbations, there is room for improvement. Our method nonetheless generates high-quality models that can be foundational for structure-based hypotheses regarding TCR recognition. PMID:27094300

  16. From the Deep Sea to Everywhere: Environmental Antigens for iNKT Cells.

    Science.gov (United States)

    Wingender, Gerhard

    2016-08-01

    Invariant natural killer T (iNKT) cells are a unique subset of innate T cells that share features with innate NK cells and adaptive memory T cells. The first iNKT cell antigen described was found 1993 in a marine sponge and it took over 10 years for other, bacterial antigens to be described. Given the paucity of known bacterial iNKT cell antigens, it appeared as if iNKT cells play a very specialist role in the protection against few, rare and unusual pathogenic bacteria. However, in the last few years several publications painted a very different picture, suggesting that antigens for iNKT cells are found almost ubiquitous in the environment. These environmental iNKT cell antigens can shape the distribution, phenotype and function of iNKT cells. Here, these recent findings will be reviewed and their implications for the field will be outlined. PMID:26703211

  17. Molecular tracking of antigen-specific T cell clones in neurological immune-mediated disorders

    Science.gov (United States)

    Muraro, Paolo A.; Wandinger, Klaus-Peter; Bielekova, Bibiana; Gran, Bruno; Marques, Adriana; Utz, Ursula; McFarland, Henry F.; Jacobson, Steve; Martin, Roland

    2016-01-01

    Summary T cells recognizing self or microbial antigens may trigger or reactivate immune-mediated diseases. Monitoring the frequency of specific T cell clonotypes to assess a possible link with the course of disease has been a difficult task with currently available technology. Our goal was to track individual candidate pathogenic T cell clones, selected on the basis of previous extensive studies from patients with immune-mediated disorders of the CNS, including multiple sclerosis, HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/ TSP) and chronic Lyme neuroborreliosis. We developed and applied a highly specific and sensitive technique to track single CD4+ and CD8+ T cell clones through the detection and quantification of T cell receptor (TCR) α or β chain complementarity-determining region 3 transcripts by real-time reverse transcriptase (RT)-PCR. We examined the frequency of the candidate pathogenic T cell clones in the peripheral blood and CSF during the course of neurological disease. Using this approach, we detected variations of clonal frequencies that appeared to be related to clinical course, significant enrichment in the CSF, or both. By integrating clono-type tracking with direct visualization of antigen-specific staining, we showed that a single T cell clone contributed substantially to the overall recognition of the viral peptide/MHC complex in a patient with HAM/ TSP. T cell clonotype tracking is a powerful new technology enabling further elucidation of the dynamics of expansion of autoreactive or pathogen-specific T cells that mediate pathological or protective immune responses in neurological disorders. PMID:12477694

  18. Expression of GABAergic receptors in mouse taste receptor cells.

    Directory of Open Access Journals (Sweden)

    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  19. The role of class I histocompatibility antigens in the regulation of T-cell activation.

    OpenAIRE

    Dasgupta, J D; Cemach, K; Dubey, D P; Yunis, E J; Amos, D. B.

    1987-01-01

    Class I major histocompatibility antigens in humans (HLA antigens) were found to participate in the regulation of T-cell activation and proliferation induced by phytohemagglutinin. W6/32, a monomorphic antibody directed against class I HLA-A,B,C antigens, significantly inhibited the phytohemagglutinin-induced cell proliferation of peripheral blood lymphocytes. Almost complete suppression of cell activation was achieved on a subfraction of peripheral blood lymphocytes enriched in Mo1+ monocyte...

  20. Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.

    Science.gov (United States)

    Lin, Jiqiang; Yang, Lu; Silva, Hernandez Moura; Trzeciak, Alissa; Choi, Yongwon; Schwab, Susan R; Dustin, Michael L; Lafaille, Juan J

    2016-01-01

    Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases. PMID:26923114

  1. Intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate the serum antibody responses induced by dietary antigen.

    Science.gov (United States)

    Tsuda, Masato; Hosono, Akira; Yanagibashi, Tsutomu; Kihara-Fujioka, Miran; Hachimura, Satoshi; Itoh, Kikuji; Hirayama, Kazuhiro; Takahashi, Kyoko; Kaminogawa, Shuichi

    2010-08-16

    Colonization of the gut by commensal bacteria modulates the induction of oral tolerance and allergy. However, how these intestinal bacteria modulate antigen-specific T cell responses induced by oral antigens remains unclear. In order to investigate this, we used germ-free (GF) ovalbumin (OVA)-specific T cell receptor transgenic (OVA23-3) mice. Conventional (CV) or GF mice were administered an OVA-containing diet. Cytokine production by CD4(+) cells from spleen (SP), mesenteric lymph nodes (MLN) and Peyer's patches (PP) was evaluated by ELISA, as was the peripheral antibody titer. T cell phenotype was assessed by flow cytometry. CD4(+) cells from the SP and MLN of CV and GF mice fed an OVA diet for 3 weeks produced significantly less IL-2 than the corresponding cells from mice receiving a control diet, suggesting that oral tolerance could be induced at the T cell level in the systemic and intestinal immune systems of both bacterial condition of mice. However, we also observed that the T cell hyporesponsiveness induced by dietary antigen was delayed in the systemic immune tissues and was weaker in the intestinal immune tissues of the GF mice. Intestinal MLN and PP CD4(+) T cells from these animals also produced lower levels of IL-10, had less activated/memory type CD45RB(low) cells, and expressed lower levels of CTLA-4 but not Foxp3 compared to their CV counterparts. Furthermore, GF mice produced higher serum levels of OVA-specific antibodies than CV animals. CD40L expression by SP CD4(+) cells from GF mice fed OVA was higher than that of CV mice. These results suggest that intestinal commensal bacteria promote T cell hyporesponsiveness and down-regulate serum antibody responses induced by dietary antigens through modulation of the intestinal and systemic T cell phenotype. PMID:20621647

  2. 嵌合抗原受体T细胞疗法在血液病中的应用:第57届美国血液学会年会报道%Application of chimeric antigen receptor-T cell therapy in hematological diseases: reports from the 57th American Society of Hematology annual meeting

    Institute of Scientific and Technical Information of China (English)

    邹怡新; 缪祎; 徐卫; 李建勇

    2016-01-01

    Chimeric antigen receptor-T cell (CAR-T) therapy is one of the effective novel immunotherapy for malignancies, and it has a great effect on leukemia and lymphoma. More researches are needed to improve the understanding of CAR-T therapy and promote the development of this technology in order to treat hematological malignancies from a novel perspective.%嵌合抗原受体T细胞(CAR-T)疗法是一种新型的免疫治疗方法,通过体外基因转移技术,于自体T细胞表面表达特异性抗体,并以非主要组织相容性复合体(MHC)限制性的方式结合对应抗原.目前,CAR-T技术是治疗恶性肿瘤的有效手段之一,在血液病中,其对部分白血病和淋巴瘤有效.未来还需要行进一步的研究,以加深对CAR-T技术的理解,促进其发展,为血液肿瘤的治疗提供新的思路.

  3. Thymic Selection of T-Cell Receptors as an Extreme Value Problem

    Science.gov (United States)

    Kosmrlj, Andrej; Chakraborty, Arup K.; Kardar, Mehran; Shakhnovich, Eugene I.

    2010-03-01

    T lymphocytes (T cells) orchestrate adaptive immune responses that clear pathogens from infected hosts. T cells recognize short peptides (p) derived from foreign proteins, which are bound to major histocompatibility complex (MHC) gene products (displayed on antigen- presenting cells). Recognition occurs when T cell receptor (TCR) proteins expressed on T cells bind sufficiently strongly to antigen- derived pMHC complexes on the surface of antigen-presenting cells. A diverse repertoire of self-tolerant TCR sequences is shaped during development of T cells in the thymus by processes called positive and negative selection. We map thymic selection processes to an extreme value problem and provide analytic expression for the amino acid composition of selected TCR sequences (which enable its recognition functions).

  4. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  5. Identification of a potent microbial lipid antigen for diverse Natural Killer T cells1

    Science.gov (United States)

    Wolf, Benjamin J.; Tatituri, Raju V. V.; Almeida, Catarina F.; Le Nours, Jérôme; Bhowruth, Veemal; Johnson, Darryl; Uldrich, Adam P.; Hsu, Fong-Fu; Brigl, Manfred; Besra, Gurdyal S.; Rossjohn, Jamie; Godfrey, Dale I.; Brenner, Michael B.

    2016-01-01

    Invariant Natural Killer T (iNKT) cells are a well-characterized CD1d-restricted T cell subset. The availability of potent antigens and tetramers for iNKT cells has allowed this population to be extensively studied and has revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse Natural Killer T (dNKT) cells are poorly understood because the lipid antigens they recognize are largely unknown. We sought to identify dNKT cell lipid antigen(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol (PG) as a microbial antigen that was significantly more potent than a previously characterized dNKT cell antigen, mammalian PG. Further, while mammalian PG loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived PG loaded tetramers did. The structure of Listeria PG was distinct from mammalian PG since it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d binding lipid displacement studies revealed that the microbial PG antigen binds significantly better to CD1d than counterparts with the same headgroup. These data reveal a highly-potent microbial lipid antigen for a subset of dNKT cells and provide an explanation for its increased antigen potency compared to the mammalian counterpart. PMID:26254340

  6. M-Type Phospholipase A2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy

    Science.gov (United States)

    Beck, Laurence H.; Bonegio, Ramon G.B.; Lambeau, Gérard; Beck, David M.; Powell, David W.; Cummins, Timothy D.; Klein, Jon B.; Salant, David J.

    2009-01-01

    BACKGROUND Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in non-reduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A2 receptor (PLA2R). Reactive serum specimens recognized recombinant PLA2R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA2R antibody. Anti-PLA2R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA2R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA2R. CONCLUSIONS A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA2R. PLA2R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA2R is a major antigen in this disease. PMID:19571279

  7. High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival.

    Science.gov (United States)

    Utzschneider, Daniel T; Alfei, Francesca; Roelli, Patrick; Barras, David; Chennupati, Vijaykumar; Darbre, Stephanie; Delorenzi, Mauro; Pinschewer, Daniel D; Zehn, Dietmar

    2016-08-22

    Chronic infections induce T cells showing impaired cytokine secretion and up-regulated expression of inhibitory receptors such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remain vaguely understood. Using newly generated recombinant antigen variant-expressing chronic lymphocytic choriomeningitis virus (LCMV) strains, we uncovered that T cell differentiation and acquisition of a chronic or exhausted phenotype depend critically on the frequency of T cell receptor (TCR) engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low-level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast with the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic and functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit, and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment.

  8. Nectin4 Is an Epithelial Cell Receptor for Canine Distemper Virus and Involved in Neurovirulence

    OpenAIRE

    Pratakpiriya, Watanyoo; Seki, Fumio; Otsuki, Noriyuki; Sakai, Kouji; FUKUHARA, HIDEO; Katamoto, Hiromu; HIRAI, Takuya; Maenaka, Katsumi; Techangamsuwan, Somporn; LAN, Nguyen Thi; Takeda, Makoto; Yamaguchi, Ryoji

    2012-01-01

    Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 i...

  9. Signal transduction by HLA class II antigens expressed on activated T cells

    DEFF Research Database (Denmark)

    Ødum, Niels; Martin, P J; Schieven, G L;

    1991-01-01

    Human T cells express HLA class II antigens upon activation. Although activated, class II+ T cells can present alloantigens under certain circumstances, the functional role of class II antigens on activated T cells remains largely unknown. Here, we report that cross-linking of HLA-DR molecules ex...

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

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1984-01-01

    We have studied the in vitro effect of the lysosomotrophic agent, chloroquine, on the presentation of soluble protein antigens by guinea pig accessory cells. Chloroquine inhibited the capacity of antigen-pulsed accessory cells to stimulate proliferation in appropriately primed T cells. The effect...

  11. NKT cell stimulation with glycolipid antigen in vivo: co-stimulation-dependent expansion, Bim-dependent contraction, and hypo-responsiveness to further antigenic challenge1

    Science.gov (United States)

    Kyparissoudis, Konstantinos; Pellicci, Daniel G.; Zhan, Yifan; Lew, Andrew M.; Bouillet, Philippe; Strasser, Andreas; Smyth, Mark J.; Godfrey, Dale I.

    2005-01-01

    Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer4) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how NKT cells respond to stimulation in vivo, which co-stimulatory pathways are important, and what factors (eg. antigen availability and activation-induced cell death) limit their response. We have explored these questions in the context of anin vivo model of NKT cell dynamics spanning activation, population expansion and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40-L co-stimulatory pathways were necessary for cytokine production by activated NKT cells, either early (2 hours) or late (3 days) following initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The pro-apoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting the pool size is regulated by apoptotic cell death in a manner similar to that of conventional T cells. Antigen availability was not the limiting factor for NKT cell expansion in vivo, and a second injection of α-GalCer induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to the NKT cells and not associated with up-regulation of inhibitory NK cell receptors such as NKG2A or Ly49 family members. Furthermore, NKT cells from mice pre-challenged with α-GalCer in vivoshowed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to α-GalCer in vivo, revealing that the full primary response depends on costimulation via the CD28 and CD40 pathways, with subsequent Bim-dependent contraction. After contraction, the NKT cells are hypo-responsive to further antigenic induced expansion. PMID:16116198

  12. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-09-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity.

  13. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    Science.gov (United States)

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  14. Prostate-specific antigen and hormone receptor expression in male and female breast carcinoma

    Directory of Open Access Journals (Sweden)

    Cohen Cynthia

    2010-09-01

    Full Text Available Abstract Background Prostate carcinoma is among the most common solid tumors to secondarily involve the male breast. Prostate specific antigen (PSA and prostate-specific acid phosphatase (PSAP are expressed in benign and malignant prostatic tissue, and immunohistochemical staining for these markers is often used to confirm the prostatic origin of metastatic carcinoma. PSA expression has been reported in male and female breast carcinoma and in gynecomastia, raising concerns about the utility of PSA for differentiating prostate carcinoma metastasis to the male breast from primary breast carcinoma. This study examined the frequency of PSA, PSAP, and hormone receptor expression in male breast carcinoma (MBC, female breast carcinoma (FBC, and gynecomastia. Methods Immunohistochemical staining for PSA, PSAP, AR, ER, and PR was performed on tissue microarrays representing six cases of gynecomastia, thirty MBC, and fifty-six FBC. Results PSA was positive in two of fifty-six FBC (3.7%, focally positive in one of thirty MBC (3.3%, and negative in the five examined cases of gynecomastia. PSAP expression was absent in MBC, FBC, and gynecomastia. Hormone receptor expression was similar in males and females (AR 74.1% in MBC vs. 67.9% in FBC, p = 0.62; ER 85.2% vs. 68.5%, p = 0.18; and PR 51.9% vs. 48.2%, p = 0.82. Conclusions PSA and PSAP are useful markers to distinguish primary breast carcinoma from prostate carcinoma metastatic to the male breast. Although PSA expression appeared to correlate with hormone receptor expression, the incidence of PSA expression in our population was too low to draw significant conclusions about an association between PSA expression and hormone receptor status in breast lesions.

  15. Antigen sampling by intestinal M cells is the principal pathway initiating mucosal IgA production to commensal enteric bacteria

    Science.gov (United States)

    Rios, D; Wood, M B; Li, J; Chassaing, B; Gewirtz, A T; Williams, I R

    2016-01-01

    Secretory IgA (SIgA) directed against gut resident bacteria enables the mammalian mucosal immune system to establish homeostasis with the commensal gut microbiota after weaning. Germinal centers (GCs) in Peyer's patches (PPs) are the principal inductive sites where naive B cells specific for bacterial antigens encounter their cognate antigens and receive T-cell help driving their differentiation into IgA-producing plasma cells. We investigated the role of antigen sampling by intestinal M cells in initiating the SIgA response to gut bacteria by developing mice in which receptor activator of nuclear factor-κB ligand (RANKL)-dependent M-cell differentiation was abrogated by conditional deletion of Tnfrsf11a in the intestinal epithelium. Mice without intestinal M cells had profound delays in PP GC maturation and emergence of lamina propria IgA plasma cells, resulting in diminished levels of fecal SIgA that persisted into adulthood. We conclude that M-cell-mediated sampling of commensal bacteria is a required initial step for the efficient induction of intestinal SIgA. PMID:26601902

  16. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    Science.gov (United States)

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. PMID:24248368

  17. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto......'s thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture...... of IgG and C3 fragments. Furthermore, the binding of Tg to B cells in preparations of normal blood cells was higher in HT serum than in serum from controls and correlated positively with the serum anti-Tg activity, as did the B and CD4+ T cell proliferation. Disruption of the three-dimensional structure...

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  20. Cutting Edge: Marginal Zone Macrophages Regulate Antigen Transport by B Cells to the Follicle in the Spleen via CD21.

    Science.gov (United States)

    Prokopec, Kajsa E; Georgoudaki, Anna-Maria; Sohn, Silke; Wermeling, Fredrik; Grönlund, Hans; Lindh, Emma; Carroll, Michael C; Karlsson, Mikael C I

    2016-09-15

    Marginal zone macrophages (MZM) are strategically located in the spleen, lining the marginal sinus where they sense inflammation and capture Ag from the circulation. One of the receptors expressed by MZM is scavenger receptor macrophage receptor with collagenous structure (MARCO), which has affinity for modified self-antigens. In this article, we show that engagement of MARCO on murine macrophages induces extracellular ATP and loss of CD21 and CD62L on marginal zone B cells. Engagement of MARCO also leads to reduction of Ag transport by marginal zone B cells and affects the subsequent immune response. This study highlights a novel function for MZM in regulating Ag transport and activation, and we suggest that MARCO-dependent ATP release regulates this through shedding of CD21 and CD62L. Because systemic lupus erythematosus patients were shown to acquire autoantibodies against MARCO, this highlights a mechanism that could affect a patient's ability to combat infections.

  1. Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells

    OpenAIRE

    Verhagen, Johan; Wraith, David C.

    2014-01-01

    Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell dif...

  2. Activation of Type II Cells into Regenerative Stem Cell Antigen-1+ Cells during Alveolar Repair

    Science.gov (United States)

    Kumar, Varsha Suresh; Zhang, Wei; Rehman, Jalees; Malik, Asrar B.

    2015-01-01

    The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa–induced lung injury, we identified a population of stem cell antigen (Sca)-1–expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1–expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C+Sca-1+ cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C+Sca-1+ cells in vitro. Thus, P. aeruginosa–mediated lung injury induces the generation of a Sca-1+ subset of type II cells. The progenitor phenotype of the Sp-C+Sca-1+ cells that mediates alveolar epithelial repair might involve Wnt signaling. PMID:25474582

  3. The 15 SCR flexible extracellular domains of human complement receptor type 2 can mediate multiple ligand and antigen interactions.

    Science.gov (United States)

    Gilbert, Hannah E; Asokan, Rengasamy; Holers, V Michael; Perkins, Stephen J

    2006-10-01

    Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (+/- 13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (+/- 0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-alpha. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d-antigen complexes with the B cell receptor.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...... in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells...

  5. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1994-01-01

    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...... the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear cells......+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens. Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity. To evaluate...

  6. IgE/FcεRI-Mediated Antigen Cross-Presentation by Dendritic Cells Enhances Anti-Tumor Immune Responses

    Directory of Open Access Journals (Sweden)

    Barbara Platzer

    2015-03-01

    Full Text Available Epidemiologic studies discovered an inverse association between immunoglobulin E (IgE-mediated allergies and cancer, implying tumor-protective properties of IgE. However, the underlying immunologic mechanisms remain poorly understood. Antigen cross-presentation by dendritic cells (DCs is of key importance for anti-tumor immunity because it induces the generation of cytotoxic CD8+ T lymphocytes (CTLs with specificity for tumor antigens. We demonstrate that DCs use IgE and FcεRI, the high-affinity IgE receptor, for cross-presentation and priming of CTLs in response to free soluble antigen at low doses. Importantly, IgE/FcεRI-mediated cross-presentation is a distinct receptor-mediated pathway because it does not require MyD88 signals or IL-12 induction in DCs. Using passive immunization with tumor antigen-specific IgE and DC-based vaccination experiments, we demonstrate that IgE-mediated cross-presentation significantly improves anti-tumor immunity and induces memory responses in vivo. Our findings suggest a cellular mechanism for the tumor-protective features of IgE and expand the known physiological functions of this immunoglobulin.

  7. Circulating human basophils lack the features of professional antigen presenting cells

    OpenAIRE

    Sharma, Meenu; Hegde, Pushpa; Aimanianda, Vishukumar; Beau, Remi; Sénéchal, Helene; Poncet, Pascal; Latgé, Jean-Paul; Kaveri, Srini V; Bayry, Jagadeesh

    2013-01-01

    Recent reports in mice demonstrate that basophils function as antigen presenting cells (APC). They express MHC class II and co-stimulatory molecules CD80 and CD86, capture and present soluble antigens or IgE-antigen complexes and polarize Th2 responses. Therefore, we explored whether human circulating basophils possess the features of professional APC. We found that unlike dendritic cells (DC) and monocytes, steady-state circulating human basophils did not express HLA-DR and co-stimulatory mo...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-10-01

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

  9. Structure of Foot-and-mouth Disease virus serotype A1061 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation

    Science.gov (United States)

    Foot-and-mouth Disease viruses (FMDVs) target epithelial cells via integrin receptors, but can acquire the capacity to bind cell-surface heparan sulphate (or alternative receptors) on passage in cell culture. Vaccine viruses must be propagated in cell culture and, hence, some rationale for the selec...

  10. Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anti-carcinoembryonic antigen bispecific adapter.

    Science.gov (United States)

    Li, Hua-Jung; Everts, Maaike; Pereboeva, Larisa; Komarova, Svetlana; Idan, Anat; Curiel, David T; Herschman, Harvey R

    2007-06-01

    Adenovirus vectors have a number of advantages for gene therapy. However, because of their lack of tumor tropism and their preference for liver infection following systemic administration, they cannot be used for systemic attack on metastatic disease. Many epithelial tumors (e.g., colon, lung, and breast) express carcinoembryonic antigen (CEA). To block the natural hepatic tropism of adenovirus and to "retarget" the virus to CEA-expressing tumors, we used a bispecific adapter protein (sCAR-MFE), which fuses the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23). sCAR-MFE untargets adenovirus-directed luciferase transgene expression in the liver by >90% following systemic vector administration. Moreover, sCAR-MFE can "retarget" adenovirus to CEA-positive epithelial tumor cells in cell culture, in s.c. tumor grafts, and in hepatic tumor grafts. The sCAR-MFE bispecific adapter should, therefore, be a powerful agent to retarget adenovirus vectors to epithelial tumor metastases.

  11. Identification of a highly antigenic linear B cell epitope within Plasmodium vivax apical membrane antigen 1 (AMA-1.

    Directory of Open Access Journals (Sweden)

    Lilian Lacerda Bueno

    Full Text Available Apical membrane antigen 1 (AMA-1 is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1 have shown a higher prevalence of specific antibodies to domain II (DII of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs. The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK, with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both, respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

  12. Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

    Science.gov (United States)

    Ruiz-Baca, Estela; Hernández-Mendoza, Gustavo; Cuéllar-Cruz, Mayra; Toriello, Conchita; López-Romero, Everardo; Gutiérrez-Sánchez, Gerardo

    2014-07-01

    The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

  13. Non-major histocompatibility complex-restricted cytotoxic activity of blood mononuclear cells stimulated with secreted mycobacterial proteins and other mycobacterial antigens

    DEFF Research Database (Denmark)

    Ravn, P; Pedersen, B K

    1994-01-01

    Several observations indicate that non-major histocompatibility complex (MHC)-restricted cytotoxicity, mediated for example by natural killer cells and lymphokine-activated killer cells, may serve as an important antimicrobial defense mechanism. The purpose of the present study was to investigate...... the influences of different mycobacterial antigens on non-MHC-restricted cytotoxicity and further to investigate the ways by which various lymphocyte subpopulations contribute to the development of this cytotoxicity. Non-MHC-restricted cytotoxicity was induced following stimulation of mononuclear...... interferon. The CD4+ cells proliferated and expressed interleukin-2 receptors following stimulation with mycobacterial antigens.Depletion studies after antigen stimulation showed that the cytotoxic effector cells were CD16+ CD56+ and CD4-; the CD4+ cells alone did not mediate non-MHC-restricted cytotoxicity...

  14. The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

    Science.gov (United States)

    Howe, Savannah E; Lickteig, Duane J; Plunkett, Kyle N; Ryerse, Jan S; Konjufca, Vjollca

    2014-01-01

    Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies.

  15. Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients.

    Science.gov (United States)

    Knights, Ashley J; Nuber, Natko; Thomson, Christopher W; de la Rosa, Olga; Jäger, Elke; Tiercy, Jean-Marie; van den Broek, Maries; Pascolo, Steve; Knuth, Alexander; Zippelius, Alfred

    2009-03-01

    The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions. PMID:18663444

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

    Science.gov (United States)

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

    2016-06-01

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

  17. Group-specific human granulocyte antigens on a chronic myelogenous leukemia cell line with a Philadelphia chromosome marker.

    Science.gov (United States)

    Drew, S I; Terasaki, P I; Billing, R J; Bergh, O J; Minowada, J; Klein, E

    1977-05-01

    Group-specific human granulocyte antigens are serologically detectable with granulocytotoxic-positive human alloantisera on a cell line, K562, of chronic myelogenous leukemia origin which bears a Philadelphia chromosomal marker. The same cell line lacks serologically detectable HLA, B2 microglobulin, and B-lymphocyte antigens. Granulocyte antigens are important cell markers for cell lines of suspected myeloid lineage.

  18. Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

    Energy Technology Data Exchange (ETDEWEB)

    Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M. (Notre)

    2012-05-08

    Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

  19. Gamma delta T cells recognize a microbial encoded B Cell antigen to initiate a rapid antigen-specific Interleukin-17 response

    Science.gov (United States)

    Gamma delta T cells contribute uniquely to host immune defense, but the way in which they do so remains an enigma. Here we show that an algae protein, phycoerythrin (PE) is recognized by gamma delta T cells from mice, bovine and humans and binds directly to specific gamma delta T cell antigen recept...

  20. Immune tolerance maintained by cooperative interactions between T cells and antigen presenting cells shapes a diverse TCR repertoire

    Directory of Open Access Journals (Sweden)

    Katharine eBest

    2015-08-01

    Full Text Available The T cell population in an individual needs to avoid harmful activation by self-peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates co-operative interactions between T cells of different specificity. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programming optimisation problem, which can be implemented as a multiplicative update algorithm which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains the diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire remains heterogeneous, and that new clones can establish themselves even when the repertoire is stable. Our study combines the salient features of the danger model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping which underlies tolerance in this model suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation.

  1. Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury.

    Directory of Open Access Journals (Sweden)

    Troy D Camarata

    Full Text Available Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1, commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI, as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor-mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.

  2. Targeting dendritic cells in lymph node with an antigen peptide-based nanovaccine for cancer immunotherapy.

    Science.gov (United States)

    Qian, Yuan; Jin, Honglin; Qiao, Sha; Dai, Yanfeng; Huang, Chuan; Lu, Lisen; Luo, Qingming; Zhang, Zhihong

    2016-08-01

    The design of peptide-based subunit vaccine formulations for the direct delivery of tumor antigen peptides (Aps) to dendritic cells (DCs) localized within draining lymph nodes (DLNs) is challenging. Mature DCs (mDCs) are abundantly distributed within DLNs but have dramatically reduced endocytic uptake and antigen-processing abilities, so their role as potential vaccine targets has been largely overlooked. Here we report an ultra-small biocompatible nanovaccine (α-Ap-FNP) functionalized by avidly targeting delivery of Ap via the scavenger receptor class B1 (SR-B1) pathway to mDCs. The self-assembly, small size (∼30 nm), SR-B1-targeting and optical properties of α-Ap-FNP resulted in its efficient Ap loading, substantial LN accumulation, targeting of mDCs and enhanced Ap presentation, and fluorescence trafficking, respectively. We also demonstrate that the α-Ap-FNP can be either used alone or encapsulated with CpG oligodeoxynucleotide as a prophylactic and therapeutic vaccine. Thus, the excellent properties of α-Ap-FNP provide it potential for clinical applications as a potent nanovaccine for cancer immunotherapy.

  3. Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

    Science.gov (United States)

    Perro, M; Tsang, J; Xue, S-A; Escors, D; Cesco-Gaspere, M; Pospori, C; Gao, L; Hart, D; Collins, M; Stauss, H; Morris, E C

    2010-06-01

    T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNgamma and TNFalpha in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells. PMID:20164855

  4. Human antibodies targeting cell surface antigens overexpressed by the hormone refractory metastatic prostate cancer cells: ICAM-1 is a tumor antigen that mediates prostate cancer cell invasion

    OpenAIRE

    Conrad, Fraser; Zhu, Xiaodong; Zhang, Xin; Chalkley, Robert J.; Burlingame, Alma L; Marks, James D.; Liu, Bin

    2009-01-01

    Transition from hormone-sensitive to hormone-refractory metastatic tumor types poses a major challenge for prostate cancer treatment. Tumor antigens that are differentially expressed during this transition are likely to play important roles in imparting prostate cancer cells with the ability to grow in a hormone-deprived environment and to metastasize to distal sites such as the bone and thus, are likely targets for therapeutic intervention. To identify those molecules and particularly cell s...

  5. SPONGIOTIC DERMATITIS WITH A MIXED INFLAMMATORY INFILTRATE OF LYMPHOCYTES, ANTIGEN PRESENTING CELLS, IMMUNOGLOBULINS AND COMPLEMENT

    Directory of Open Access Journals (Sweden)

    Abreu Velez Ana Maria

    2011-04-01

    Full Text Available Background: The clinical and histological presentation of spongiotic dermatitis and its inflammatory infiltrates warrant further investigation. In this case documentation of a patient with cutaneous spongiotic reactivity, we aim to characterize antigen presenting cells, as well as the skin-specific cutaneous lymphocyte antigen population by multiple techniques. Case report: A 30 year old Caucasian female presented with a two week history of blistering and erosions around the vaginal, rectal and axillary areas. Material and Methods: We utilized hematoxylin and eosin histology, direct immunofluorescence, immunohistochemistry and confocal microscopy methods to evaluate the immune reaction patterns of the cutaneous inflammatory cells. Results: In the primary histologic areas of spongiotic dermatitis, a mixed population of B and T lymphocytes was seen. Ki-67 antigen proliferative index staining was accentuated in these areas, correlating with the presence of large numbers of epidermal and dermal antigen presenting cells. Among the antigen presenting cell population, we detected strong positivities with CD1a, Factor XIIIa, myeloid/hystoid antigen, S100, HAM-56, and CD68. Interestingly, immunoglobulins G, D and M and Complement factors C1q and C3 were also strongly expressed in antigen presenting cell areas, including positivity within the spongiotic epidermis and around dermal vessels. Conclusions: We document a heterogeneous population of B and T lymphocytes and the presence of multiple classes of antigen presenting cells, immunoglobulins and complement in and surrounding histologically spongiotic areas; these findings further correlated with increased levels of expression of Ki-67.

  6. Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

    Science.gov (United States)

    Choi, Bongseo; Moon, Hyojin; Hong, Sung Joon; Shin, Changsik; Do, Yoonkyung; Ryu, Seongho; Kang, Sebyung

    2016-08-23

    In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

  7. Epithelial membrane antigen in cells from the uterine cervix: immunocytochemical staining of cervical smears.

    OpenAIRE

    Valkova, B; Ormerod, M G; Moncrieff, D.; Coleman, D V

    1984-01-01

    Smears made from cervical scrapes have been stained immunocytochemically for epithelial membrane antigen using a polyclonal antiserum and two monoclonal antibodies. With the polyclonal antiserum malignant cells and those showing dysplasia consistently expressed the antigen. Normal cells were generally negative, with the exception of some metaplastic cells. The monoclonal antibodies, although they stained the abnormal cells less consistently, gave the same pattern of staining. All three antibo...

  8. Progress of chimeric antigen receptor T cells in the treatment of hematologic malignancies:reports from the 57th American Society of Hematology annual meeting%嵌合抗原受体T细胞的应用进展与挑战:第57届美国血液学会年会报道

    Institute of Scientific and Technical Information of China (English)

    张旭华; 宋永平

    2016-01-01

    Chimeric antigen receptor T cells (CAR-T), one of the most promising cancer immunotherapy, has attracted much attention in the 57th American Society of Hematology (ASH) annual meeting. CAR-T therapy has obtained significant effect on leukemia and lymphoma and the latest research results are also inspiring in the 57th ASH annual meeting. It is an important task that how to combine CAR-T therapy with the traditional methods of treatment and the immune checkpoint blocking antibodies and small-molecule-targeted drugs to achieve the best effect. This paper will review the progress of CAR-T in the treatment of hematologic malignancies.%嵌合抗原受体T细胞(CAR-T)是目前最有发展前景的肿瘤免疫疗法之一,在第57届美国血液学会(ASH)年会上受到了极大关注。CAR-T在白血病和淋巴瘤中已经取得显著的疗效,该次大会上的最新研究结果也令人鼓舞。 CAR-T疗法如何与传统的治疗方法、免疫靶点阻断剂以及小分子靶向药物等联合应用,以达到最佳的疗效,也是面临的一个重要任务。文章就CAR-T在血液肿瘤中的应用进展进行介绍。

  9. Diminished Memory T-Cell Expansion Due to Delayed Kinetics of Antigen Expression by Lentivectors.

    Directory of Open Access Journals (Sweden)

    Karina Furmanov

    Full Text Available Memory CD8(+ T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+ T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+ T-cell expansion.

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

    Science.gov (United States)

    Weerkamp, Anton H.; Jacobs, Ton

    1982-01-01

    Three cell wall-associated protein antigens (antigens b, c, and d) were isolated from mutanolysin-solubilized cell walls of Streptococcus salivarius HB and purified to apparent homogeneity by a combination of ion-exchange chromatography, gel filtration, and immunoadsorption chromatography. Antigens b and c were also isolated from culture supernatants. Antigen b consisted of more than 80% protein and had an apparent molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 320,000. Antigen c consisted of 57% protein, about 30% neutral sugar, and about 13% amino sugar, and its glycoprotein nature was confirmed by specific staining techniques. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis antigen c resolved into two or more bands, depending on the source or the isolation procedure, in the molecular weight range from 220,000 to 280,000. Antigen d consisted of 95% protein and was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with molecular weights of 129,000 and 121,000. Under nondenaturing conditions all three antigens had molecular weights in the range from 1 × 106 to 3 × 106 as determined by gel filtration. The amino acid compositions of antigens b, c, and d were characterized by low amounts of basic amino acids and relatively high levels of nonpolar amino acids. Among oral streptococcal species antigens b and c were virtually restricted to strains of S. salivarius and most often to serotype I strains. Antigen b was recognized as the factor that mediates coaggregation of S. salivarius with Veillonella strains. The purified protein retained its biological activity. Antigen c could be linked to functions relating to adhesion of the streptococci to host tissues on the basis of its absence in mutant strains and blocking by specific antisera. The purified molecule had no detectable biological activity. Antigen d could not be linked to an established adhesion function. Images

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

    Science.gov (United States)

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    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. PMID:26788525

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

    Directory of Open Access Journals (Sweden)

    Martin Kreutz

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

  13. Cell death sensitization of leukemia cells by opioid receptor activation

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  14. 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.

  15. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore...... to examine whether human bladder tumor cells express VDR. Tumor biopsies were obtained from 26 patients with TCC. Expression of VDR was examined by immunohistochemical experiments. All tumors expressed VDR. Biopsies from advanced disease contained more VDR positive cells than low stage disease (p ....05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  16. Towards efficient cancer immunotherapy: advances in developing artificial antigen-presenting cells

    NARCIS (Netherlands)

    Eggermont, L.J.; Paulis, L.E.M.; Tel, J.; Figdor, C.G.

    2014-01-01

    Active anti-cancer immune responses depend on efficient presentation of tumor antigens and co-stimulatory signals by antigen-presenting cells (APCs). Therapy with autologous natural APCs is costly and time-consuming and results in variable outcomes in clinical trials. Therefore, development of artif

  17. Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

    Science.gov (United States)

    Geigerseder, Christof; Doepner, Richard FG; Thalhammer, Andrea; Krieger, Annette; Mayerhofer, Artur

    2004-01-01

    The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment. PMID:15040802

  18. Stimulation of TM3 Leydig cell proliferation via GABAA receptors: A new role for testicular GABA

    Directory of Open Access Journals (Sweden)

    Krieger Annette

    2004-03-01

    Full Text Available Abstract The neurotransmitter gamma-aminobutyric acid (GABA and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD, as well as GABAA and GABAB receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABAA receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABAA receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABAA receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA we demonstrated that GABA or the GABAA agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABAA antagonist bicuculline, implying a role for GABAA receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABAA receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.

  19. Regulation of dendritic cell differentiation and function by estrogen receptor ligands

    OpenAIRE

    Kovats, Susan; Carreras, Esther

    2008-01-01

    Estrogen receptor (ER) ligands can modulate innate and adaptive immunity and hematopoiesis, which may explain the clear sex differences in immune responses during autoimmunity, infection or trauma. Dendritic cells (DC) are antigen-presenting cells important for initiation of innate and adaptive immunity, as well as immune tolerance. DC progenitors and terminally differentiated DC express ER, indicating the ER ligands may regulate DC at multiple developmental and functional stages. Although th...

  20. Formaldehyde treatment of proteins can constrain presentation to T cells by limiting antigen processing.

    OpenAIRE

    Di Tommaso, A; De Magistris, M T; Bugnoli, M.; Marsili, I; Rappuoli, R; Abrignani, S.

    1994-01-01

    Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (p...

  1. Variability in expression of cell surface antigens of Candida albicans during morphogenesis.

    OpenAIRE

    Brawner, D L; Cutler, J. E.

    1986-01-01

    The location and expression of two different cell surface antigens on germinating and nongerminating Candida albicans cells was examined by using transmission electron microscopy after labeling with monoclonal antibodies (H9 or C6) and immunocolloidal gold. Immunodeterminant expression of the two carbohydrate antigens was followed from early germination events through 20 h of development. The determinant detected by H9 antibody, which was initially lost from the mother cell surface and prefer...

  2. Regulation of murine macrophage Ia-antigen expression by products of activated spleen cells

    OpenAIRE

    1980-01-01

    This investigation examined the effects of mediators derived form activated spleen cells on macrophage Ia-antigen expression and function. Incubation of adherent thioglycollate-induced murine peritoneal macrophages(> 90% Ia-) with concanavalin A (Con A)- stimulated spleen cell supernate (Con A sup) resulted in a dose- dependent increase in the percentage of Ia-containing (Ia+) phagocytic cells, as detected by antiserum-and-complement-mediated cytotoxicity. The Ia-antigen expression of macroph...

  3. Genetic Evidence for O-Specific Antigen as Receptor of Pseudomonas aeruginosa Phage K8 and Its Genomic Analysis

    Directory of Open Access Journals (Sweden)

    Xuewei ePan

    2016-03-01

    Full Text Available Phage therapy requires the comprehensive understanding of the mechanisms underlying the host-phage interactions. In this work, to identify the genes related to Pseudomonas aeruginosa phage K8 receptor synthesis, 16 phage-resistant mutants were selected from a Tn5G transposon mutant library of strain PAK. The disrupted genetic loci were identified and they were related to O-specific antigen (OSA synthesis, including gene wbpR, ssg, wbpV, wbpO, and Y880_RS05480, which encoded a putative O-antigen polymerase Wzy. The LPS profile of the Y880_RS05480 mutant was analyzed and shown to lack the O-antigen. Therefore, the data from characterization of Y880_RS05480 by TMHMM and SDS-PAGE silver staining analysis suggest that this locus might encode Wzy. The complete phage K8 genome was characterized as 93879 bp in length and contained identical 1188-bp terminal direct repeats. Comparative genomic analysis showed that phage K8 was highly homologous to members of the genus PaP1-like phages. On the basis of our genetic findings, OSA of P. aeruginosa PAK is proven to be the receptor of phage K8. The highly conserved structural proteins among the genetic closely related phages suggest that they may recognize the same receptor.

  4. Interaction of proliferation cell nuclear antigen (PCNA with c-Abl in cell proliferation and response to DNA damages in breast cancer.

    Directory of Open Access Journals (Sweden)

    Huajun Zhao

    Full Text Available Cell proliferation in primary and metastatic tumors is a fundamental characteristic of advanced breast cancer. Further understanding of the mechanism underlying enhanced cell growth will be important in identifying novel prognostic markers and therapeutic targets. Here we demonstrated that tyrosine phosphorylation of the proliferating cell nuclear antigen (PCNA is a critical event in growth regulation of breast cancer cells. We found that phosphorylation of PCNA at tyrosine 211 (Y211 enhanced its association with the non-receptor tyrosine kinase c-Abl. We further demonstrated that c-Abl facilitates chromatin association of PCNA and is required for nuclear foci formation of PCNA in cells stressed by DNA damage as well as in unperturbed cells. Targeting Y211 phosphorylation of PCNA with a cell-permeable peptide inhibited the phosphorylation and reduced the PCNA-Abl interaction. These results show that PCNA signal transduction has an important impact on the growth regulation of breast cancer cells.

  5. Peptide-beta2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells.

    Science.gov (United States)

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-09-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.

  6. Peptide-β2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells

    Science.gov (United States)

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-01-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-β2-microglobulin–MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide–MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9·2 × 108 antigen-specific cytotoxic CD8+ T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8+ T cells, which could be used for adoptive T-cell therapy. PMID:17472719

  7. Human parvovirus B19 induced apoptotic bodies contain altered self-antigens that are phagocytosed by antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Kanoktip Thammasri

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

  8. Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

    OpenAIRE

    Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G.; Nghiem, Paul; DeCaprio, James A.

    2013-01-01

    Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes...

  9. Lung adenocarcinoma with clear cell features producing carbohydrate antigen 19-9.

    Science.gov (United States)

    Goto, Taichiro; Hada, Masao; Oyama, Toshio

    2015-10-01

    A 76-year-old man underwent surgery for lung cancer. Histopathologically, most of the resected tumor was composed of polygonal cells with foamy cytoplasm, and the cells were arranged predominantly in acinar patterns. In this case, although the carbohydrate antigen 19-9 level was high before surgery, it normalized after resection. The tumor was considered a carbohydrate antigen 19-9-producing tumor, which was further supported by the results of immunohistochemical analysis. Adenocarcinoma with clear cell features, producing carbohydrate antigen 19-9, is an exceedingly rare entity.

  10. Pollen-induced antigen presentation by mesenchymal stem cells and T cells from allergic rhinitis.

    Science.gov (United States)

    Desai, Mauli B; Gavrilova, Tatyana; Liu, Jianjun; Patel, Shyam A; Kartan, Saritha; Greco, Steven J; Capitle, Eugenio; Rameshwar, Pranela

    2013-10-01

    Mesenchymal stem cells (MSCs) are promising cellular suppressor of inflammation. This function of MSCs is partly due to their licensing by inflammatory mediators. In cases with reduced inflammation, MSCs could become immune-enhancer cells. MSCs can suppress the inflammatory response of antigen-challenged lymphocytes from allergic asthma. Although allergic rhinitis (AR) is also an inflammatory response, it is unclear if MSCs can exert similar suppression. This study investigated the immune effects (suppressor vs enhancer) of MSCs on allergen-stimulated lymphocytes from AR subjects (grass or weed allergy). In contrast to subjects with allergic asthma, MSCs caused a significant (Pcells (antigen-presenting cells (APCs)). This correlated with increased production of inflammatory cytokines from T cells, and increased expressions of major histocompatibility complex (MHC)-II and CD86 on MSCs. The specificity of APC function was demonstrated in APC assay using MSCs that were knocked down for the master regulator of MHC-II transcription, CIITA. The difference in the effects of MSCs on allergic asthma and AR could not be explained by the sensitivity to the allergen, based on skin tests. Thus, we deduced that the contrasting immune effects of MSCs for antigen-challenged lymphocytes on AR and allergic asthma could be disease specific. It is possible that the enhanced inflammation from asthma might be required to license the MSCs to become suppressor cells. This study underscores the need for robust preclinical studies to effectively translate MSCs for any inflammatory disorder. PMID:25505949

  11. The Mertk receptor tyrosine kinase promotes T-B interaction stimulated by IgD B-cell receptor cross-linking.

    Science.gov (United States)

    Shao, Wen-Hai; Zhen, Yuxuan; Finkelman, Fred D; Cohen, Philip L

    2014-09-01

    The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation. PMID:24768065

  12. Red Blood Cell Antigen Genotyping for Sickle Cell Disease, Thalassemia, and Other Transfusion Complications.

    Science.gov (United States)

    Fasano, Ross M; Chou, Stella T

    2016-10-01

    Since the discovery of the ABO blood group in the early 20th century, more than 300 blood group antigens have been categorized among 35 blood group systems. The molecular basis for most blood group antigens has been determined and demonstrates tremendous genetic diversity, particularly in the ABO and Rh systems. Several blood group genotyping assays have been developed, and 1 platform has been approved by the Food and Drug Administration as a "test of record," such that no phenotype confirmation with antisera is required. DNA-based red blood cell (RBC) phenotyping can overcome certain limitations of hemagglutination assays and is beneficial in many transfusion settings. Genotyping can be used to determine RBC antigen phenotypes in patients recently transfused or with interfering allo- or autoantibodies, to resolve discrepant serologic typing, and/or when typing antisera are not readily available. Molecular RBC antigen typing can facilitate complex antibody evaluations and guide RBC selection for patients with sickle cell disease (SCD), thalassemia, and autoimmune hemolytic anemia. High-resolution RH genotyping can identify variant RHD and RHCE in patients with SCD, which have been associated with alloimmunization. In the future, broader access to cost-efficient, high-resolution RBC genotyping technology for both patient and donor populations may be transformative for the field of transfusion medicine. PMID:27345938

  13. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    Science.gov (United States)

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

  14. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    Science.gov (United States)

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients. PMID:27019998

  15. Serum antibodies to whole-cell and recombinant antigens of Borrelia burgdorferi in cottontail rabbits.

    Science.gov (United States)

    Magnarelli, Louis A; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985-86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37, or VlsE) during different seasons, but there was no reactivity to outer surface protein (Osp)A or OspB. Seventy-six of the 102 sera (75%) analyzed were reactive with one or more of the antigens; 61 of the positive samples (80%) reacted to whole-cell antigens, followed by results for the p35 (58%, 44/76), VlsE (43%, 33/76), and p37 (29%, 22/ 76) antigens. Fifty-eight sera (76%) contained antibodies to the VlsE or p35 antigens with or without reactivity to whole-cell antigens. High antibody titers (≥1:2,560) recorded for 52 sera indicate robust antibody production. In analyses for IgM antibodies in an ELISA containing whole-cell antigens, there were 30 positive sera; titers ranged from 1:160 to 1:640. There was minimal cross-reactivity when rabbit antisera to Treponema pallidum or four serovars of Leptospira interrogans were screened against B. burgdorferi antigens. Based on more-specific results, VlsE and p35 antigens appear to be useful markers for detecting possible B. burgdorferi infections. PMID:22247369

  16. Malassezia yeasts activate the NLRP3 inflammasome in antigen-presenting cells via Syk-kinase signalling.

    Science.gov (United States)

    Kistowska, Magdalena; Fenini, Gabriele; Jankovic, Dragana; Feldmeyer, Laurence; Kerl, Katrin; Bosshard, Philipp; Contassot, Emmanuel; French, Lars E

    2014-12-01

    Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro-inflammatory cytokine IL-1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL-1β secretion in human antigen-presenting cells. In contrast, keratinocytes were not able to secrete IL-1β when exposed to Malassezia spp. Moreover, we demonstrate that IL-1β secretion in antigen-presenting cells was dependent on Syk-kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro-inflammatory responses when taken up by antigen-presenting cells and identify C-type lectin receptors and the NLRP3 inflammasome as crucial actors in this process. PMID:25267545

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

    Directory of Open Access Journals (Sweden)

    Eric F Tewalt

    2009-05-01

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

  18. Measuring receptor recycling in polarized MDCK cells.

    Science.gov (United States)

    Gallo, Luciana; Apodaca, Gerard

    2015-01-01

    Recycling of proteins such as channels, pumps, and receptors is critical for epithelial cell function. In this chapter we present a method to measure receptor recycling in polarized Madin-Darby canine kidney cells using an iodinated ligand. We describe a technique to iodinate transferrin (Tf), we discuss how (125)I-Tf can be used to label a cohort of endocytosed Tf receptor, and then we provide methods to measure the rate of recycling of the (125)I-Tf-receptor complex. We also show how this approach, which is easily adaptable to other proteins, can be used to simultaneously measure the normally small amount of (125)I-Tf transcytosis and degradation.

  19. Immune Responses of Dendritic Cells Loaded with Antigens from Apoptotic Cholangiocarcinoma Cells Caused by γ-Irradation

    Institute of Scientific and Technical Information of China (English)

    WUGang; HANBenli; PEIXuetao

    2002-01-01

    Objective:To investigate the induction cytotoxic T cells(CTLs) with antitumor activity and therapeutic efficacy after dendritic cells(DCs) acquired antigen from apoptotic cholangiocarcinoma cells caused by γ-irradiation. Methods:DCs from peripheral blood mononuclear cells (PBMC) that maintain the antigen capturing and processing capacity charateristic of immature cells have been established in vitro, using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then, in cholangiocarcinoma cells apoptosis was induced by γ-irradiation. The experimental groups were as follows:(1)coculture of DCs and apoptotic cancer cells and T cells;(2)coculture of DCs and necrotic cancer cells and T cells;(3)coculture of DCs, cultured cancer cell and T cells. They are cocultured for 7 days.DCs and T cells were riched, isolated and their antitumor response was tested. Results:The cells had typical dendritic morphology, expressed high levels of CDla and B7, acquired antigen from apoptotic cells caused by γ-irradiation and induced an increased T cell stimulatory capacity in mixed lymphocyte reactions (MLR). Conclusion:DCs obtained from PBMCs using GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by γ-irradiation and efficiently induce T cells.This strategy, therefore, may present an effective approach to transduce DCs with antigen.

  20. Surface expression of Mo3e antigen by activated human monocytes and U-937 cells

    Energy Technology Data Exchange (ETDEWEB)

    Todd R.F. III; Bury, M.J.; Liu, D.Y.

    1986-03-05

    The surface expression of a protease-sensitive antigen, Mo3e, by activated human monocytes and U-937 cells is a plasma membrane feature of the activated state. Mo3e, which is an 80 kD protein on Western blot analysis, may represent the surface receptor for migration inhibitory factor (MIF), as evidenced by inhibition of MIF responsiveness produced by anti-Mo3e monoclonal antibody. Mo3e is barely detectable (by surface immunofluorescence) on freshly isolated monocytes but becomes expressed in high antigen density during 18-24 hrs culture in medium containing E. coli lipopolysaccharide (> 1 ng/ml), 4..beta..-phorbol 12-myristate 13-acetate (PMA) (5-10 nM), or muramyl dipeptide (0.1-1 ..mu..M). In U-937 cells, Mo3e surface expression is detectable after 24 hrs exposure to PMA and other pharmacological activators of protein kinase C: 4..beta..-phorbol 12, 13 dibutyrate, 4..beta..-phorbol 12, 13 didecanoate, mezerein, or Sn-1,2-dioctanoylglycerol. The biologically-inactivate phorbol compounds, 4..cap alpha..-phorbol 12, 13 didecanoate and 4/sub ..beta../-phorbol do not stimulate Mo3e expression. The calcium ionophore, ionomycin, has a synergistic effect on Mo3e expression stimulated by PMA; conversely, calcium antagonists block PMA-induced Mo3e expression. These results suggest the involvement of protein kinase C activation and intracellular calcium mobilization in the stimulated expression of Mo3e by activated human mononuclear phagocytes.

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

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar

    2007-09-01

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  2. Oncogenic cancer/testis antigens

    DEFF Research Database (Denmark)

    Gjerstorff, Morten F; Andersen, Mads H; Ditzel, Henrik J

    2015-01-01

    /testis antigens are immunogenic, highly cancer-specific, and frequently expressed in various types of cancer, which make them promising candidate targets for cancer immunotherapy, including cancer vaccination and adoptive T-cell transfer with chimeric T-cell receptors. Our current understanding of tumor...

  3. Merkel cell polyomavirus large T antigen has growth-promoting and inhibitory activities.

    Science.gov (United States)

    Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G; Nghiem, Paul; DeCaprio, James A

    2013-06-01

    Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region. PMID:23514892

  4. Expression and functional role of 1F7 (CD26) antigen on peripheral blood and synovial fluid T cells in rheumatoid arthritis patients.

    Science.gov (United States)

    Muscat, C; Bertotto, A; Agea, E; Bistoni, O; Ercolani, R; Tognellini, R; Spinozzi, F; Cesarotti, M; Gerli, R

    1994-11-01

    The expression and the functional role of the CD26 (1F7) T cell surface molecule, an ectoenzyme which seems to represent a functional collagen receptor of T lymphocytes and to have a role in T cell activation, were analysed in both peripheral blood (PB) and synovial fluid (SF) T cell samples from patients with active and inactive rheumatoid arthritis (RA). Although patients with active disease displayed higher percentages of PB CD26+ CD4+ T cells than inactive RA and control subjects, CD26 antigen expression on RA SF T lymphocytes was low. The anti-1F7 binding to the T cell surface, that led to CD26 antigen modulation and enhancement of both IL-2 synthesis by, and 3H-TdR incorporation of, anti-CD3- or anti-CD2-triggered PB T cells in RA and control subjects, was unable to affect significantly both expression and functional activity of RA SF T lymphocytes. Since the 1F7 antigen spontaneously reappeared on the surface of unstimulated SF T cells after 2-5 days of culturing, the low 1F7 antigen expression of anti-1F7 in the SF T cell compartment may be the result of in vivo molecule modulation exerted by the natural ligand in the joint, with important implications for T cell activation and lymphokine synthesis. PMID:7955530

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

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P

    2012-10-01

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

  6. Proliferating cell nuclear antigen and Ki-67 immunohistochemistry of oligodendrogliomas with special reference to prognosis

    DEFF Research Database (Denmark)

    HEEGAARD, S.; Sommer, Helle Mølgaard; BROHOLM, H.;

    1995-01-01

    Background. The biologic behavior of oligodendrogliomas is somewhat unpredictable. A supplementary prognostic factor is, therefore, desirable. Methods. Thirty-two pure supratentorial oligodendrogliomas were investigated using proliferating cell nuclear antigen (PCNA) and Ki-67 immunohistochemical...

  7. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4.

    Science.gov (United States)

    Newton, Kim; Sun, Xiaoqing; Dixit, Vishva M

    2004-02-01

    RIP3 is a member of the RIP kinase family. It is expressed in the embryo and in multiple adult tissues, including most hemopoietic cell lineages. Several studies have implicated RIP3 in the regulation of apoptosis and NF-kappa B signaling, but whether RIP3 promotes or attenuates activation of the NF-kappa B family of transcription factors has been controversial. We have generated RIP3-deficient mice by gene targeting and find RIP3 to be dispensable for normal mouse development. RIP3-deficient cells showed normal sensitivity to a variety of apoptotic stimuli and were indistinguishable from wild-type cells in their ability to activate NF-kappa B signaling in response to the following: human tumor necrosis factor (TNF), which selectively engages mouse TNF receptor 1; cross-linking of the B- or T-cell antigen receptors; peptidoglycan, which activates Toll-like receptor 2; and lipopolysaccharide (LPS), which stimulates Toll-like receptor 4. Consistent with these observations, RIP3-deficient mice exhibited normal antibody production after immunization with a T-dependent antigen and normal interleukin-1 beta (IL-1 beta), IL-6, and TNF production after LPS treatment. Thus, we can exclude RIP3 as an essential modulator of NF-kappa B signaling downstream of several receptor systems.

  8. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    Science.gov (United States)

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.

  9. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape.

    Science.gov (United States)

    Hegde, Meenakshi; Mukherjee, Malini; Grada, Zakaria; Pignata, Antonella; Landi, Daniel; Navai, Shoba A; Wakefield, Amanda; Fousek, Kristen; Bielamowicz, Kevin; Chow, Kevin K H; Brawley, Vita S; Byrd, Tiara T; Krebs, Simone; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Dotti, Gianpietro; Mamonkin, Maksim; Brenner, Malcolm K; Orange, Jordan S; Ahmed, Nabil

    2016-08-01

    In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape. PMID:27427982

  10. Interleukin 2 receptors on human B cells. Implications for the role of interleukin 2 in human B cell function

    OpenAIRE

    1985-01-01

    In the present study, we examined the expression of interleukin 2 (IL- 2) receptors on normal human B cells as well as established B cell lines. Anti-Tac monoclonal antibody did not bind to freshly separated normal human B cells. Unexpectedly, with the appropriate activation of the normal B cells by anti-mu antibody, phorbol myristate acetate, or Staphylococcus aureus Cowan I (SAC), Tac antigen was induced on the activated B cells. Anti-Tac antibody showed consistent reactivity with two B cel...

  11. Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells.

    Science.gov (United States)

    Pouniotis, Dodie; Tang, Choon-Kit; Apostolopoulos, Vasso; Pietersz, Geoffrey

    2016-08-01

    Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations. PMID:27138940

  12. The liver X receptor agonist T0901317 acts as androgen receptor antagonist in human prostate cancer cells

    International Nuclear Information System (INIS)

    T0901317 is a potent non-steroidal synthetic liver X receptor (LXR) agonist. T0901317 blocked androgenic stimulation of the proliferation of androgen-dependent LNCaP 104-S cells and androgenic suppression of the proliferation of androgen-independent LNCaP 104-R2 cells, inhibited the transcriptional activation of an androgen-dependent reporter gene by androgen, and suppressed gene and protein expression of prostate specific antigen (PSA), a target gene of androgen receptor (AR) without affecting gene and protein expression of AR. T0901317 also inhibited binding of a radiolabeled androgen to AR, but inhibition was much weaker compared to the effect of the antiandrogens, bicalutamide and hydroxyflutamide. The LXR agonist T0901317, therefore, acts as an antiandrogen in human prostate cancer cells

  13. Location of T cell and major histocompatibility complex antigens in the human thymus

    OpenAIRE

    1980-01-01

    A series of monoclonal antibodies were used to study the intrathymic distribution of T cell-specific antigens, Ia antigens, and beta 2- microglobulin in frozen sections of human thymus by immunofluorescence and immunoperoxidase techniques. Most of the cortical thymocytes reacted with anti-T4, anti-T5, anti-T6, anti-T8, and anti-T10 antibodies, thus indicating coexpression of multiple antigens on cortical lymphocytes. The staining of cells in the medulla was most satisfactorily judged in secti...

  14. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

    OpenAIRE

    Zuber, M; Tan, E M; Ryoji, M

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replicatio...

  15. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    OpenAIRE

    Magnarelli, Louis A.; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37...

  16. Quantitative interrelations of Lewis antigens in normal mucosa and transitional cell bladder carcinomas.

    OpenAIRE

    Limas, C

    1991-01-01

    The factors regulating the expression of the Lewis blood group related antigens in tissues have yet to be clarified. In an attempt to resolve some of the existing controversies the quantitative interrelationship of the Le(a), Le(b), X and Y antigens in normal urothelium and transitional cell carcinomas (TCC) was studied using biopsy specimens derived from 22 patients whose ABO and Lewis red blood cell phenotype was known. A quantitative scale was devised to encompass both the extent and inten...

  17. Activation of Cdc42 is necessary for sustained oscillations of Ca2+ and PIP2 stimulated by antigen in RBL mast cells

    Directory of Open Access Journals (Sweden)

    Marcus M. Wilkes

    2014-07-01

    Full Text Available Antigen stimulation of mast cells via FcεRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca2+ mobilization for exocytosis of secretory granules during the allergic response. To characterize the role of Rho GTPases in FcεRI signaling, we utilized a mutant RBL cell line, B6A4C1, that is deficient in antigen-stimulated Cdc42 activation important for these processes. Recently the importance of stimulated intracellular oscillations has emerged, and we find that B6A4C1 cells exhibit severely attenuated Ca2+ oscillations in response to antigen, which are restored to wild-type RBL-2H3 levels by expression of constitutively active Cdc42 G12V or by a GEF for Cdc42, DOCK7, but not when the C-terminal di-arginine motif of active Cdc42 is mutated to di-glutamine. We found that antigen-stimulated FcεRI endocytosis, which occurs independently of Ca2+ mobilization, is also defective in B6A4C1 cells, and Cdc42 G12V reconstitutes this response as well. Thus, activation of Cdc42 occurs prior to and is critical for antigen-stimulated pathways leading separately to both Ca2+ mobilization and receptor endocytosis. Accounting for these downstream functional consequences, we show that Cdc42 G12V reconstitutes antigen-stimulated oscillations of phosphatidylinositol 4,5-bisphosphate (PIP2 at the plasma membrane in mutant B6A4C1 cells, pointing to Cdc42 participation in the regulation of stimulated PIP2 synthesis.

  18. Thymic selection of T-cell receptors as an extreme value problem

    CERN Document Server

    Kosmrlj, Andrej; Kardar, Mehran; Shakhnovich, Eugene I

    2009-01-01

    T lymphocytes (T cells) orchestrate adaptive immune responses upon activation. T cell activation requires sufficiently strong binding of T cell receptors (TCRs) on their surface to short peptides (p) derived from foreign proteins, which are bound to major histocompatibility (MHC) gene products (displayed on antigen presenting cells). A diverse and self-tolerant T cell repertoire is selected in the thymus. We map thymic selection processes to an extreme value problem and provide an analytic expression for the amino acid compositions of selected TCRs (which enable its recognition functions).

  19. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

    Wewer, U M; Taraboletti, G; Sobel, M E;

    1987-01-01

    Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986...... in vivo exhibited a marked cytoplasmic immunoreactivity for the receptor antigen. Together these findings indicate a specific role for the laminin receptor in laminin-mediated migration and that the ligand binding of the laminin receptor is encompassed in the COOH-terminal end of the protein....

  20. Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans.

    Science.gov (United States)

    Pavia, J; Aguado, C; Mormeneo, S; Sentandreu, R

    2001-07-01

    The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls. PMID:11429475

  1. Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately.

    Science.gov (United States)

    Gorskaya, Yu F; Danilova, T A; Grabko, V I; Nesterenko, V G

    2015-12-01

    Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

  2. Neural Crest Cells Isolated from the Bone Marrow of Transgenic Mice Express JCV T-Antigen.

    Directory of Open Access Journals (Sweden)

    Jennifer Gordon

    Full Text Available JC virus (JCV, a common human polyomavirus, is the etiological agent of the demyelinating disease, progressive multifocal leukoencephalopathy (PML. In addition to its role in PML, studies have demonstrated the transforming ability of the JCV early protein, T-antigen, and its association with some human cancers. JCV infection occurs in childhood and latent virus is thought to be maintained within the bone marrow, which harbors cells of hematopoietic and non-hematopoietic lineages. Here we show that non-hematopoietic mesenchymal stem cells (MSCs isolated from the bone marrow of JCV T-antigen transgenic mice give rise to JCV T-antigen positive cells when cultured under neural conditions. JCV T-antigen positive cells exhibited neural crest characteristics and demonstrated p75, SOX-10 and nestin positivity. When cultured in conditions typical for mesenchymal cells, a population of T-antigen negative cells, which did not express neural crest markers arose from the MSCs. JCV T-antigen positive cells could be cultured long-term while maintaining their neural crest characteristics. When these cells were induced to differentiate into neural crest derivatives, JCV T-antigen was downregulated in cells differentiating into bone and maintained in glial cells expressing GFAP and S100. We conclude that JCV T-antigen can be stably expressed within a fraction of bone marrow cells differentiating along the neural crest/glial lineage when cultured in vitro. These findings identify a cell population within the bone marrow permissible for JCV early gene expression suggesting the possibility that these cells could support persistent viral infection and thus provide clues toward understanding the role of the bone marrow in JCV latency and reactivation. Further, our data provides an excellent experimental model system for studying the cell-type specificity of JCV T-antigen expression, the role of bone marrow-derived stem cells in the pathogenesis of JCV-related diseases

  3. Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

    Institute of Scientific and Technical Information of China (English)

    Peng Yang; Bo Li; Ping Lv; Yan Zhang; XiaoMing Gao

    2007-01-01

    Systemic lupus erythematosus (SLE) is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells (APCs) and an autoreactive T cell (ATL1) clone obtained from lupus-prone BXSB mice. ATL1 cells, either before or after γ-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL1 cells at a responder/stimulator (R/S) ratio of 1/2.5. Dendritic cells (DCs) were much more powerful stimulators for ATL1 cells on a per cell basis. The T cell stimulating ability of macrophages and B cells, but not DCs, was sensitive toγ-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱand CD4 were able to block DC-mediated stimulation of ATL1 proliferation, indicating cognate recognition between ATL1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.

  4. The generation and antigen-specificity of CD4+CD25+ regulatory T cells.

    Science.gov (United States)

    Taams, Leonie S; Curnow, S John; Vukmanovic-Stejic, M; Akbar, Arne N

    2006-09-01

    CD4+CD25+ regulatory T cells are essential components of the immune system. They help to maintain immune tolerance by exerting suppressive effects on cells of the adaptive and innate immune system. In the last few years there has been an abundance of papers addressing the suppressive effects of CD4+CD25+ regulatory T cells and their putative role in various experimental disease models and human diseases. Despite the enormous amounts of data on these cells a number of controversial issues still exists. CD4+CD25+ regulatory T cells were originally described as thymus-derived anergic/suppressive T cells. Recent papers however indicate that these cells might also be generated in the periphery. Due to the thymic development of CD4+CD25+ regulatory T cells it was thought that these cells were specific for self-antigens. Indeed it was shown that CD4+CD25+ regulatory T cells could be positively selected upon high affinity interaction with self-antigens. However, evidence is accumulating that these cells might also interact with non-self antigens. Finally, in the literature there is conflicting evidence regarding the role of soluble factors versus cell-contact in the mechanism of suppression. The aim of this review is to summarize the evidence supporting these opposing viewpoints and to combine them into a general model for the origin, function and antigen-specificity of CD4+CD25+ regulatory T cells. PMID:16918478

  5. Single molecule analysis of B cell receptor motion during signaling activation

    Science.gov (United States)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  6. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    Science.gov (United States)

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies. PMID:17855129

  7. 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...... for cytokine expression. They were co-cultured with primed T cells to measure induction of T cell proliferation and cytokine response. RESULTS: The number of CD11c+ microglia cells increased dramatically in EAE. They expressed equivalent levels of major histocompatibility complex and co-stimulatory ligands CD...

  8. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies.

    Science.gov (United States)

    Tang, Jonathan Cy; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. PMID:27205882

  9. The T-cell anergy induced by Leishmania amazonensis antigens is related with defective antigen presentation and apoptosis

    Directory of Open Access Journals (Sweden)

    Roberta O. Pinheiro

    2004-09-01

    Full Text Available Leishmania amazonensis is the main agent of diffuse cutaneous leishmaniasis, a disease associated with anergic immune responses. In this study we show that the crude antigen of Leishmania amazonensis (LaAg but not L. braziliensis promastigotes (LbAg contains substances that suppress mitogenic and spontaneous proliferative responses of T cells. The suppressive substances in LaAg are thermoresistant (100ºC/1h and partially dependent on protease activity. T cell anergy was not due to a decreased production of growth factors as it was not reverted by addition of exogenous IL-2, IL-4, IFN-gamma or IL-12. LaAg did not inhibit anti-CD3-induced T cell activation, suggesting that anergy was due to a defect in antigen presentation. It was also not due to cell necrosis, but was accompanied by expressive DNA fragmentation in lymph node cells, indicative of apoptosis. Although pre-incubation of macrophages with LaAg prevented their capacity to present antigens, this effect was not due to apoptosis of the former. These results suggest that the T cell anergy found in diffuse leishmaniasis may be the result of parasite antigen-driven apoptosis of those cells following defective antigen presentation.A Leishmania amazonensis é o principal agente etiológico da leishmaniose cutânea difusa, uma doença associada a respostas imunes anérgicas. Neste estudo nós mostramos que o extrato bruto de promastigotas de Leishmania amazonensis (LaAg, mas não de L. braziliensis (LbAg, contém substâncias que suprimem respostas proliferativas, espontâneas e mitogênicas, de células T. As substâncias supressoras no LaAg são termo-resistentes (100°C/1h e parcialmente dependentes da atividade de proteases. A anergia de células T não foi devida à diminuição na produção de fatores de crescimento, uma vez que não foi revertida pela adição de: IL-2, IL-4, IFN-gama ou IL-12. O LaAg não inibiu a ativação de células T induzida por anti-CD3, sugerindo que a anergia

  10. INVESTIGATION OF INDUCING EFFECT OF SPECIFIC CYTOTOXICITY OF CTLS BY ANTIGEN PEPTIDES FROM T LYMPHOCYTIC LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    张桂梅; 黄波; 李东; 王洪涛; 冯作化

    2003-01-01

    Objective: To investigate the characteristics of specific antitumor immunity induced by antigen peptides mixture from T lymphocytic leukemia cells. Method: Antigen peptides mixtures were prepared from different leukemia cell lines and then bound with Hsp70 in vitro. Human peripheral blood mononuclear cells (PBMC) were cultured in vitro, and activated with Hsp70-antigen peptides. The activated PBMC was cultured continuously in vitro, and used as effector cells in vitro test of cytotoxicity to different target cells. Results: The antigen peptides from different leukemia cell lines were peptides mixture and could activate PBMC effectively if they were presented by Hsp70. The activated PBMC could proliferate in the presence of IL-2 and Hsp70-antigen peptides. The proliferative PBMC had specific cytotoxicity to leukemia cells corresponding to the antigen peptides. PBMC activated by antigen peptides from T lymphocytic leukemia cell lines could effectively kill T lymphocytic leukemia cells, and the cytotoxicity of these PBMC to T lymphocytic leukemia cells was significantly stronger than that of PBMC activated by antigen peptides from other leukemia cells (P < 0.05). PBMC activated by either Hut78-peptides or Molt 4-peptides could effectively kill Jurkat cells. And the cytotoxicity of PBMC activated by Hut78/Molt-4-peptides to Jurkat cells was significantly stronger than that of PBMC activated by either Hut78-peptides or Molt-4-peptides alone (P<0.05).Conclusion: Antigen peptides mixture from T lymphocytic leukemia cell lines can induce specific cytotoxic effect to T lymphocytic leukemia cells. There exists cross-reactivity among antigen peptides mixture from different T lymphocytic leukemia cell lines. The cross-reactivity could be amplified by blending of different antigen peptides from different T lymphocytic leukemia cell lines, suggesting that it is possible to prepare broad-spectrum antigen peptide vaccine against T lymphocytic leukemia by using multiple leukemia

  11. Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells*

    Science.gov (United States)

    Wittmann, Alexandra; Lamprinaki, Dimitra; Bowles, Kristian M.; Katzenellenbogen, Ewa; Knirel, Yuriy A.; Whitfield, Chris; Nishimura, Takashi; Matsumoto, Naoki; Yamamoto, Kazuo; Iwakura, Yoichiro; Saijo, Shinobu; Kawasaki, Norihito

    2016-01-01

    LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2. PMID:27358401

  12. Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells.

    Science.gov (United States)

    Wittmann, Alexandra; Lamprinaki, Dimitra; Bowles, Kristian M; Katzenellenbogen, Ewa; Knirel, Yuriy A; Whitfield, Chris; Nishimura, Takashi; Matsumoto, Naoki; Yamamoto, Kazuo; Iwakura, Yoichiro; Saijo, Shinobu; Kawasaki, Norihito

    2016-08-19

    LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2. PMID:27358401

  13. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

    Directory of Open Access Journals (Sweden)

    Morteza Abouzaripour

    2016-02-01

    Full Text Available Objective: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone marrow have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1 positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs in order to investigate their differentiation potential for future use in cell therapy. Materials and Methods: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS followed by characterization with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR, immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. Results: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4 detected by immunocytochemistry and C-X-C chemokine receptor type 4 (CXCR4 and stem cell antigen-1 (SCA-1 detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors, Ngn3 (endocrine progenitor marker, Insulin1 and Insulin2 (pancreaticβ-cell markers. Additionally, our results demonstrate expression of PDX1 and GLUT2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. Conclusion: Our study clearly demonstrates the potential of SSEA-1

  14. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    Directory of Open Access Journals (Sweden)

    Rocio Flores-Fernández

    2016-01-01

    Full Text Available Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE. In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease.

  15. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    Science.gov (United States)

    Flores-Fernández, Rocio; Blanco-Favela, Francisco; Fuentes-Pananá, Ezequiel M.; Chávez-Sánchez, Luis; Gorocica-Rosete, Patricia; Pizaña-Venegas, Alberto; Chávez-Rueda, Adriana Karina

    2016-01-01

    Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. PMID:27314053

  16. Hepatitis C virus and ethanol alter antigen presentation in liver cells

    Institute of Scientific and Technical Information of China (English)

    Natalia A Osna

    2009-01-01

    Alcoholic patients have a high incidence of hepatitis Cvirus (HCV) infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCVinduced inability of the immune system to recognizeinfected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex (MHC) classⅠ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC classⅠand class Ⅱ in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) predominance,preventing cell maturation and allostimulation capacity.The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC classⅠ-restricted antigen presentation.Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.

  17. Identification of the Hair Cell Soma-1 Antigen, HCS-1, as Otoferlin

    OpenAIRE

    Goodyear, Richard J.; Legan, P. Kevin; Christiansen, Jeffrey R.; Xia, Bei; Korchagina, Julia; Gale, Jonathan E.; Mark E Warchol; Corwin, Jeffrey T.; Richardson, Guy P.

    2010-01-01

    Hair cells, the mechanosensitive receptor cells of the inner ear, are critical for our senses of hearing and balance. The small number of these receptor cells in the inner ear has impeded the identification and characterization of proteins important for hair cell function. The binding specificity of monoclonal antibodies provides a means for identifying hair cell-specific proteins and isolating them for further study. We have generated a monoclonal antibody, termed hair cell soma-1 (HCS-1), w...

  18. The effect of 125I labeled anti-androgen receptor agent on the proliferation of prostate cancer cells

    International Nuclear Information System (INIS)

    Objective: Based on the previous experience of using anti-androgen receptor triple helix forming oligonucleotide (TFO) to inhibit proliferation of prostate cancer cells, a 125I labeled TFO was prepared and tested in this experiment as an androgen receptor targeted antigene radiotherapy. Methods: 125I-TFO was labeled through Iodogen and then transfected LNCaP prostate cancer cells via liposome. The unlabeled TFO, 125I, and naturally cultured cells served as controls. The cellular proliferation was detected by methyl thiazolium tetrazolium (MTT) method, the expression of androgen receptor gene was carried out by RT-PCR and immunohistochemical study. Results: The radiolabeling efficiency, radiochemical purity and specific activity of 125I-TFO were 63.7%, 95.6% and 80.1 kBq/μg, respectively. At the same TFO concentration, the androgen receptor expression level in 125I-TFO treated cells was markedly lower than that of TFO group (P125I-TFO on cellular proliferation was significantly higher (P< 0.01). Conclusion: The inhibitory effect on androgen receptor expression and cell proliferation of prostate cancer cells of antigene therapy with radio-labeled TFO were significantly more obvious than that of classical antigene therapy. (authors)

  19. Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

    Science.gov (United States)

    Nashida, Tomoko; Sato, Ritsuko; Haga-Tsujimura, Maiko; Yoshie, Sumio; Yoshimura, Ken; Imai, Akane; Shimomura, Hiromi

    2013-02-01

    Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

  20. Inhibition of Ly-6A antigen expression prevents T cell activation

    OpenAIRE

    1990-01-01

    Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, ...

  1. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

    Directory of Open Access Journals (Sweden)

    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

  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. Purification and characterization of fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA)

    DEFF Research Database (Denmark)

    Hokland, P; Rosenthal, P; Griffin, J D;

    1983-01-01

    Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual fluore...... that these cells are relatively immature lymphoid cells, CALLA+ cells do not appear to contain either myeloid precursor cells (CFU-G/M) or the earliest lymphoid stem cells. Udgivelsesdato: 1983-Jan-1......Fetal hematopoietic cells that express the common acute lymphoblastic leukemia antigen (CALLA) were purified from both fetal liver and fetal bone marrow by immune rosetting with sheep erythrocytes coated with rabbit anti-mouse immunoglobulin and by fluorescence-activated cell sorting. Dual...... fluorescence techniques disclosed that these cells were heterogenous with respect to the expression of a series of differentiation and activation antigens defined by monoclonal antibodies. Thus, whereas all CALLA+ cells were Ia+ and expressed two activation antigens, J2 and T10, only 30-50% expressed B1...

  4. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

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

    Science.gov (United States)

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

    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. PMID:25778870

  6. T Cell Receptor Engineering and Analysis Using the Yeast Display Platform.

    Science.gov (United States)

    Smith, Sheena N; Harris, Daniel T; Kranz, David M

    2015-01-01

    The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g., a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g., T cell activation by as few as 1-3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with K D values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

  7. Antigen-activated dendritic cells ameliorate influenza A infections

    OpenAIRE

    Boonnak, Kobporn; Vogel, Leatrice; Orandle, Marlene; Zimmerman, Daniel; Talor, Eyal; Subbarao, Kanta

    2013-01-01

    Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecu...

  8. Identification of alpha beta and gamma delta T cell receptor-positive cells

    DEFF Research Database (Denmark)

    Geisler, C; Larsen, J K; Plesner, T

    1988-01-01

    distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study......Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue...... was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR...

  9. Human T cell responses to dengue virus antigens. Proliferative responses and interferon gamma production.

    OpenAIRE

    Kurane, I; Innis, B L; Nisalak, A; Hoke, C; Nimmannitya, S; Meager, A.; Ennis, F A

    1989-01-01

    The severe complications of dengue virus infections, hemorrhagic manifestations and shock, are more commonly observed during secondary dengue virus infections than during primary infections. It has been speculated that these complications are mediated by cross-reactive host-immune responses. We have begun to analyze human T cell responses to dengue antigens in vitro to explain the possible role of T lymphocytes in the pathogenesis of these complications. Dengue antigens induce proliferative r...

  10. Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

    OpenAIRE

    Bezek, D M; Baker, J. C.; Kaneene, J B

    1988-01-01

    A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood...

  11. Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.

    Science.gov (United States)

    Xu, Yingping; Liu, Yi; Yang, Chunqing; Kang, Li; Wang, Meixiang; Hu, Jingxia; He, Hao; Song, Wengang; Tang, Hua

    2016-10-01

    Defects in rapid clearance of apoptotic cells lead to an accumulation of dead cells (late apoptotic or secondary necrotic cells), which results in an aberrant immune response. However, little is known about whether and how macrophages (Mφs) cooperate with dendritic cells (DCs) in the presentation of dead-cell-associated antigens in this process. By transferring high numbers of dead cells to mimic a failure of apoptotic cell clearance in vivo, we found that Mφs and neutrophils were the predominant phagocytes in the uptake of dead cells in the spleen. Moreover, both Mφs and DCs were required for an optimal CD4(+) T-cell response triggered by dead-cell-associated antigens. Importantly, although Mφs alone had a poor capacity for antigen presentation, they could transfer phagocytosed antigens to DCs for potent antigen presentation to enhance T-cell responses. Finally, we found that exosomes released from Mφs acted as a transmitter to convey antigens to DCs partially in a ceramide-dependent manner, since treatment with the neutral sphingomyelinase inhibitor GW4869 and spiroepoxide resulted in a significant reduction of T-cell proliferation in vitro and in vivo. These findings point to a novel pathway of cross-talk between Mφs and DCs, which will be helpful to explain possible mechanisms for autoimmune diseases characterized by increased rates of apoptosis.

  12. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion.

    OpenAIRE

    Klein, L M; Lavker, R M; Matis, W L; Murphy, G F

    1989-01-01

    To understand better the role of mast cell secretory products in the genesis of inflammation, a system was developed for in vitro degranulation of human mast cells in skin organ cultures. Within 2 hr after morphine sulfate-induced degranulation, endothelial cells lining microvessels adjacent to affected mast cells expressed an activation antigen important for endothelial-leukocyte adhesion. Identical results were obtained when other mast cell secretagogues (anti-IgE, compound 48/80, and calci...

  13. Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

    Science.gov (United States)

    Goodman, C H; Russell, B J; Velez, J O; Laven, J J; Nicholson, W L; Bagarozzi, D A; Moon, J L; Bedi, K; Johnson, B W

    2014-11-01

    Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

  14. Antigen dynamics govern the induction of CD4(+) T cell tolerance during autoimmunity.

    Science.gov (United States)

    Challa, Dilip K; Mi, Wentao; Lo, Su-Tang; Ober, Raimund J; Ward, E Sally

    2016-08-01

    Antigen-specific T cell tolerance holds great promise for the treatment of autoimmune diseases. However, strategies to induce durable tolerance using high doses of soluble antigen have to date been unsuccessful, due to lack of efficacy and the risk of hypersensitivity. In the current study we have overcome these limitations by developing a platform for tolerance induction based on engineering the immunoglobulin Fc region to modulate the dynamic properties of low doses (1 μg/mouse; ∼50 μg/kg) of Fc-antigen fusions. Using this approach, we demonstrate that antigen persistence is a dominant factor governing the elicitation of tolerance in the model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), induced by immunizing B10.PL mice with the N-terminal epitope of myelin basic protein. Unexpectedly, our analyses reveal a stringent threshold of antigen persistence for both prophylactic and therapeutic treatments, although distinct mechanisms lead to tolerance in these two settings. Importantly, the delivery of tolerogenic Fc-antigen fusions during ongoing disease results in the downregulation of T-bet and CD40L combined with amplification of Foxp3(+) T cell numbers. The generation of effective, low dose tolerogens using Fc engineering has potential for the regulation of autoreactive T cells. PMID:27236506

  15. Leishmania chagasi T-cell antigens identified through a double library screen.

    Science.gov (United States)

    Martins, Daniella R A; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2006-12-01

    Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines. PMID:17000724

  16. Systems Pharmacology Modeling of Prostate‐Specific Antigen in Patients With Prostate Cancer Treated With an Androgen Receptor Antagonist and Down‐Regulator

    Science.gov (United States)

    Mistry, HB; Young, J; Clack, G; Dickinson, PA

    2016-01-01

    First‐in‐human (FIH) studies with AZD3514, a selective androgen receptor (AR) down‐regulator, showed decreases of >30% in the prostate‐specific antigen (PSA) in some patients. A modeling approach was adopted to understand these observations and define the optimum clinical use hypothesis for AZD3514 for clinical testing. Initial empirical modeling showed that only baseline PSA correlated significantly with this biological response, whereas drug concentration did not. To identify the mechanistic cause of this observation, a mechanism‐based model was first developed, which described the effects of AZD3514 on AR protein and PSA mRNA levels in LNCaP cells with and without dihydrotestosterone (DHT). Second, the mechanism‐based model was linked to a population pharmacokinetic (PK) model; PSA effects of clinical doses were subsequently simulated under different clinical conditions. This model was used to adjust the design of the ongoing clinical FIH study and direct the backup program. PMID:27299938

  17. Overexpression of Hepatitis B Virus-binding Protein, Squamous Cell Carcinoma Antigen 1, Extends Retention of Hepatitis B Virus in Mouse Liver

    Institute of Scientific and Technical Information of China (English)

    Hong-Bin XIA; Xi-Gu CHEN

    2006-01-01

    How receptors mediate the entry of hepatitis B virus (HBV) into the target liver cells is poorly understood. Recently, human squamous cell carcinoma antigen 1 (SCCA1) has been found to mediate binding and internalization of HBV to liver-derived cell lines in vitro. In this report, we investigate if SCCA1 is able to function as an HBV receptor and mediate HBV entry into mouse liver. SCCA1 transgene under the control of Rous sarcoma virus promoter was constructed in a minicircle DNA vector that was delivered to NOD/SCID mouse liver using the hydrodynamic technique. Subsequently, HBV-positive human serum was injected intravenously. We demonstrated that approximately 30% of the mouse liver cells expressed a high level of recombined SCCA1 protein for at least 37 d. The HBV surface antigen was found to persist in mouse liver for up to 17 d. Furthermore, HBV genome also persisted in mouse liver, as determined by polymerase chain reaction, for up to 17 d, and in mouse circulation for 7 d. These results suggest that SCAA1 might serve as an HBV receptor or co-receptor and play an important role in mediating HBV entry into hepatocytes, although its role in human HBV infection remains to be determined.

  18. bcRep: R Package for Comprehensive Analysis of B Cell Receptor Repertoire Data.

    Science.gov (United States)

    Bischof, Julia; Ibrahim, Saleh M

    2016-01-01

    Immunoglobulins, as well as T cell receptors, play a key role in adaptive immune responses because of their ability to recognize antigens. Recent advances in next generation sequencing improved also the quality and quantity of individual B cell receptors repertoire sequencing. Unfortunately, appropriate software to exhaustively analyze repertoire data from NGS platforms without limitations of the number of sequences are lacking. Here we introduce a new R package, bcRep, which offers a platform for comprehensive analyses of B cell receptor repertoires, using IMGT/HighV-QUEST formatted data. Methods for gene usage statistics, clonotype classification, as well as diversity measures, are included. Furthermore, functions to filter datasets, to do summary statistics about mutations, as well as visualization methods, are available. To compare samples in respect of gene usage, diversity, amino acid proportions, similar sequences or clones, several functions including also distance measurements, as well as multidimensional scaling methods, are provided. PMID:27551775

  19. bcRep: R Package for Comprehensive Analysis of B Cell Receptor Repertoire Data

    Science.gov (United States)

    Ibrahim, Saleh M.

    2016-01-01

    Immunoglobulins, as well as T cell receptors, play a key role in adaptive immune responses because of their ability to recognize antigens. Recent advances in next generation sequencing improved also the quality and quantity of individual B cell receptors repertoire sequencing. Unfortunately, appropriate software to exhaustively analyze repertoire data from NGS platforms without limitations of the number of sequences are lacking. Here we introduce a new R package, bcRep, which offers a platform for comprehensive analyses of B cell receptor repertoires, using IMGT/HighV-QUEST formatted data. Methods for gene usage statistics, clonotype classification, as well as diversity measures, are included. Furthermore, functions to filter datasets, to do summary statistics about mutations, as well as visualization methods, are available. To compare samples in respect of gene usage, diversity, amino acid proportions, similar sequences or clones, several functions including also distance measurements, as well as multidimensional scaling methods, are provided. PMID:27551775

  20. The Human Laminin Receptor is a Member of the Integrin Family of Cell Adhesion Receptors

    Science.gov (United States)

    Gehlsen, Kurt R.; Dillner, Lena; Engvall, Eva; Ruoslahti, Erkki

    1988-09-01

    A receptor for the adhesive basement membrane protein, laminin, was isolated from human glioblastoma cells by affinity chromatography on laminin. This receptor has a heterodimeric structure similar to that of receptors for other extracellular matrix proteins such as fibronectin and vitronectin. Incorporation of the laminin receptor into liposomal membranes makes it possible for liposomes to attach to surfaces coated with laminin. The receptor liposomes also attached to some extent to surfaces coated with fibronectin, but not with other matrix proteins. These properties identify the laminin receptor as a member of the integrin family of cell adhesion receptors.

  1. A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

    Science.gov (United States)

    Arora, Pooja; Baena, Andres; Yu, Karl O A; Saini, Neeraj K; Kharkwal, Shalu S; Goldberg, Michael F; Kunnath-Velayudhan, Shajo; Carreño, Leandro J; Venkataswamy, Manjunatha M; Kim, John; Lazar-Molnar, Eszter; Lauvau, Gregoire; Chang, Young-tae; Liu, Zheng; Bittman, Robert; Al-Shamkhani, Aymen; Cox, Liam R; Jervis, Peter J; Veerapen, Natacha; Besra, Gurdyal S; Porcelli, Steven A

    2014-01-16

    Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

  2. Self-antigen recognition by TGFβ1-deficient T cells causes their activation and systemic inflammation

    OpenAIRE

    Bommireddy, Ramireddy; Pathak, Leena J; Martin, Jennifer; Ormsby, Ilona; Engle, Sandra J; Gregory P. Boivin; Babcock, George F.; Eriksson, Anna U.; Singh, Ram R; DOETSCHMAN, THOMAS

    2006-01-01

    To investigate whether the multifocal inflammatory disease in TGFβ1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1−/− and Tgfb1−/− Rag1−/− mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1−/− DO11.10 mice develop a milder inflammation than do Tgfb1−/− mice, and their T cells display a less acti...

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

    Directory of Open Access Journals (Sweden)

    Cleo Goyvaerts

    2015-01-01

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

  4. Modulation of innate antigen-presenting cell function by pre-patent schistosome infection.

    Directory of Open Access Journals (Sweden)

    Christine E Ferragine

    Full Text Available Schistosomes are intravascular helminths that infect over 200 million people worldwide. Deposition of eggs by adult schistosomes stimulates Th2 responses to egg antigens and induces granulomatous pathology that is a hallmark of schistosome infection. Paradoxically, schistosomes require host immune function for their development and reproduction and for egress of parasite eggs from the host. To identify potential mechanisms by which immune cells might influence parasite development prior to the onset of egg production, we assessed immune function in mice infected with developing schistosomes. We found that pre-patent schistosome infection is associated with a loss of T cell responsiveness to other antigens and is due to a diminution in the ability of innate antigen-presenting cells to stimulate T cells. Diminution of stimulatory capacity by schistosome worms specifically affected CD11b(+ cells and did not require concomitant adaptive responses. We could not find evidence for production of a diffusible inhibitor of T cells by innate cells from infected mice. Rather, inhibition of T cell responsiveness by accessory cells required cell contact and only occurred when cells from infected mice outnumbered competent APCs by more than 3∶1. Finally, we show that loss of T cell stimulatory capacity may in part be due to suppression of IL-12 expression during pre-patent schistosome infection. Modulation of CD4(+ T cell and APC function may be an aspect of host immune exploitation by schistosomes, as both cell types influence parasite development during pre-patent schistosome infection.

  5. Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Rebekka Müller

    Full Text Available Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM. Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

  6. New progress of adoptive immunotherapy in the treatment of hematologic malignancies based on chimeric antigen receptor T-cells: reports from the 56th American Society of Hematology annual meeting%基于嵌合抗原受体修饰T细胞的血液肿瘤过继性免疫治疗新进展:第56届美国血液学会年会报道

    Institute of Scientific and Technical Information of China (English)

    吴秀丽; 李扬秋

    2015-01-01

    靶向识别特异抗原的嵌合抗原受体修饰T细胞(CAR-T细胞)已成为治疗恶性血液病的有效手段.临床研究涉及多种多样的CAR设计、不同的生产工艺及研究群体等.CAR-T细胞治疗慢性淋巴细胞白血病(CLL)和急性淋巴细胞白血病(ALL)已取得了令人瞩目的疗效,这将有可能改变所有类型血液系统恶性肿瘤的治疗模式.第56届美国血液学会(ASH)年会中关于CAR-T细胞的研究结果鼓舞了人们对这类新型治疗策略的信心.文章介绍CAR-T细胞治疗的临床研究结果和应用前景.虽然CAR-T细胞治疗恶性血液病还存在着许多有待解决的问题,但其治疗的高反应率促使开展了更多的临床研究.%T cells redirected to specific antigen targets with engineered chimeric antigen receptors (CARs) are emerging as powerful therapies in hematologic malignancies.Various CAR designs,manufacturing processes,and study populations,among other variables,have been studied and reported in clinical trials.The accumulating data supporting CAR-T cells therapy for disease such as chronic lymphocytic leukemia (CLL)and acute lymphocytic leukemia (ALL) highlight what may well become the next sea change in the care of patients with all types of hematologic neoplasia.The scientific symposium on CAR-T cells therapy inspires the mounting enthusiasm regarding this new treatment strategy.Here,the results of the reported clinical trials and the outlook for CAR-T cells therapies were reviewed and discussed.Many questions remain in the field of CAR-T cells directed to hematologic malignancies,but the encouraging response rates pave a wide road for future investigation.

  7. Serum squamous cell carcinoma antigen and CYFRA 21-1 in cervical cancer treatment

    International Nuclear Information System (INIS)

    Purpose: To analyze whether serum squamous cell carcinoma (SCC) antigen and cytokeratin-19 fragments (CYFRA) levels can assist in selecting patients with locally advanced cervical cancer who will benefit from combined treatment or additive surgery. Methods and Materials: Of 114 patients with cervical cancer Stage IB-IV, the first 39 patients received radiotherapy, the following 75 patients received identical radiotherapy plus concomitant chemotherapy (3 cycles of carboplatin and 5-fluorouracil). SCC antigen and CYFRA 21-1 serum levels were measured before treatment, after therapy, and during follow-up. Baseline tumor markers were related to tumor stage and size and clinical outcome. Results: Before treatment, SCC antigen was elevated (>1.9 μg/L) in 60% and CYFRA 21-1 (>2.2 μg/L) in 46% of patients. For all patients, disease-free survival (DFS) was better after combined treatment (67% vs. 43%, p<0.0005). For patients with elevated baseline SCC antigen, DFS was better after combination therapy (67% vs. 27%, p=0.001) which resulted more frequently in a normal SCC antigen (93% vs. 65%, p=0.004). In contrast, in those with a normal baseline CYFRA 21-1, combined therapy resulted in a better DFS (p=0.04). Patients who achieved a normal SCC antigen or CYFRA 21-1 after treatment had a better DFS (respectively 63 vs. 17% and 64 vs. 30%). Elevated SCC antigen posttreatment indicated residual tumor in 11/12 patients (92%), elevated CYFRA 21-1 in 7/10 patients (70%). Forty-seven patients had a tumor recurrence. At recurrence, SCC antigen was raised in 70% and CYFRA 21-1 in 69%. Conclusions: In patients with an elevated pretreatment SCC antigen, SCC antigen normalized more frequently with combined treatment and those patients had a better DFS. Elevated SCC antigen or CYFRA 21-1 levels after treatment completion indicated residual tumor in respectively 92% and 70%. The presence of elevated posttreatment levels of SCC antigen or CYFRA 21-1 indicates the need for additional

  8. The Mertk Receptor Tyrosine Kinase Promotes T-B interaction Stimulated by IgD B-cell Receptor Cross-linking

    OpenAIRE

    Shao, Wen-Hai; Zhen, Yuxuan; Finkelman, Fred D.; Cohen, Philip L.

    2014-01-01

    The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat a...

  9. Use of Heteropolymeric Monoclonal Antibodies to Attach Antigens to the C3b Receptor of Human Erythrocytes: A Potential Therapeutic Treatment

    Science.gov (United States)

    Taylor, Ronald P.; Sutherland, William M.; Reist, Craig J.; Webb, Donna J.; Wright, Eleanor L.; Labuguen, Ronald H.

    1991-04-01

    We have prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine γ globulin) to human RBCs under conditions that either allow or preclude complement activation. Quantitative analyses of this binding agree well with the number of complement receptors per RBC. In vitro "whole-blood" model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37^circC. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  10. The clinical value of squamous cell carcinoma antigen in cancer of the uterine cervix

    NARCIS (Netherlands)

    de Bruijn, HWA; Duk, JM; van der Zee, AGJ; Pras, E; Willemse, PHB; Hollema, H; Mourits, MJE; de Vries, EGE; Aalders, JG; Boonstra, J.

    1998-01-01

    A review is given of the clinical use and interpretation of serum tumor marker levels during the treatment of patients with cancer of the uterine cervix, Pretreatment serum squamous cell carcinoma (SCC) antigen provides a new prognostic factor in early stage squamous cell carcinoma of the uterine ce

  11. Discovering naturally processed antigenic determinants that confer protective T cell immunity

    DEFF Research Database (Denmark)

    Gilchuk, Pavlo; Spencer, Charles T; Conant, Stephanie B;

    2013-01-01

    CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infectio...

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

    Science.gov (United States)

    Furusawa, Chikara; Yamaguchi, Tomoyuki

    2016-01-01

    The immune response by T cells usually discriminates self and non-self antigens, even though the negative selection of self-reactive T cells is imperfect and a certain fraction of T cells can respond to self-antigens. In this study, we construct a simple mathematical model of T cell populations to analyze how such self/non-self discrimination is possible. The results demonstrate that the control of the immune response by regulatory T cells enables a robust and accurate discrimination of self and non-self antigens, even when there is a significant overlap between the affinity distribution of T cells to self and non-self antigens. Here, the number of regulatory T cells in the system acts as a global variable controlling the T cell population dynamics. The present study provides a basis for the development of a quantitative theory for self and non-self discrimination in the immune system and a possible strategy for its experimental verification. PMID:27668873

  13. Squamous cell carcinoma antigen isoforms in serum from cervical cancer patients

    NARCIS (Netherlands)

    Roijer, E; de Bruijn, HWA; Dahlen, U; ten Hoor, K; Lundin, M; Nilsson, K; Soderstrom, K; Nilsson, O

    2006-01-01

    Squamous cell carcinoma antigen (SCCA) is a serological marker of squamous cell carcinomas (SCC). To study whether any of the SCCA isoforms would provide additional and more specific/sensitive clinical information than total SCCA, immunoassays specific for the different forms of SCCA (free SCCA2, to

  14. A rendezvous before rejection: Where do T cells meet transplant antigens?

    OpenAIRE

    Briscoe, David M.; Sayegh, Mohamed H.

    2002-01-01

    Interactions between recipient T cells and donor endothelial graft cells may be an important mechanism for both acute and chronic rejection of vascularized allografts. This finding provides a starting point for investigations to develop novel ways of inducing long-lasting immunologic tolerance to donor antigens.

  15. Serological survey of normal humans for natural antibody to cell surface antigens of melanoma.

    Science.gov (United States)

    Houghton, A N; Taormina, M C; Ikeda, H; Watanabe, T; Oettgen, H F; Old, L J

    1980-01-01

    Sera of 106 normal adult men were tested for antibodies reacting with cell surface antigens of three established lines of cultured malignant melanoma. Positive reactions with a protein A assay for IgG antibodies were extremely rare (1-2%). The frequency of positive reactions with assays for IgM antibodies was higher: 5-15% in immune adherence assays and 55-82% in anti-C3 mixed hemadsorption assays. After low-titered sera and sera reacting with fetal calf serum components, conventional alloantigens, and widely distributed class 3 antigens were excluded, sera from seven individuals (one with IgG antibody and six with IgM antibodies) were selected for detailed analysis. The serum containing the IgG antibody came from a healthy 65-year-old Caucasian man; titers of antibody in his serum ranged from < 1/10 to 1/40,000 in tests with different melanoma cell lines. This IgG antibody identifies a differentiation antigen of melanocytes, provisionally designated Mel 1, that distinguishes two classes of melanomas: 22 melanoma cell lines typed Mel 1+ and 17 types Mel 1-. Mel 1 is expressed by fetal fibroblasts but not adult fibroblasts and can be found on a proportion of cultured epithelial cancer cell lines (5 out of 23) but not on glioma or B-cell lines. The melanoma antigens detected by the naturally occurring IgM antibodies are serologically unrelated to Mel 1 but, like Mel 1, appear to be differentiation antigens that distinguish subsets of melanoma. These IgM antibodies detect antigens that are identical or closely related to the AH antigen, a melanoma surface antigen that was initially defined by autologous antibody in a patient with melanoma. In view of the immunogenicity of both Mel 1 and the AH antigens in humans and their occurrence on more than 50% of melanomas, it remains to be seen whether antibody to these antigens can be elicited by specific vaccination of seronegative melanoma patients and whether this will have an influence on the clinical course of the disease

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

    Science.gov (United States)

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

    2016-01-01

    CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages. PMID:27003360

  17. Antigen presentation by murine epidermal langerhans cells and its alteration by ultraviolet B light

    International Nuclear Information System (INIS)

    Mice that are chronically exposed in vivo to ultraviolet B light (UV-B) display altered immunologic reactivity to various antigenic stimuli. A possible mode of UV-B action is that it exerts adverse effects on antigen-presenting cell function. Because the epidermis is the only tissue that is naturally subject to UV exposure we investigated if murine epidermal cells (EC) could perform an antigen presentation function and, if so, could this function be altered by UV-B irradiation. For this purpose, T cells immune to purified protein derivative of tuberculin (PPD) and dinitrophenylated ovalbumin (DNP6-OVA) from either BALB/c or C3H/He mice were incubated with syngeneic, semisyngeneic, or allogeneic EC or, for control purposes, with peritoneal exudate cells (PEC) that had been pulse-exposed to either the immunizing antigens or, as controls, left unpulsed, or pulsed to human serum albumin (HSA). After 4 days of culture, T cell proliferation was assessed by 3H-thymidine incorporation. PPD- and DNP/6-OVA pulsed, but not HSA-pulsed EC and PEC, induced vigorous proliferation of syngeneic and semisyngeneic, but not allogeneic, immune T cells. Pretreatment of stimulator cells with specific anti-Ia serum and complement virtually abolished this response, which indicated that among EC, Ia-bearing Langerhans cells are the critical stimulators. Exposure of EC either before or after pulsing to UV-B resulted in a dose-dependent impairment of antigen-specific T cell proliferation; the T proliferative response was abolished after administration of 20 mJ/cm2 UV-B. UV-B in the dose range employed did not produce immediate lethal cell damage, premature death of cultured EC, or toxic factors inhibitory for T cell proliferation

  18. Merkel Cell Polyomavirus Small T Antigen Targets the NEMO Adaptor Protein To Disrupt Inflammatory Signaling

    OpenAIRE

    Griffiths, David A.; Abdul-Sada, Hussein; Knight, Laura M.; Jackson, Brian R.; Richards, Kathryn; Prescott, Emma L.; Peach, A. Howard S.; Blair, G. Eric; MacDonald, Andrew; Whitehouse, Adrian

    2013-01-01

    Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST)...

  19. Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents.

    OpenAIRE

    Eldrige, S R; Butterworth, B E; Goldsworthy, T L

    1993-01-01

    Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellula...

  20. Tumorigenic activity of Merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice

    Science.gov (United States)

    Spurgeon, Megan E.; Cheng, Jingwei; Bronson, Roderick T.; Lambert, Paul F.; DeCaprio, James A.

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contain wild type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiological role in human cancer. PMID:25596282

  1. Tumorigenic activity of merkel cell polyomavirus T antigens expressed in the stratified epithelium of mice.

    Science.gov (United States)

    Spurgeon, Megan E; Cheng, Jingwei; Bronson, Roderick T; Lambert, Paul F; DeCaprio, James A

    2015-03-15

    Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contains wild-type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads, and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiologic role in human cancer. PMID:25596282

  2. Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

    Science.gov (United States)

    Frelinger, J A; Hibbler, F J; Hill, S W

    1978-12-01

    Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.

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

    Energy Technology Data Exchange (ETDEWEB)

    Nisbet-Brown, E.; Diener, E. (Univ. of Alberta, Edmonton (Canada))

    1986-01-01

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

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

    International Nuclear Information System (INIS)

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

  5. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Carlos M. Carballosa

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal...... gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for...... MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

  7. HCA520, A NOVEL TUMOR ASSOCIATED ANTIGEN, INVOLVED IN CELL PROLIFERATION AND APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    杨美香; 曲迅; 刘福利; 郑广娟

    2003-01-01

    Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis. Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Shayla K Shorter

    Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

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

    Directory of Open Access Journals (Sweden)

    Benencia Fabian

    2008-04-01

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

  10. The Short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction

    OpenAIRE

    Chen, Lanfen; Chen, Zhangguo; Baker, Kristi; Halvorsen, E lizabeth M.; da Cunha, Andre Pires; Flak, Magdalena B.; Gerber, Georg; Huang, Yu-Hwa; Hosomi, Shuhei; Arthur, J anelle C.; Dery, Ken J.; Nagaishi, Takashi; Beauchemin, Nicole; Kathryn V Holmes; Joshua W K Ho

    2012-01-01

    Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in ti...

  11. Interferon-induced changes in expression of antigens defined by monoclonal antibodies on malignant and nonmalignant mononuclear hematopoietic cells

    DEFF Research Database (Denmark)

    Hokland, M; Ritz, J; Hokland, P

    1983-01-01

    The effect of alpha interferon (alpha IFN) on the expression of histocompatibility--as well as differentiation antigens on normal and malignant hematopoietic mononuclear cells--were investigated by cell cytofluorometry using a panel of monoclonal antibodies (MoAbs). An increase in the expression...... of HLA-antigens detected by beta 2-Microglobulin (beta 2-M) could be demonstrated for peripheral blood mononuclear cells, non-T cells, Null cells, activated T cells, fetal thymocytes, adherent cells, and on four malignant non-T lymphoblastoid cell lines. In contrast, no significant differences were...... observed in the expression of antigens specific for B-lymphocytes (B1), T-lymphocytes (T3, T4, T6, T8, T11), NK-cells (901) and adherent cells (Mo1-4). Likewise, the expression of Ia-antigens remained unaltered on non-T cells, Null cells, and monocytes. The only other effect of IFN was an increase...

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

    Directory of Open Access Journals (Sweden)

    Warren L Denning

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

  13. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Directory of Open Access Journals (Sweden)

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  14. Evidence for an androgen receptor in porcine Leydig cells

    International Nuclear Information System (INIS)

    Cytosol and nuclear androgen receptor concentrations were measured in freshly prepared and cultured Leydig cells of immature pig testis with exchange assays using (3H)methyltrienolone a