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Sample records for partial t-cell receptor

  1. T-Cell Receptor-Transduced T Cells: Clinical Experience.

    PubMed

    Robbins, Paul F

    2015-01-01

    The large number of T-cell epitopes that have been found to be processed and presented on human tumors, now numbering in the hundreds, provides a rich source of targets for therapeutic interventions aimed at inducing durable tumor regression. Vaccination strategies aimed at inducing responses to these antigens have been largely ineffective, and it has been challenging to generate large numbers of T cells with the functional capacity to mediate durable tumor regressions in adoptive immunotherapy strategies in patients who have common epithelial malignancies. The ability to generate T-cell receptors that recognize shared as well as unique antigens expressed in a wide variety of common tumor types that include lung, breast, ovarian, gastrointestinal, urothelial, and genitourinary cancers provides an opportunity to develop widely applicable therapies based on the adoptive transfer of autologous T cells transduced with those receptors.

  2. Changing T cell specificity by retroviral T cell receptor display

    PubMed Central

    Kessels, Helmut W. H. G.; van den Boom, Marly D.; Spits, Hergen; Hooijberg, Erik; Schumacher, Ton N. M.

    2000-01-01

    The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generated in vitro and introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequent in vitro selection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resulting in vitro selected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than the in vivo generated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes. PMID:11121060

  3. Preferential expansion of human virus-specific multifunctional central memory T cells by partial targeting of the IL-2 receptor signaling pathway: the key role of CD4+ T cells.

    PubMed

    Schmueck, Michael; Fischer, Annika M; Hammoud, Ben; Brestrich, Gordon; Fuehrer, Henrike; Luu, Si-Hong; Mueller, Karin; Babel, Nina; Volk, Hans-Dieter; Reinke, Petra

    2012-05-15

    Effector memory T cells are effective in controlling acute infections, but central memory T cells play a key role in long-lasting protection against viruses and tumors. In vivo/in vitro challenge by Ag commonly supports the generation of effector memory T cells with limited longevity. To our knowledge, this study demonstrates for the first time in the human system and under rechallenge conditions that targeting IL-2R by partial mammalian target of rapamycin inhibition or blocking IL-2Rα enriches human CD4(+)/CD8(+) central memory T cells within the virus-specific T cell product associated with enhanced functionality (i.e., multicytokine secretors, including IL-2; enhanced CD137 and CD107a expression on CD8(+) and CD4(+) T cells, respectively; and killing infected target cells). Remarkably, the effects on CD8(+) T cells are mainly mediated via the enhancement of CD4(+) T cell function. The data reveal new insights into the role of CD4(+) T cell support for the quality of CD8(+) T cell memory, even under rechallenge conditions. Moreover, our method offers a new approach to improve the long-lasting efficacy of adoptive T cell therapy in patients.

  4. T Cell Receptor-Engineered T Cells to Treat Solid Tumors: T Cell Processing Toward Optimal T Cell Fitness

    PubMed Central

    van Steenbergen-Langeveld, Sabine; van Brakel, Mandy; Groot-van Ruijven, Corrien M.; van Elzakker, Pascal M.M.L.; van Krimpen, Brigitte; Sleijfer, Stefan; Debets, Reno

    2014-01-01

    Abstract Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for carbonic anhydrase IX (CAIX), we observed toxicities that (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have redefined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene-modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or after gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+, CD27+, CD62L+, CD45RA+T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors. PMID:25423330

  5. Genetic engineering with T cell receptors.

    PubMed

    Zhang, Ling; Morgan, Richard A

    2012-06-01

    In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers.

  6. T-cell receptor variable region gene usage in T-cell populations.

    PubMed Central

    Garman, R D; Ko, J L; Vulpe, C D; Raulet, D H

    1986-01-01

    We have examined T-cell receptor alpha- and beta-chain variable (V) region gene usage in T-cell populations predicted to have different major histocompatibility complex-restriction specificities. Using a sensitive ribonuclease protection assay to measure T-cell receptor mRNA levels, we found no striking differences in the usage of three V alpha genes and three V beta genes in T-cell populations from three congeneic H-2-disparate strains of mice and between the mutually exclusive Ly2+ L3T4- and Ly2- L3T4+ T-cell subpopulations. These results suggest that major histocompatibility complex restriction cannot be explained by the differential usage of nonoverlapping V alpha or V beta gene pools. In contrast, striking but unpredictable differences were seen in V gene usage in populations of T cells selected by activation with particular alloantigens. Images PMID:3487085

  7. Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

    PubMed Central

    Davey, Gayle M.; Schober, Sonya L.; Endrizzi, Bart T.; Dutcher, Angela K.; Jameson, Stephen C.; Hogquist, Kristin A.

    1998-01-01

    During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes. PMID:9815264

  8. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells.

    PubMed

    Farber, D L; Acuto, O; Bottomly, K

    1997-08-01

    Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3zeta subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3zeta in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4 T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.

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

    PubMed Central

    Newick, Kheng; Moon, Edmund; Albelda, Steven M

    2016-01-01

    Chimeric antigen receptor (CAR) T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias). This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment. PMID:27162934

  10. Vaccination against Experimental Allergic Encephalomyelitis with T Cell Receptor Peptides

    NASA Astrophysics Data System (ADS)

    Howell, Mark D.; Winters, Steven T.; Olee, Tsaiwei; Powell, Henry C.; Carlo, Dennis J.; Brostoff, Steven W.

    1989-11-01

    Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the β chain VDJ region and Jα regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell--mediated pathogenesis.

  11. SNX17 Affects T Cell Activation by Regulating T Cell Receptor and Integrin Recycling

    PubMed Central

    Osborne, Douglas G.; Piotrowski, Joshua T.; Dick, Christopher J.; Zhang, Jin-San; Billadeau, Daniel D.

    2015-01-01

    A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and antigen recognition. One protein potentially involved in T cell receptor transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 co-localizes with TCR and localizes to the immune synapse in T-APC conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared to control T cells. Lastly, we identified the FERM-domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse. PMID:25825439

  12. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation

    PubMed Central

    Au-Yeung, Byron B.; Zikherman, Julie; Mueller, James L.; Ashouri, Judith F.; Matloubian, Mehrdad; Cheng, Debra A.; Chen, Yiling; Shokat, Kevan M.; Weiss, Arthur

    2014-01-01

    T-cell antigen receptor (TCR) signaling is essential for activation, proliferation, and effector function of T cells. Modulation of both intensity and duration of TCR signaling can regulate these events. However, it remains unclear how individual T cells integrate such signals over time to make critical cell-fate decisions. We have previously developed an engineered mutant allele of the critical T-cell kinase zeta-chain-associated protein kinase 70 kDa (Zap70) that is catalytically inhibited by a small molecule inhibitor, thereby blocking TCR signaling specifically and efficiently. We have also characterized a fluorescent reporter Nur77–eGFP transgenic mouse line in which T cells up-regulate GFP uniquely in response to TCR stimulation. The combination of these technologies unmasked a sharp TCR signaling threshold for commitment to cell division both in vitro and in vivo. Further, we demonstrate that this threshold is independent of both the magnitude of the TCR stimulus and Interleukin 2. Similarly, we identify a temporal threshold of TCR signaling that is required for commitment to proliferation, after which T cells are able to proliferate in a Zap70 kinase-independent manner. Taken together, our studies reveal a sharp threshold for the magnitude and duration of TCR signaling required for commitment of T cells to proliferation. These results have important implications for understanding T-cell responses to infection and optimizing strategies for immunomodulatory drug delivery. PMID:25136127

  13. “The role of T cell receptor signaling thresholds in guiding T cell fate decisions”

    PubMed Central

    Zikherman, Julie; Au-Yeung, Byron

    2015-01-01

    Canonical T cell receptor signal transduction has been extensively studied and dissected in cell lines and primary lymphocytes. However, a static depiction of this signaling cascade fails to capture the complex and dynamic process by which individual T cells discriminate TCR:peptide-MHC affinity, then integrate signals over time to drive discrete cellular behaviors such as thymic selection, proliferation, and cytokine production. Recent technological advances have made it possible to study complex lymphocyte behavior on a single cell level and are revealing how T cells interpret information about affinity and abundance of antigen in order to make life-and-death cell fate decisions individually and collectively. PMID:25660212

  14. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy

    PubMed Central

    Sha, Huan-huan; Wang, Dan-dan; Yan, Da-li; Hu, Yong; Yang, Su-jin; Liu, Si-wen

    2017-01-01

    Chimaeric antigen receptor (CAR) T-cell therapies, as one of the cancer immunotherapies, have heralded a new era of treating cancer. The accumulating data, especially about CAR-modified T cells against CD19 support that CAR T-cell therapy is a highly effective immune therapy for B-cell malignancies. Apart from CD19, there have been many trials of CAR T cells directed other tumour specific or associated antigens (TSAs/TAAs) in haematologic malignancies and solid tumours. This review will briefly summarize basic CAR structure, parts of reported TSAs/TAAs, results of the clinical trials of CAR T-cell therapies as well as two life-threatening side effects. Experiments in vivo or in vitro, ongoing clinical trials and the outlook for CAR T-cell therapies also be included. Our future efforts will focus on identification of more viable cancer targets and more strategies to make CAR T-cell therapy safer. PMID:28053197

  15. Design of T cell receptor libraries with diverse binding properties to examine adoptive T cell responses

    PubMed Central

    Chervin, A.S.; Stone, J.D.; Soto, C.M.; Engels, B.; Schreiber, H.; Roy, E.J.; Kranz, D.M.

    2017-01-01

    Adoptive T cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, that introduces antigen-specific T cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T cell repertoires. Studies have suggested that use of higher affinity TCRs against class I MHC antigens could drive the activity of both CD4+ and CD8+ T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here we describe a high-throughput platform of “reverse biochemistry” whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8+ T cells expressing the highest affinity TCR variants were deleted in both the tumor infiltrating lymphocyte population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4+ T cells in the tumor, suggesting they played a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR binding properties that promote peripheral T cell survival and tumor elimination. PMID:23052828

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

    NASA Astrophysics Data System (ADS)

    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.

  17. Multimolecular associations of the T-cell antigen receptor.

    PubMed

    Beyers, A D; Spruyt, L L; Williams, A F

    1992-09-01

    T cells are activated when the T-cell receptor for antigen (TCR) interacts with an antigenic peptide bound to a major histocompatibility complex (MHC) molecule on the surface of another cell. It is often assumed that T-cell activation is induced by the crosslinking of TCRs. In this article, Albertus Beyers, Louise Spruyt and Alan Williams argue that this mechanism is not generally applicable. They hypothesize that the key event in T-cell activation is the formation of multimolecular complexes consisting of the TCR and several other polypeptides, including CD4 or CD8, CD2, CD5 and the associated tyrosine kinases p59(fyn) and p56(lck).

  18. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    PubMed

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy.

  19. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy

    PubMed Central

    Dai, Hanren; Wang, Yao; Lu, Xuechun

    2016-01-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  20. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    PubMed

    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.

  1. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    PubMed Central

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

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

    PubMed

    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.

  3. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    PubMed

    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.

  4. Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

    PubMed Central

    Thompson, S D; Pelkonen, J; Hurwitz, J L

    1990-01-01

    A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development. Images PMID:2164690

  5. Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

    PubMed

    Shank, Brandon R; Do, Bryan; Sevin, Adrienne; Chen, Sheree E; Neelapu, Sattva S; Horowitz, Sandra B

    2017-03-01

    Patients with B-cell hematologic malignancies who progress through first- or second-line chemotherapy have a poor prognosis. Early clinical trials with autologous anti-CD19 chimeric antigen receptor (CAR) T cells have demonstrated promising results for patients who have relapsed or refractory disease. Lymphodepleting conditioning regimens, including cyclophosphamide, fludarabine, pentostatin, bendamustine, interleukin-2, and total body irradiation, are often administered before the infusion of CAR T cells, allowing for greater T-cell expansion. The major toxicity associated with CAR T-cell infusions is cytokine release syndrome (CRS), a potentially life-threatening systemic inflammatory disorder. The quick onset and progression of CRS require rapid detection and intervention to reduce treatment-related mortality. Management with tocilizumab can help ameliorate the symptoms of severe CRS, allowing steroids, which diminish the expansion and persistence of CAR T cells, to be reserved for tocilizumab-refractory patients. Other toxicities of CAR T-cell therapy include neutropenia and/or febrile neutropenia, infection, tumor lysis syndrome, neurotoxicity and nausea/vomiting. A review of patients' medications is imperative to eliminate medications that may contribute to treatment-related toxicities. Studies are ongoing to help optimize patient selection, preparation, safety, and management of individuals receiving CAR T cells. Long-term follow-up will help establish the place of CAR T cells in therapy.

  6. Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells.

    PubMed

    Qiao, Yu; Zhu, Lingqiao; Sofi, Hanief; Lapinski, Philip E; Horai, Reiko; Mueller, Kristen; Stritesky, Gretta L; He, Xi; Teh, Hung-Sia; Wiest, David L; Kappes, Dietmar J; King, Philip D; Hogquist, Kristin A; Schwartzberg, Pamela L; Sant'Angelo, Derek B; Chang, Cheong-Hee

    2012-10-02

    MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

  7. SHARPIN controls regulatory T cells by negatively modulating the T cell antigen receptor complex

    PubMed Central

    Park, Yoon; Jin, Hyung-seung; Lopez, Justine; Lee, Jeeho; Liao, Lujian; Elly, Chris; Liu, Yun-Cai

    2016-01-01

    SHARPIN forms a linear-ubiquitin-chain-assembly complex that promotes signaling via the transcription factor NF-κB. SHARPIN deficiency leads to progressive multi-organ inflammation and immune system malfunction, but how SHARPIN regulates T cell responses is unclear. Here we found that SHARPIN deficiency resulted in a substantial reduction in the number of and defective function of regulatory T cells (Treg cells). Transfer of SHARPIN-sufficient Treg cells into SHARPIN-deficient mice considerably alleviated their systemic inflammation. SHARPIN-deficient T cells displayed enhanced proximal signaling via the T cell antigen receptor (TCR) without an effect on the activation of NF-κB. SHARPIN conjugated with Lys63 (K63)-linked ubiquitin chains, which led to inhibition of the association of TCRζ with the signaling kinase Zap70; this affected the generation of Treg cells. Our study therefore identifies a role for SHARPIN in TCR signaling whereby it maintains immunological homeostasis and tolerance by regulating Treg cells. PMID:26829767

  8. T cell receptor repertoires after adoptive transfer of expanded allogeneic regulatory T cells.

    PubMed

    Theil, A; Wilhelm, C; Kuhn, M; Petzold, A; Tuve, S; Oelschlägel, U; Dahl, A; Bornhäuser, M; Bonifacio, E; Eugster, A

    2017-02-01

    Regulatory T cell (Treg ) therapy has been exploited in autoimmune disease, solid organ transplantation and in efforts to prevent or treat graft-versus-host disease (GVHD). However, our knowledge on the in-vivo persistence of transfused Treg is limited. Whether Treg transfusion leads to notable changes in the overall Treg repertoire or whether longevity of Treg in the periphery is restricted to certain clones is unknown. Here we use T cell receptor alpha chain sequencing (TCR-α-NGS) to monitor changes in the repertoire of Treg upon polyclonal expansion and after subsequent adoptive transfer. We applied TCR-α-NGS to samples from two patients with chronic GVHD who received comparable doses of stem cell donor derived expanded Treg . We found that in-vitro polyclonal expansion led to notable repertoire changes in vitro and that Treg cell therapy altered the peripheral Treg repertoire considerably towards that of the infused cell product, to different degrees, in each patient. Clonal changes in the peripheral blood were transient and correlated well with the clinical parameters. We suggest that T cell clonotype analyses using TCR sequencing should be considered as a means to monitor longevity and fate of adoptively transferred T cells.

  9. Effect of partially hydrolyzed soluble glucan produced by glucosyltrasferases of Streptococcus mutans on stimulating human T cell.

    PubMed

    Choi, Inwook; Jung, Changhwa; Han, Yeook; Lee, Eunjoo H

    2006-01-01

    Soluble glucan, which was obtained from action of glucosyltransferases (GTFs) of Streptococcus mutans on sucrose, was partially hydrolyzed by acetic acid and examined for human T lymphoblast (MOLT-4) stimulating activity. Addition of the partially hydrolyzed glucan (15-60 microg/ml) stimulated human T cell (39-65%) in a dose dependant manner according to MTT assay. Production of interleukine-2 (IL-2) and interleukine-2 receptor (IL-2R) from T cell was increased by 44.5 and 25%, respectively, by addition of partially hydrolyzed glucan (15 microg/ml). These results indicate that stimulation of human T cells by hydrolyzed glucan is probably caused by its effects on stimulating gene expression of IL-2 and IL-2R of human T cell.

  10. Optimizing T-cell receptor gene therapy for hematologic malignancies

    PubMed Central

    Morris, Emma C.

    2016-01-01

    Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator–like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients. PMID:27207802

  11. The T-cell receptor as immunoglobulin: paradigm regained.

    PubMed

    Marchalonis, J J; Schluter, S F; Edmundson, A B

    1997-12-01

    The quest to determine the molecular nature of T-lymphocyte receptors for antigen was a "holy grail" to immunologists for over 25 years. This paper updates a review written 15 years ago (Marchalonis JJ, Hunt JC. Proc Soc Exp Biol Med 171:127-145, 1982), which proposed that "these molecules apparently do not bear determinants specified by the major histocompatibility complex, but express Ig-related variable regions and constant regions unique to T-cell products." We review subsequent contributions from molecular biology, protein chemistry, peptide immunochemistry, and structural biology establishing that T-cell receptors (TCRs) are members of the immunoglobulin family restricted to T cells that share 3-dimensional structural features, sequence homology, antigenic cross-reactivity, and common mechanisms of diversification with conventional immunoglobulins. These molecules and their light- and heavy-chain siblings appeared contemporaneously in vertebrate evolution with the emergence of sharks. We illustrate how extrapolation of concepts from immunoglobulin to T-cell receptors has aided in the understanding of these often enigmatic molecules, and, conversely, how concepts derived for T-cell receptors such as the role of "superantigens" can be directly applied to conventional immunoglobulins. A second precept that follows from the symmetry of the combining sites of Igs and TCRs is that MHC-restricted antibodies should exist. Such molecules have in fact been reported, and the x-ray crystallography for T-cell receptors suggests that the combining sites recognizing simultaneously MHC and peptide epitopes resemble the combining sites of antibodies directed against protein determinants. Additional immunoglobulin molecules of nonmammalian species have been detected and characterized based upon conserved homology to TCR and Igs, and it is anticipated that further study will enable the identification of more antigen-specific members of the family in mammals as well.

  12. CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage

    PubMed Central

    Fuchs, Yannick F.; Eugster, Anne; Dietz, Sevina; Sebelefsky, Christian; Kühn, Denise; Wilhelm, Carmen; Lindner, Annett; Gavrisan, Anita; Knoop, Jan; Dahl, Andreas; Ziegler, Anette-G.; Bonifacio, Ezio

    2017-01-01

    CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and β-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen. PMID:28300170

  13. Optimal T-cell receptor affinity for inducing autoimmunity

    PubMed Central

    Koehli, Sabrina; Naeher, Dieter; Galati-Fournier, Virginie; Zehn, Dietmar; Palmer, Ed

    2014-01-01

    T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity. PMID:25411315

  14. Finding Balance: T cell Regulatory Receptor Expression during Aging.

    PubMed

    Cavanagh, Mary M; Qi, Qian; Weyand, Cornelia M; Goronzy, Jörg J

    2011-10-01

    Aging is associated with a variety of changes to immune responsiveness. Reduced protection against infection, reduced responses to vaccination and increased risk of autoimmunity are all hallmarks of advanced age. Here we consider how changes in the expression of regulatory receptors on the T cell surface contribute to altered immunity during aging.

  15. NOD1 Cooperates with TLR2 to Enhance T Cell Receptor-Mediated Activation in CD8 T Cells

    PubMed Central

    Mercier, Blandine C.; Debaud, Anne-Laure; Tomkowiak, Martine; Marvel, Jacqueline; Bonnefoy, Nathalie

    2012-01-01

    Pattern recognition receptors (PRR), like Toll-like receptors (TLR) and NOD-like receptors (NLR), are involved in the detection of microbial infections and tissue damage by cells of the innate immune system. Recently, we and others have demonstrated that TLR2 can additionally function as a costimulatory receptor on CD8 T cells. Here, we establish that the intracytosolic receptor NOD1 is expressed and functional in CD8 T cells. We show that C12-iEDAP, a synthetic ligand for NOD1, has a direct impact on both murine and human CD8 T cells, increasing proliferation and effector functions of cells activated via their T cell receptor (TCR). This effect is dependent on the adaptor molecule RIP2 and is associated with an increased activation of the NF-κB, JNK and p38 signaling pathways. Furthermore, we demonstrate that NOD1 stimulation can cooperate with TLR2 engagement on CD8 T cells to enhance TCR-mediated activation. Altogether our results indicate that NOD1 might function as an alternative costimulatory receptor in CD8 T cells. Our study provides new insights into the function of NLR in T cells and extends to NOD1 the recent concept that PRR stimulation can directly control T cell functions. PMID:22848741

  16. Unravelling the association of partial T-cell immunodeficiency and immune dysregulation.

    PubMed

    Liston, Adrian; Enders, Anselm; Siggs, Owen M

    2008-07-01

    Partial T-cell immunodeficiencies constitute a heterogeneous cluster of disorders characterized by an incomplete reduction in T-cell number or activity. The immune deficiency component of these diseases is less severe than that of the severe T-cell immunodeficiencies and therefore some ability to respond to infectious organisms is retained. Unlike severe T-cell immunodeficiencies, however, partial immunodeficiencies are commonly associated with hyper-immune dysregulation, including autoimmunity, inflammatory diseases and elevated IgE production. This causative association is counter-intuitive--immune deficiencies are caused by loss-of-function changes to the T-cell component, whereas the coincident autoimmune symptoms are the consequence of gain-of-function changes. This Review details the genetic basis of partial T -cell immunodeficiencies and draws on recent advances in mouse models to propose mechanisms by which a reduction in T-cell numbers or function may disturb the population-dependent balance between activation and tolerance.

  17. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    NASA Astrophysics Data System (ADS)

    Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

    1994-06-01

    IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

  18. Prospects and limitations of T cell receptor gene therapy.

    PubMed

    Jorritsma, Annelies; Schotte, Remko; Coccoris, Miriam; de Witte, Moniek A; Schumacher, Ton N M

    2011-08-01

    Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer. First, it circumvents the demanding process of in vitro generation of large numbers of specific immune cells. Second, it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments, mouse model systems and phase I clinical trials. Furthermore, we discuss the prospects of widespread clinical application of this gene therapy approach for the treatment of human cancer.

  19. Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8(+) T Cell Receptor Signaling.

    PubMed

    Gonzalez-Perez, Gabriela; Lamousé-Smith, Esi S N

    2017-01-01

    We recently reported that maternal antibiotic treatment (MAT) of mice in the last days of pregnancy and during lactation dramatically alters the density and composition of the gastrointestinal microbiota of their infants. MAT infants also exhibited enhanced susceptibility to a systemic viral infection and altered adaptive immune cell activation phenotype and function. CD8(+) effector T cells from MAT infants consistently demonstrate an inability to sustain interferon gamma (IFN-γ) production in vivo following vaccinia virus infection and in vitro upon T cell receptor (TCR) stimulation. We hypothesize that T cells developing in infant mice with gastrointestinal microbiota dysbiosis and insufficient toll-like receptor (TLR) exposure alters immune responsiveness associated with intrinsic T cell defects in the TCR signaling pathway and compromised T cell effector function. To evaluate this, splenic T cells from day of life 15 MAT infant mice were stimulated in vitro with anti-CD3 and anti-CD28 antibodies prior to examining the expression of ZAP-70, phosphorylated ZAP-70, phospho-Erk-1/2, c-Rel, total protein tyrosine phosphorylation, and IFN-γ production. We determine that MAT infant CD8(+) T cells fail to sustain total protein tyrosine phosphorylation and Erk1/2 activation. Lipopolysaccharide treatment in vitro and in vivo, partially restored IFN-γ production in MAT effector CD8(+) T cells and reduced mortality typically observed in MAT mice following systemic viral infection. Our results demonstrate a surprising dependence on the gastrointestinal microbiome and TLR ligand stimulation toward shaping optimal CD8(+) T cell function during infancy.

  20. Gastrointestinal Microbiome Dysbiosis in Infant Mice Alters Peripheral CD8+ T Cell Receptor Signaling

    PubMed Central

    Gonzalez-Perez, Gabriela; Lamousé-Smith, Esi S. N.

    2017-01-01

    We recently reported that maternal antibiotic treatment (MAT) of mice in the last days of pregnancy and during lactation dramatically alters the density and composition of the gastrointestinal microbiota of their infants. MAT infants also exhibited enhanced susceptibility to a systemic viral infection and altered adaptive immune cell activation phenotype and function. CD8+ effector T cells from MAT infants consistently demonstrate an inability to sustain interferon gamma (IFN-γ) production in vivo following vaccinia virus infection and in vitro upon T cell receptor (TCR) stimulation. We hypothesize that T cells developing in infant mice with gastrointestinal microbiota dysbiosis and insufficient toll-like receptor (TLR) exposure alters immune responsiveness associated with intrinsic T cell defects in the TCR signaling pathway and compromised T cell effector function. To evaluate this, splenic T cells from day of life 15 MAT infant mice were stimulated in vitro with anti-CD3 and anti-CD28 antibodies prior to examining the expression of ZAP-70, phosphorylated ZAP-70, phospho-Erk-1/2, c-Rel, total protein tyrosine phosphorylation, and IFN-γ production. We determine that MAT infant CD8+ T cells fail to sustain total protein tyrosine phosphorylation and Erk1/2 activation. Lipopolysaccharide treatment in vitro and in vivo, partially restored IFN-γ production in MAT effector CD8+ T cells and reduced mortality typically observed in MAT mice following systemic viral infection. Our results demonstrate a surprising dependence on the gastrointestinal microbiome and TLR ligand stimulation toward shaping optimal CD8+ T cell function during infancy. PMID:28337207

  1. New Insights into How Trafficking Regulates T Cell Receptor Signaling

    PubMed Central

    Lou, Jieqiong; Rossy, Jérémie; Deng, Qiji; Pageon, Sophie V.; Gaus, Katharina

    2016-01-01

    There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. PMID:27508206

  2. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  3. New Strategies in Engineering T-Cell Receptor Gene-Modified T Cells to More Effectively Target Malignancies

    PubMed Central

    Schmitt, Thomas M.; Stromnes, Ingunn M.; Chapuis, Aude G.; Greenberg, Philip D.

    2016-01-01

    The immune system, and T cells in particular, have the ability to target and destroy malignant cells. However, anti-tumor immune responses induced from the endogenous T cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune-evasion. PMID:26463711

  4. New Strategies in Engineering T-cell Receptor Gene-Modified T cells to More Effectively Target Malignancies.

    PubMed

    Schmitt, Thomas M; Stromnes, Ingunn M; Chapuis, Aude G; Greenberg, Philip D

    2015-12-01

    The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion.

  5. Direct Measurement of T Cell Receptor Affinity and Sequence from Naïve Anti-Viral T Cells

    PubMed Central

    Zhang, Shuqi; Parker, Patricia; Ma, Keyue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad; Wendel, Ben S.; Meriwether, Amanda; Salazar, Mary A.; Jiang, Ning

    2016-01-01

    T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCR). The affinity of TCR to cognate antigen is of high interest in adoptive T cell transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. Here, we introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8+ T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications. PMID:27252176

  6. A T-cell-directed chimeric antigen receptor for the selective treatment of T-cell malignancies.

    PubMed

    Mamonkin, Maksim; Rouce, Rayne H; Tashiro, Haruko; Brenner, Malcolm K

    2015-08-20

    Options for targeted therapy of T-cell malignancies remain scarce. Recent clinical trials demonstrated that chimeric antigen receptors (CARs) can effectively redirect T lymphocytes to eradicate lymphoid malignancies of B-cell origin. However, T-lineage neoplasms remain a more challenging task for CAR T cells due to shared expression of most targetable surface antigens between normal and malignant T cells, potentially leading to fratricide of CAR T cells or profound immunodeficiency. Here, we report that T cells transduced with a CAR targeting CD5, a common surface marker of normal and neoplastic T cells, undergo only limited fratricide and can be expanded long-term ex vivo. These CD5 CAR T cells effectively eliminate malignant T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma lines in vitro and significantly inhibit disease progression in xenograft mouse models of T-ALL. These data support the therapeutic potential of CD5 CAR in patients with T-cell neoplasms.

  7. Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells.

    PubMed

    Tsatsanis, Christos; Vaporidi, Katerina; Zacharioudaki, Vassiliki; Androulidaki, Ariadne; Sykulev, Yuri; Margioris, Andrew N; Tsichlis, Philip N

    2008-02-26

    The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2(-/-) genetic background. Surprisingly, the TCR2C(tg/tg)/Tpl2(-/-) mice developed T cell lymphomas with a latency of 4-6 months. The tumor cells were consistently TCR2C(+)CD8(+)CD4(-), suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8(+) ISP cells was not expanded in the thymus of TCR2C(tg/tg)/Tpl2(-/-) mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2(-/-) mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

  8. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    SciTech Connect

    Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  9. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  10. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells.

    PubMed

    Otáhal, Pavel; Průková, Dana; Král, Vlastimil; Fabry, Milan; Vočková, Petra; Latečková, Lucie; Trněný, Marek; Klener, Pavel

    2016-04-01

    Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use of LEN for

  11. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells

    PubMed Central

    Otáhal, Pavel; Průková, Dana; Král, Vlastimil; Fabry, Milan; Vočková, Petra; Latečková, Lucie; Trněný, Marek; Klener, Pavel

    2016-01-01

    ABSTRACT Tumor immunotherapy based on the use of chimeric antigen receptor modified T cells (CAR T cells) is a promising approach for the treatment of refractory hematological malignancies. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable.Lenalidomide (LEN) is an immunomodulatory drug currently approved for the treatment of multiple myeloma (MM) and mantle cell lymphoma, while it is clinically tested in the therapy of diffuse large B-cell lymphoma of activated B cell immunophenotype. LEN was shown to increase antitumor immune responses at least partially by modulating the activity of E3 ubiquitin ligase Cereblon, which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors, which in turn results in changed expression of various receptors on the surface of tumor cells. In order to enhance the effectiveness of CAR-based immunotherapy, we assessed the anti-lymphoma efficacy of LEN in combination with CAR19 T cells or CAR20 T cells in vitro and in vivo using various murine models of aggressive B-cell non-Hodgkin lymphomas (B-NHL).Immunodeficient NSG mice were transplanted with various human B-NHL cells followed by treatment with CAR19 or CAR20 T cells with or without LEN. Next, CAR19 T cells were subjected to series of tests in vitro to evaluate their response and signaling capacity following recognition of B cell in the presence or absence of LEN.Our data shows that LEN significantly enhances antitumor functions of CAR19 and CAR20 T cells in vivo. Additionally, it enhances production of interferon gamma by CAR19 T cells and augments cell signaling via CAR19 protein in T cells in vitro. Our data further suggests that LEN works through direct effects on T cells but not on B-NHL cells. The biochemical events underlying this costimulatory effect of LEN are currently being investigated. In summary, our data supports the use

  12. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

  13. Two distinct T-cell receptor alpha-chain transcripts in a rabbit T-cell line: implications for allelic exclusion in T cells.

    PubMed Central

    Marche, P N; Kindt, T J

    1986-01-01

    Information relevant to allelic exclusion in T cells has been obtained by a study of cDNA clones corresponding to alpha-chain genes of the T-cell receptor in the rabbit T-cell line RL-5. One clone contains a variable-joining-constant (VJC) sequence encoding a complete alpha chain of the T-cell receptor. A second has an identical constant region and includes a distinct variable-joining (VJ) sequence. However, a single-base deletion in the variable region places the remainder of the second transcript out-of-phase and appears to be the product of a rearrangement involving a variable region of the T-cell receptor alpha-chain pseudogene. Presence of two variable-joining-constant (VJC) transcripts in the same cell line indicates that alpha-chain gene rearrangement is not affected by transcription of a complete alpha-chain mRNA and suggests that steps after mRNA synthesis are involved in the allelic exclusion process for alpha-chain genes. Comparison of rabbit alpha-chain sequences with those of man and mouse revealed interspecies conservation in constant and variable regions. Genomic Southern blot analyses using a rabbit constant region of the T-cell receptor alpha-chain probe revealed the presence of a single constant region gene. Hybridization with variable region probes defined two distinct multigenic subfamilies. Homology between certain rabbit and murine variable regions of the T-cell receptor alpha-chain sequences suggests that the existence of subfamilies predated divergence of these species. Images PMID:3485798

  14. Unusual features of Self-Peptide/MHC Binding by Autoimmune T Cell Receptors

    SciTech Connect

    Nicholson,M.; Hahn, M.; Wucherpfennig, K.

    2005-01-01

    Structural studies on T cell receptors (TCRs) specific for foreign antigens demonstrated a remarkably similar topology characterized by a central, diagonal TCR binding mode that maximizes interactions with the MHC bound peptide. However, three recent structures involving autoimmune TCRs demonstrated unusual interactions with self-peptide/MHC complexes. Two TCRs from multiple sclerosis patients bind with unconventional topologies, and both TCRs are shifted toward the peptide N terminus and the MHC class II {beta} chain helix. A TCR from the experimental autoimmune encephalomyelitis (EAE) model binds in a conventional orientation, but the structure is unusual because the self-peptide only partially fills the binding site. For all three TCRs, interaction with the MHC bound self-peptide is suboptimal, and only two or three TCR loops contact the peptide. Optimal TCR binding modes confer a competitive advantage for antimicrobial T cells during an infection, whereas altered binding properties may permit survival of a subset of autoreactive T cells during thymic selection.

  15. Crammed signaling motifs in the T-cell receptor.

    PubMed

    Borroto, Aldo; Abia, David; Alarcón, Balbino

    2014-09-01

    Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering.

  16. Generation of CD8+ T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy

    PubMed Central

    Höfflin, Sandra; Prommersberger, Sabrina; Uslu, Ugur; Schuler, Gerold; Schmidt, Christopher W; Lennerz, Volker; Dörrie, Jan; Schaft, Niels

    2015-01-01

    Adoptive T-cell therapy of cancer often fails due to the tumor cells' immune escape mechanisms, like antigen loss or down-regulation. To anticipate immune escape by loss of a single antigen, it would be advantageous to equip T cells with multiple specificities. To study the possible interference of 2 T-cell receptors (TCRs) in one cell, and to examine how to counteract competing effects, we generated TETARs, CD8+ T cells expressing two additional T-cell receptors by simultaneous transient transfection with 2 TCRs using RNA electroporation. The TETARs were equipped with one TCR specific for the common melanoma antigen gp100 and one TCR recognizing a patient-specific, individual mutation of CCT6A (chaperonin containing TCP1, subunit 6A) termed “CCT6Am TCR.” These CD8+ T cells proved functional in cytokine secretion and lytic activity upon stimulation with each of their cognate antigens, although some reciprocal inhibition was observed. Murinisation of the CCT6Am TCR increased and prolonged its expression and increased the lytic capacity of the dual-specific T cells. Taken together, we generated functional, dual-specific CD8+ T cells directed against a common melanoma-antigen and an individually mutated antigen for the use in personalised adoptive T-cell therapy of melanoma. The intended therapy would involve repetitive injections of the RNA-transfected cells to overcome the transiency of TCR expression. In case of autoimmunity-related side effects, a cessation of treatment would result in a disappearance of the introduced receptors, which increases the safety of this approach. PMID:26178065

  17. Mouse T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    All mouse T-cell receptor {alpha}/{delta}, {beta}, and {gamma} variable (Tcra/d-, b-, and g-V) gene segments were aligned to compare the sequences with one another, to group them into subfamilies, and to derive a name which complies with the standard nomenclature. it was necessary to change the names of some V gene segments because they conflicted with those of other segments. The traditional classification into subfamilies was re-evaluated using a much larger pool of sequences. In the mouse, most V gene segments can be grouped into subfamilies of closely related genes with significantly less similarity between different subfamilies. 118 refs., 11 figs., 4 tabs.

  18. T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

    PubMed Central

    Karas, Michael; Zaks, Tal Z.; JL, Liu; LeRoith, Derek

    1999-01-01

    Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle. PMID:10588669

  19. Regulatory T cells play a role in T-cell receptor CDR2 peptide regulation of experimental autoimmune encephalomyelitis.

    PubMed

    Buenafe, Abigail C; Andrew, Shayne; Offner, Halina; Vandenbark, Arthur A

    2012-02-01

    Eliciting T-cell receptor (TCR) -specific responsiveness has been known to provide an effective autoregulatory mechanism for limiting inflammation mediated by T effector cells. Our previous use of TCR peptides derived from the CDR3 regions of a pathogenic TCR effectively reversed ongoing experimental autoimmune encephalomyelitis (EAE) in a humanized TCR transgenic model. In this study, we use the TCR BV8S2 CDR2 peptide in the non-transgenic C57BL/6 EAE model to down-regulate the heterogeneous TCR BV8S2(+)  MOG-35-55-specific pathogenic T-cell population and demonstrate successful treatment of EAE after disease onset. Suppression of disease was associated with reduced MOG-35-55-specific and non-specific T-cell production of interleukin-17a and interferon-γ in the central nervous system, as well as reduced numbers of CD4(+) and Foxp3(+) T cells in the central nervous system. With the use of Foxp3-GFP and Foxp3 conditional knockout mice, we demonstrate that the TCR CDR2 peptide treatment effect is dependent on the presence of Foxp3(+) regulatory T cells and that regulatory T cell numbers are significantly expanded in the periphery of treated mice. Hence, TCR CDR2 peptide therapy is effective in regulating heterogeneous, pathogenic T-cell populations through the activity of the Foxp3(+) regulatory T cell population.

  20. Tumor necrosis factor receptor superfamily costimulation couples T cell receptor signal strength to thymic regulatory T cell differentiation

    PubMed Central

    Mahmud, Shawn A.; Manlove, Luke S.; Schmitz, Heather M.; Xing, Yan; Wang, Yanyan; Owen, David L.; Schenkel, Jason M.; Boomer, Jonathan S.; Green, Jonathan M.; Yagita, Hideo; Chi, Hongbo; Hogquist, Kristin A.; Farrar, Michael A.

    2014-01-01

    Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL-2R/STAT5 responsiveness. GITR-ligand costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire. PMID:24633226

  1. Reconciling views on T cell receptor germline bias for MHC.

    PubMed

    Garcia, K Christopher

    2012-09-01

    Whether MHC restriction by the T cell receptor (TCR) is a product of evolutionary pressures leading to germline-encoded 'rules of engagement' remains avidly debated. Structural results derived from analysis of TCR-peptide-MHC complexes appear to support a model of physical specificity between TCR germline V regions and MHC. Yet, some recent evidence suggests that thymic selection, and co-receptors may have misled us into thinking the TCR is exclusively MHC-specific, when in fact, TCRs can robustly engage non-MHC ligands when given the chance. Here, I propose that seemingly contradictory data and hypotheses for, and against, germline bias are, in fact, compatible and can be reconciled into a unifying model.

  2. Chemokine receptor expression by leukemic T cells of cutaneous T-cell lymphoma: clinical and histopathological correlations.

    PubMed

    Capriotti, Elisabetta; Vonderheid, Eric C; Thoburn, Christopher J; Bright, Emilie C; Hess, Allan D

    2007-12-01

    Chemokine receptors expressed by normal and neoplastic lymphocytes provide an important mechanism for cells to traffic into the skin and skin-associated lymph nodes. The goal of this study was to correlate chemokine receptor and CD62L expression by circulating neoplastic T cells with the clinical and pathological findings of the leukemic phase of cutaneous T-cell lymphoma, primarily Sézary syndrome (SS). Chemokine receptor mRNA transcripts were found in the majority of leukemic cells for CCR1, CCR4, CCR7, CCR10, CXCR3, and CD62L and in 20-50% of the samples for CXCR5. In patients with SS, relatively high expression levels of CCR7 and CCR10 by circulating neoplastic T cells correlated with epidermotropism, CXCR5 expression correlated with density of the dermal infiltrate, and CD62L correlated with extent of lymphadenopathy. Of note, CXCR5 expression and a dense dermal infiltrate correlated with a poor prognosis. The chemokine receptor profile supports the concept that neoplastic T cells are central memory T cells, and that CCR10 and CD62L play a fundamental role respectively in epidermotropism and lymphadenopathy that is observed in SS.

  3. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia

    PubMed Central

    Maude, Shannon L.; Frey, Noelle; Shaw, Pamela A.; Aplenc, Richard; Barrett, David M.; Bunin, Nancy J.; Chew, Anne; Gonzalez, Vanessa E.; Zheng, Zhaohui; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, Jan J.; Rheingold, Susan R.; Shen, Angela; Teachey, David T.; Levine, Bruce L.; June, Carl H.; Porter, David L.; Grupp, Stephan A.

    2014-01-01

    BACKGROUND Relapsed acute lymphoblastic leukemia (ALL) is difficult to treat despite the availability of aggressive therapies. Chimeric antigen receptor–modified T cells targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease. METHODS We infused autologous T cells transduced with a CD19-directed chimeric antigen receptor (CTL019) lentiviral vector in patients with relapsed or refractory ALL at doses of 0.76×106 to 20.6×106 CTL019 cells per kilogram of body weight. Patients were monitored for a response, toxic effects, and the expansion and persistence of circulating CTL019 T cells. RESULTS A total of 30 children and adults received CTL019. Complete remission was achieved in 27 patients (90%), including 2 patients with blinatumomab-refractory disease and 15 who had undergone stem-cell transplantation. CTL019 cells proliferated in vivo and were detectable in the blood, bone marrow, and cerebrospinal fluid of patients who had a response. Sustained remission was achieved with a 6-month event-free survival rate of 67% (95% confidence interval [CI], 51 to 88) and an overall survival rate of 78% (95% CI, 65 to 95). At 6 months, the probability that a patient would have persistence of CTL019 was 68% (95% CI, 50 to 92) and the probability that a patient would have relapse-free B-cell aplasia was 73% (95% CI, 57 to 94). All the patients had the cytokine-release syndrome. Severe cytokine-release syndrome, which developed in 27% of the patients, was associated with a higher disease burden before infusion and was effectively treated with the anti–interleukin-6 receptor antibody tocilizumab. CONCLUSIONS Chimeric antigen receptor–modified T-cell therapy against CD19 was effective in treating relapsed and refractory ALL. CTL019 was associated with a high remission rate, even among patients for whom stem-cell transplantation had failed, and durable remissions up to 24 months were observed. (Funded by

  4. The promise of γδ T cells and the γδ T cell receptor for cancer immunotherapy.

    PubMed

    Legut, Mateusz; Cole, David K; Sewell, Andrew K

    2015-11-01

    γδ T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human γδ T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the γδ T cell compartment in cancer immunosurveillance and paving the way for the use of γδ TCRs in cancer therapy. Ligand recognition by the γδ TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike αβ T cells, γδ T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human γδ T cells and discuss the promise of harnessing these cells for cancer treatment.

  5. Coupling of T cell receptor specificity to natural killer T cell development by bivalent histone H3 methylation.

    PubMed

    Dobenecker, Marc-Werner; Kim, Jong Kyong; Marcello, Jonas; Fang, Terry C; Prinjha, Rab; Bosselut, Remy; Tarakhovsky, Alexander

    2015-03-09

    The fidelity of T cell immunity depends greatly on coupling T cell receptor signaling with specific T cell effector functions. Here, we describe a chromatin-based mechanism that enables integration of TCR specificity into definite T cell lineage commitment. Using natural killer T cells (iNKT cell) as a model of a T cell subset that differentiates in response to specific TCR signaling, we identified a key role of histone H3 lysine 27 trimethylation (H3K27me3) in coupling iNKT cell TCR specificity with the generation of iNKT cells. We found that the Zbtb16/PLZF gene promoter that drives iNKT cell differentiation possesses a bivalent chromatin state characterized by the simultaneous presence of negative and positive H3K27me3 and H3K4me3 modifications. Depletion of H3K27me3 at the Zbtb16/PLZF promoter leads to uncoupling of iNKT cell development from TCR specificity and is associated with accumulation of iNKT-like CD4(+) cells that express a non-iNKT cell specific T cell repertoire. In turn, stabilization of H3K27me3 leads to a drastic reduction of the iNKT cell population. Our data suggest that H3K27me3 levels at the bivalent Zbtb16/PLZF gene define a threshold enabling precise coupling of TCR specificity to lineage commitment.

  6. Immunophenotypic and antigen receptor gene rearrangement analysis in T cell neoplasia.

    PubMed Central

    Knowles, D. M.

    1989-01-01

    The author reviews the immunophenotypic profiles displayed by the major clinicopathologic categories of T cell neoplasia, the immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia, and the contributions made by antigen receptor gene rearrangement analysis to the understanding of T cell neoplasia. Neoplasms belonging to distinct clinicopathologic categories of T cell neoplasia often exhibit characteristic immunophenotypic profiles. Approximately 80% of lymphoblastic lymphomas and 20% of acute lymphoblastic leukemias express phenotypes consistent with prethymic and intrathymic stages of T cell differentiation, including intranuclear terminal deoxynucleotidyl transferase. Cutaneous T cell lymphomas of mycosis fungoides type usually express pan-T cell antigens CD2, CD5, and CD3, often lack the pan-T cell antigen CD7, and usually express the mature, peripheral helper subset phenotype, CD4+ CD8-. Cutaneous T cell lymphomas of nonmycosis fungoides type and peripheral T cell lymphomas often lack one or more pan-T cell antigens and, in addition, occasionally express the anomalous CD4+ CD8+ or CD4- CD8- phenotypes. T gamma-lymphoproliferative disease is divisable into two broad categories: those cases that are CD3 antigen positive and exhibit clonal T cell receptor beta chain (TCR-beta) gene rearrangements and those cases that are CD3 antigen negative and exhibit the TCR-beta gene germline configuration. Human T cell lymphotropic virus-I (HTLV-I) associated Japanese, Carribean, and sporadic adult T cell leukemia/lymphomas usually express pan-T cell antigens, the CD4+ CD8- phenotype, and various T cell-associated activation antigens, including the interleukin-2 receptor (CD25). Immunophenotypic criteria useful in the immunodiagnosis of T cell neoplasia include, in increasing order of utility, T cell predominance, T cell subset antigen restriction, anomalous T cell subset antigen expression, and deletion of one or more pan-T cell antigens. Only in

  7. Peripheral T Cell Survival Requires Continual Ligation of the T Cell Receptor to Major Histocompatibility Complex–Encoded Molecules

    PubMed Central

    Kirberg, Jörg; Berns, Anton; Boehmer, Harald von

    1997-01-01

    In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2b mice expressing a transgenic TCR restricted by I-Ed class II MHC molecules. After engraftment with Rag−/− H-2d fetal thymi, CD4+8− peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2b or H-2d haplotype, some of the latter being common cytokine receptor γ chain deficient to exclude rejection of H-2b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells. PMID:9334366

  8. Human Th17 cells share major trafficking receptors with both polarized effector T cells and FOXP3+ regulatory T cells.

    PubMed

    Lim, Hyung W; Lee, Jeeho; Hillsamer, Peter; Kim, Chang H

    2008-01-01

    It is a question of interest whether Th17 cells express trafficking receptors unique to this Th cell lineage and migrate specifically to certain tissue sites. We found several Th17 cell subsets at different developing stages in a human secondary lymphoid organ (tonsils) and adult, but not in neonatal, blood. These Th17 cell subsets include a novel in vivo-stimulated tonsil IL17+ T cell subset detected without any artificial stimulation in vitro. We investigated in depth the trafficking receptor phenotype of the Th17 cell subsets in tonsils and adult blood. The developing Th17 cells in tonsils highly expressed both Th1- (CCR2, CXCR3, CCR5, and CXCR6) and Th2-associated (CCR4) trafficking receptors. Moreover, Th17 cells share major non-lymphoid tissue trafficking receptors, such as CCR4, CCR5, CCR6, CXCR3, and CXCR6, with FOXP3+ T regulatory cells. In addition, many Th17 cells express homeostatic chemokine receptors (CD62L, CCR6, CCR7, CXCR4, and CXCR5) implicated in T cell migration to and within lymphoid tissues. Expression of CCR6 and CCR4 by some Th17 cells is not a feature unique to Th17 cells but shared with FOXP3+ T cells. Interestingly, the IL17+IFN-gamma+ Th17 cells have the features of both IL17-IFN-gamma+ Th1 and IL17+IFN-gamma- Th17 cells in expression of trafficking receptors. Taken together, our results revealed that Th17 cells are highly heterogeneous, in terms of trafficking receptors, and programmed to share major trafficking receptors with other T cell lineages. These findings have important implications in their distribution in the human body in relation to other regulatory T cell subsets.

  9. T Cell Receptor Signaling in the Control of Regulatory T Cell Differentiation and Function

    PubMed Central

    Li, Ming O.; Rudensky, Alexander Y.

    2016-01-01

    Regulatory T cells (TReg cells), a specialized T cell lineage, have a pivotal function in the control of self-tolerance and inflammatory responses. Recent studies have revealed a discrete mode of TCR signaling that regulates Treg cell differentiation, maintenance and function and that impacts on gene expression, metabolism, cell adhesion and migration of these cells. Here, we discuss the emerging understanding of TCR-guided differentiation of Treg cells in the context of their function in health and disease. PMID:27026074

  10. Enhancement of the interleukin 2 receptor expression on T cells by multiple B-lymphotropic lymphokines.

    PubMed

    Noma, T; Mizuta, T; Rosén, A; Hirano, T; Kishimoto, T; Honjo, T

    1987-07-01

    Three new human lymphokines, interleukin-5, BSF-2 and BSF-MP6, were shown to be active in the enhancement of the IL-2 receptor expression on T cells, although they do not stimulate growth of the T cells.

  11. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor.

    PubMed

    Schreiner, Jens; Thommen, Daniela S; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A; Umana, Pablo; Pisa, Pavel; von Bergwelt-Baildon, M; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-02-01

    T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1(hi) TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1(hi) TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1(int) and PD-1(neg) T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1(hi) expressing cells; in contrast, patients with abundance of PD-1(hi) expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1(hi) T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction.

  12. CTLA4 blockade broadens the peripheral T cell receptor repertoire

    PubMed Central

    Robert, Lidia; Tsoi, Jennifer; Wang, Xiaoyan; Emerson, Ryan; Homet, Blanca; Chodon, Thinle; Mok, Stephen; Huang, Rong Rong; Cochran, Alistair J.; Comin-Anduix, Begonya; Koya, Richard C.; Graeber, Thomas G.; Robins, Harlan; Ribas, Antoni

    2014-01-01

    Purpose To evaluate the immunomodulatory effects of CTLA-4 blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design We used next generation sequencing to study the complementarity determining region 3 (CDR3) from the rearranged T cell receptor (TCR) variable beta (V-beta) in PBMC of 21 patients, at baseline and 30–60 days after receiving tremelimumab. Results After receiving tremelimumab there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (p=0.01) and for Shannon index diversity (p=0.04). In comparison, serially collected PBMC from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over one year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared to patients without toxicity (p=0.05). No relevant differences were noted between clinical responders and non-responders. Conclusions CTLA4 blockade with tremelimumab diversifies the peripheral T cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. PMID:24583799

  13. Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

    PubMed

    Ahsan, Nagib; Salomon, Arthur R

    2017-01-01

    TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.

  14. Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy

    PubMed Central

    Liu, Lingfeng; Sommermeyer, Daniel; Cabanov, Alexandra; Kosasih, Paula; Hill, Tyler; Riddell, Stanley R

    2016-01-01

    The tactical introduction of Strep-tag II into synthetic antigen receptors provides engineered T cells with a marker for identification and rapid purification, and a functional element for selective antibody coated microbead-driven large-scale expansion. Such receptor designs can be applied to chimeric antigen receptors of different ligand specificities and costimulatory domains, and to T cell receptors to facilitate cGMP manufacturing of adoptive T cell therapies to treat cancer and other diseases. PMID:26900664

  15. Fetal liver T cell receptor gamma/delta+ T cells as cytotoxic T lymphocytes specific for maternal alloantigens

    PubMed Central

    1992-01-01

    We have established fetal liver-derived T cell receptor (TCR) gamma/delta+, CD3+ T cell lines that are cytotoxic for maternal T cells. Fetal liver-derived lymphoid progenitors yielded predominantly TCR-gamma/delta+ cell clusters when cultured on fetal bone marrow- derived stromal cells in the presence of a cytokine cocktail under magnetic force. These tightly adherent clusters were cloned by limiting dilution and the resulting cell lines analyzed for phenotype and function. Six of eight TCR-gamma/delta lines from 8-9.5-wk gestation fetuses were V delta 2+ as compared with zero of eight lines from later stages of gestation (10 and 15 wk), where all the lines were V delta 1+. In cytotoxicity assays, these TCR-gamma/delta+, CD3+, CD4-, and CD8+ or CD8- long-term cultured lymphoid cells (LLC) were killer cells active against the class I antigens on maternal T cells. Of the cell lines, the CD8+ TCR-gamma/delta+ LLC had the highest levels of killer activity. Thus fetal liver TCR-gamma/delta+ T cells may play a crucial role in protection against invading maternal T cells generated in the feto-maternal interaction. PMID:1535364

  16. The α4 Nicotinic Receptor Promotes CD4+ T-Cell Proliferation and a Helper T-Cell Immune Response

    PubMed Central

    Nordman, Jacob C.; Muldoon, Pretal; Clark, Sarah; Damaj, M. Imad

    2014-01-01

    Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell-based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4+CD3+CD4+) and show that this group of cells is responsive to sustained nicotine exposure. In the circulation, spleen, bone marrow, and thymus, we find that nicotine promotes an increase in CD3+CD4+ cells via its activation of the α4 nAChR and regulation of G protein subunit o, G protein regulated–inducer of neurite outgrowth, and CDC42 signaling within T cells. In particular, nicotine is found to promote a helper T cell 2 adaptive immunologic response within T cells that is absent in α4−/− mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T cells, possibly accounting for the effect of smoking on the immune system. PMID:24107512

  17. Linking the T cell receptor to the single cell transcriptome in antigen-specific human T cells.

    PubMed

    Eltahla, Auda A; Rizzetto, Simone; Pirozyan, Mehdi R; Betz-Stablein, Brigid D; Venturi, Vanessa; Kedzierska, Katherine; Lloyd, Andrew R; Bull, Rowena A; Luciani, Fabio

    2016-07-01

    Heterogeneity of T cells is a hallmark of a successful adaptive immune response, harnessing the vast diversity of antigen-specific T cells into a coordinated evolution of effector and memory outcomes. The T cell receptor (TCR) repertoire is highly diverse to account for the highly heterogeneous antigenic world. During the response to a virus multiple individual clones of antigen specific CD8+ (Ag-specific) T cells can be identified against a single epitope and multiple epitopes are recognised. Advances in single-cell technologies have provided the potential to study Ag-specific T cell heterogeneity at both surface phenotype and transcriptome levels, thereby allowing investigation of the diversity within the same apparent sub-population. We propose a new method (VDJPuzzle) to reconstruct the native TCRαβ from single cell RNA-seq data of Ag-specific T cells and then to link these with the gene expression profile of individual cells. We applied this method using rare Ag-specific T cells isolated from peripheral blood of a subject who cleared hepatitis C virus infection. We successfully reconstructed productive TCRαβ in 56 of a total of 63 cells (89%), with double α and double β in 18, and 7% respectively, and double TCRαβ in 2 cells. The method was validated via standard single cell PCR sequencing of the TCR. We demonstrate that single-cell transcriptome analysis can successfully distinguish Ag-specific T cell populations sorted directly from resting memory cells in peripheral blood and sorted after ex vivo stimulation. This approach allows a detailed analysis of the TCR diversity and its relationship with the transcriptional profile of different clones.

  18. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into.../057272 and foreign equivalents thereof entitled ``Anti-MAGE-A3 T cell receptors and related materials and... Patent Application No. PCT/US2011/051537 and foreign equivalents thereof entitled ``Anti-SSX-2 T...

  19. Apoptosis in a Fas-resistant, T-cell receptor-sensitive human leukaemic T-cell clone.

    PubMed Central

    Delehanty, L L; Payne, J A; Farrow, S N; Brown, R; Champion, B R

    1997-01-01

    The Fas (CD95) antigen plays a key role in regulating T-cell activation and survival. We have generated a Fas-resistant subclone of the human T-cell leukaemia line, H9, which is still able to undergo apoptosis in response to T-cell receptor ligation. Molecular analyses revealed that resistance to Fas-mediated apoptosis was due to a heterozygous mutation in the death domain of the Fas gene which generates a stop codon, and thus encodes a truncated Fas molecule. Fas ligation was able to induce apoptosis in the presence of cycloheximide, indicating that the mutant Fas molecule retained some signalling capability, which is death-domain independent. These cells will provide a useful tool for dissecting the complexities of Fas signalling pathways. Images Figure 5 PMID:9155645

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

    NASA Astrophysics Data System (ADS)

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

    2009-08-01

    T lymphocytes (T cells) orchestrate adaptive immune responses upon activation. T-cell activation requires sufficiently strong binding of T-cell receptors on their surface to short peptides (p) derived from foreign proteins, which are bound to major histocompatibility 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 T-cell receptors (which enable its recognition functions).

  1. Sustained interactions between T cell receptors and antigens promote the differentiation of CD4⁺ memory T cells.

    PubMed

    Kim, Chulwoo; Wilson, Theodore; Fischer, Kael F; Williams, Matthew A

    2013-09-19

    During CD4⁺ T cell activation, T cell receptor (TCR) signals impact T cell fate, including recruitment, expansion, differentiation, trafficking, and survival. To determine the impact of TCR signals on the fate decision of activated CD4⁺ T cells to become end-stage effector or long-lived memory T helper 1 (Th1) cells, we devised a deep-sequencing-based approach that allowed us to track the evolution of TCR repertoires after acute infection. The transition of effector Th1 cells into the memory pool was associated with a significant decrease in repertoire diversity, and the major histocompatibility complex (MHC) class II tetramer off rate, but not tetramer avidity, was a key predictive factor in the representation of individual clonal T cell populations at the memory stage. We conclude that stable and sustained interactions with antigens during the development of Th1 responses to acute infection are a determinative factor in promoting the differentiation of Th1 memory cells.

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

    PubMed Central

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.; Dustin, Michael L.; Groves, Jay

    2011-01-01

    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. Here 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. This threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower. PMID:21576490

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

    SciTech Connect

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.; Dustin, Michael L.; Groves, Jay

    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.

  4. Vav1 regulates T cell activation through a feedback mechanism and crosstalk between the T cell receptor and CD28

    PubMed Central

    Helou, Ynes A.; Petrashen, Anna P.; Salomon, Arthur R.

    2015-01-01

    Vavl, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T cell receptor (TCR) signaling cascade, is rapidly tyrosine phosphorylated in response to T cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca2+ responses, and actin cytoskeleton regulation, however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck, and ZAP-70 is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways. PMID:26043137

  5. Engineered secreted T-cell receptor alpha beta heterodimers.

    PubMed Central

    Grégoire, C; Rebaï, N; Schweisguth, F; Necker, A; Mazza, G; Auphan, N; Millward, A; Schmitt-Verhulst, A M; Malissen, B

    1991-01-01

    We have produced a soluble form of a mouse alpha beta T-cell antigen receptor (TCR) by shuffling its variable (V) and constant (C) domains to the C region of an immunoglobulin kappa light chain. These chimeric molecules composed of V alpha C alpha C kappa and V beta C beta C kappa chains were efficiently secreted (up to 1 micrograms/ml) by transfected myeloma cells as noncovalent heterodimers of about 95-kDa molecular mass. In the absence of direct binding measurement, we have refined the epitopic analysis of the soluble V alpha C alpha C kappa-V beta C beta C kappa dimers and shown that they react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TCR alpha and beta chains. Conversely, we have raised three distinct monoclonal antibodies against the soluble TCR heterodimers and shown that they recognize surface-expressed TCRs. Two of these antibodies were found to react specifically with the products of the V alpha 2 (V delta 8) and V beta 2 gene segments, respectively. When considered together, these data suggest that these soluble TCR molecules are folded in a conformation indistinguishable from that which they assume at the cell surface. Images PMID:1716770

  6. Human T-cell receptor variable gene segment families

    SciTech Connect

    Arden, B.; Kabelitz, D.; Clark, S.P.; Mak, T.W.

    1995-10-01

    Multiple DNA and protein sequence alignments have been constructed for the human T-cell receptor {alpha}/{delta}, {beta}, and {gamma} (TCRA/D, B, and G) variable (V) gene segments. The traditional classification into subfamilies was confirmed using a much larger pool of sequences. For each sequence, a name was derived which complies with the standard nomenclature. The traditional numbering of V gene segments in the order of their discovery was continued and changed when in conflict with names of other segments. By discriminating between alleles at the same locus versus genes from different loci, we were able to reduce the number of more than 150 different TCRBV sequences in the database to a repertoire of only 47 functional TCRBV gene segments. An extension of this analysis to the over 100 TCRAV sequences results in a predicted repertoire of 42 functional TCRAV gene segments. Our alignment revealed two residues that distinguish between the highly homologous V{delta} and V{alpha}, one at a site that in V{sub H} contacts the constant region, the other at the interface between immunoglobulin V{sub H} and V{sub L}. This site may be responsible for restricted pairing between certain V{delta} and V{gamma} chains. On the other hand, V{beta} and V{gamma} appear to be related by the fact that their CDR2 length is increased by four residues as compared with that of V{alpha}/{delta} peptides. 150 refs., 12 figs., 5 tabs.

  7. Dopamine and T cells: dopamine receptors and potent effects on T cells, dopamine production in T cells, and abnormalities in the dopaminergic system in T cells in autoimmune, neurological and psychiatric diseases.

    PubMed

    Levite, M

    2016-01-01

    Dopamine, a principal neurotransmitter, deserves upgrading to 'NeuroImmunotransmitter' thanks to its multiple, direct and powerful effects on most/all immune cells. Dopamine by itself is a potent activator of resting effector T cells (Teffs), via two independent ways: direct Teffs activation, and indirect Teffs activation by suppression of regulatory T cells (Tregs). The review covers the following findings: (i) T cells express functional dopamine receptors (DRs) D1R-D5R, but their level and function are dynamic and context-sensitive, (ii) DR membranal protein levels do not necessarily correlate with DR mRNA levels, (iii) different T cell types/subtypes have different DR levels and composition and different responses to dopamine, (iv) autoimmune and pro-inflammatory T cells and T cell leukaemia/lymphoma also express functional DRs, (v) dopamine (~10(-8) M) activates resting/naive Teffs (CD8(+) >CD4(+) ), (vi) dopamine affects Th1/Th2/Th17 differentiation, (vii) dopamine inhibits already activated Teffs (i.e. T cells that have been already activated by either antigen, mitogen, anti-CD3 antibodies cytokines or other molecules), (viii) dopamine inhibits activated Tregs in an autocrine/paracrine manner. Thus, dopamine 'suppresses the suppressors' and releases the inhibition they exert on Teffs, (ix) dopamine affects intracellular signalling molecules and cascades in T cells (e.g. ERK, Lck, Fyn, NF-κB, KLF2), (x) T cells produce dopamine (Tregs>Teffs), can release dopamine, mainly after activation (by antigen, mitogen, anti-CD3 antibodies, PKC activators or other), uptake extracellular dopamine, and most probably need dopamine, (xi) dopamine is important for antigen-specific interactions between T cells and dendritic cells, (xii) in few autoimmune diseases (e.g. multiple sclerosis/SLE/rheumatoid arthritis), and neurological/psychiatric diseases (e.g. Parkinson disease, Alzheimer's disease, Schizophrenia and Tourette), patient's T cells seem to have abnormal DRs

  8. Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells

    PubMed Central

    1992-01-01

    The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double- negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E- expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection. PMID:1512537

  9. CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development

    PubMed Central

    1996-01-01

    CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti- CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3- mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal. PMID:8760821

  10. Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

    PubMed Central

    Deniger, Drew C; Switzer, Kirsten; Mi, Tiejuan; Maiti, Sourindra; Hurton, Lenka; Singh, Harjeet; Huls, Helen; Olivares, Simon; Lee, Dean A; Champlin, Richard E; Cooper, Laurence JN

    2013-01-01

    Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR+γδ T cells were expanded on CD19+ artificial antigen-presenting cells (aAPC), which resulted in >109 CAR+γδ T cells from <106 total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR+γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19+ tumor cell lines compared with CARnegγδ T cells. CD19+ leukemia xenografts in mice were reduced with CAR+γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells. PMID:23295945

  11. Interleukin-1 receptors are differentially expressed in normal and psoriatic T cells.

    PubMed

    Bebes, Attila; Kovács-Sólyom, Ferenc; Prihoda, Judit; Kui, Róbert; Kemény, Lajos; Gyulai, Rolland

    2014-01-01

    This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4(+)CD25(-) effector and CD4(+)CD25(+)CD127(low) regulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis.

  12. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells

    PubMed Central

    Boddupalli, Chandra Sekhar; Bar, Noffar; Kadaveru, Krishna; Krauthammer, Michael; Pornputtapong, Natopol; Ariyan, Stephan; Narayan, Deepak; Kluger, Harriet; Deng, Yanhong; Verma, Rakesh; Das, Rituparna; Bacchiocchi, Antonella; Halaban, Ruth; Sznol, Mario; Dhodapkar, Madhav V.; Dhodapkar, Kavita M.

    2016-01-01

    Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site. PMID:28018970

  13. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    NASA Astrophysics Data System (ADS)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  14. Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE).

    PubMed

    Kumar, V; Stellrecht, K; Sercarz, E

    1996-11-01

    T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.

  15. A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development

    PubMed Central

    Mothe-Satney, Isabelle; Murdaca, Joseph; Sibille, Brigitte; Rousseau, Anne-Sophie; Squillace, Raphaëlle; Le Menn, Gwenaëlle; Rekima, Akila; Larbret, Frederic; Pelé, Juline; Verhasselt, Valérie; Grimaldi, Paul A.; Neels, Jaap G.

    2016-01-01

    Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor β (PPARβ) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARβ activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4−CD8− double-negative stage 4 (DN4) thymocytes. These results support a model where PPARβ activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αβ T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells. PMID:27680392

  16. Autoreactive T cells in a partially humanized murine model of T1D.

    PubMed

    Gebe, John A; Falk, Ben; Unrath, Kellee; Nepom, Gerald T

    2007-04-01

    Glutamic acid decarboxylase (GAD65) and insulin are implicated as target antigens in the pathogenesis of human diabetes through correlative measurements of humoral and cellular reactivity to them in diabetics and at-risk diabetic individuals. Recently, an age-dependent loss of tolerance to one of several naturally processed epitopes of GAD65 (555-567) has been observed to precede diabetes in diabetes-prone mice transgenic for diabetes-correlated human class II genes. Extended studies in these mice (RIP-B7/DR0404) now show that tolerance is maintained to another DR4-restricted naturally processed region within GAD65. While tolerance is lost to GAD65 (555-567) in B7/DR0404 mice prior to diabetes, these mice remain T cell-tolerant to GAD65 (273-286). Prediabetes loss of tolerance to GAD65 (555-567) has now been shown to correlate with an impaired response to exogenous glucose in an intraperitoneal (i.p.) glucose tolerance test. In addition, these mice exhibit a T cell response to insulin A(6-21) at the hyperglycemic state. Investigating a possible cause-and-effect relationship between T cell reactivity to GAD65 and diabetes pathogenesis, GAD65 (555-567) T cell receptor (TcR) transgenic mice have been generated and future work is aimed at understanding the importance of T cell GAD65 reactivity and its role in diabetes progression.

  17. Secretion of a chimeric T-cell receptor-immunoglobulin protein.

    PubMed Central

    Gascoigne, N R; Goodnow, C C; Dudzik, K I; Oi, V T; Davis, M M

    1987-01-01

    To produce sufficient quantities of soluble T-cell receptor protein for detailed biochemical and biophysical analyses we have explored the use of immunoglobulin--T-cell receptor gene fusions. In this report we describe a chimeric gene construct containing a T-cell receptor alpha-chain variable (V) domain and the constant (C) region coding sequences of an immunoglobulin gamma 2a molecule. Cells transfected with the chimeric gene synthesize a stable protein product that expresses immunoglobulin and T-cell receptor antigenic determinants as well as protein A binding sites. We show that the determinant recognized by the anticlonotypic antibody A2B4.2 resides on the V alpha domain of the T-cell receptor. The chimeric protein associates with a normal lambda light chain to form an apparently normal tetrameric (H2L2, where H = heavy and L = light) immunoglobulin molecule that is secreted. Also of potential significance is the fact that a T-cell receptor V beta gene in the same construct is neither assembled nor secreted with the lambda light chain, and when expressed with a C kappa region it does not assemble with the chimeric V alpha C gamma 2a protein mentioned above. This indicates that not all T-cell receptor V regions are similar enough to immunoglobulin V regions for them to be completely interchangeable. Images PMID:3472243

  18. CD8(+) T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing.

    PubMed

    Schneider-Hohendorf, Tilman; Mohan, Hema; Bien, Christian G; Breuer, Johanna; Becker, Albert; Görlich, Dennis; Kuhlmann, Tanja; Widman, Guido; Herich, Sebastian; Elpers, Christiane; Melzer, Nico; Dornmair, Klaus; Kurlemann, Gerhard; Wiendl, Heinz; Schwab, Nicholas

    2016-04-04

    Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8(+) T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vβ genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8(+) lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS.

  19. Avidity of human T cell receptor engineered CD4+ T cells drives T-helper differentiation fate

    PubMed Central

    Adair, Patrick; Kim, Yong Chan; Pratt, Kathleen P.; Scott, David W.

    2016-01-01

    The role of the T cell receptor (TCR) in antigen recognition and activation of T lymphocytes is well established. However, how the TCR affects T-helper differentiation/skewing is less well understood, particularly for human CD4+ (CD4) T cell subsets. Here we investigate the role of TCR specific antigen avidity in differentiation and maintenance of human Th1, Th2 and Th17 subsets. Two human TCRs, both specific for the same peptide antigen but with different avidities, were cloned and expressed in human CD4 T cells. These TCR engineered cells were then stimulated with specific antigen in unskewed and T-helper skewed conditions. We show that TCR avidity can control the percentage of IL-4 and IFN-γ co-expression in unskewed TCR engineered cells, that effector function can be maintained in a TCR avidity-dependent manner in skewed TCR engineered cells, and that increased TCR avidity can accelerate Th1 skewing of TCR engineered cells. PMID:26653006

  20. CD8+ T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing

    PubMed Central

    Schneider-Hohendorf, Tilman; Mohan, Hema; Bien, Christian G.; Breuer, Johanna; Becker, Albert; Görlich, Dennis; Kuhlmann, Tanja; Widman, Guido; Herich, Sebastian; Elpers, Christiane; Melzer, Nico; Dornmair, Klaus; Kurlemann, Gerhard; Wiendl, Heinz; Schwab, Nicholas

    2016-01-01

    Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8+ T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vβ genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8+ lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS. PMID:27040081

  1. T cell receptor-dependent tyrosine phosphorylation of beta2-chimaerin modulates its Rac-GAP function in T cells.

    PubMed

    Siliceo, María; Mérida, Isabel

    2009-04-24

    The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported beta2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of beta2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of beta2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of beta2-chimaerin and identifies a novel mechanism for its inactivation.

  2. T Cell Mineralocorticoid Receptor Controls Blood Pressure by Regulating Interferon Gamma.

    PubMed

    Sun, Xue Nan; Li, Chao; Liu, Yuan; Du, Lin-Juan; Zeng, Meng-Ru; Zheng, Xiao Jun; Zhang, Wu Chang; Liu, Yan; Zhu, Mingjiang; Kong, Deping; Zhou, Li; Lu, Limin; Shen, Zhu-Xia; Yi, Yi; Du, Lili; Qin, Mu; Liu, Xu; Hua, Zichun; Sun, Shuyang; Yin, Huiyong; Zhou, Bin; Yu, Ying; Zhang, Zhiyuan; Duan, Sheng-Zhong

    2017-03-15

    Rationale: Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T cell-derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T cell MR in blood pressure (BP) regulation has not been elucidated. Objective: We aim to determine the role of T cell MR in BP regulation and to explore the mechanism. Methods and Results: Using T cell MR knockout (TMRKO) mouse in combination with angiotensin II (AngII)-induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP, and attenuated renal and vascular damage. Flow cytometric analysis showed that TMRKO mitigated AngII-induced accumulation of interferon-gamma (IFNγ)-producing T cells, particularly CD8(+) population, in both kidneys and aortas. Similarly, eplerenone attenuated AngII-induced elevation of BP and accumulation of IFNγ-producing T cells in wild type mice. In cultured CD8(+) T cells, TMRKO suppressed IFNγ expression whereas T cell MR overexpression and aldosterone both enhanced IFNγ expression. At the molecular level, MR interacted with nuclear factor of activated T-cells 1 (NFAT1) and activator protein-1 (AP-1) in T cells. Finally, T cell MR overexpressing mice manifested more elevated BP compared to control mice after AngII infusion and such difference was abolished by IFNγ-neutralizing antibodies. Conclusions: MR may interact with NFAT1 and AP-1 to control IFNγ in T cells, and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment.

  3. Identifying Individual T Cell Receptors of Optimal Avidity for Tumor Antigens

    PubMed Central

    Hebeisen, Michael; Allard, Mathilde; Gannon, Philippe O.; Schmidt, Julien; Speiser, Daniel E.; Rufer, Nathalie

    2015-01-01

    Cytotoxic T cells recognize, via their T cell receptors (TCRs), small antigenic peptides presented by the major histocompatibility complex (pMHC) on the surface of professional antigen-presenting cells and infected or malignant cells. The efficiency of T cell triggering critically depends on TCR binding to cognate pMHC, i.e., the TCR–pMHC structural avidity. The binding and kinetic attributes of this interaction are key parameters for protective T cell-mediated immunity, with stronger TCR–pMHC interactions conferring superior T cell activation and responsiveness than weaker ones. However, high-avidity TCRs are not always available, particularly among self/tumor antigen-specific T cells, most of which are eliminated by central and peripheral deletion mechanisms. Consequently, systematic assessment of T cell avidity can greatly help distinguishing protective from non-protective T cells. Here, we review novel strategies to assess TCR–pMHC interaction kinetics, enabling the identification of the functionally most-relevant T cells. We also discuss the significance of these technologies in determining which cells within a naturally occurring polyclonal tumor-specific T cell response would offer the best clinical benefit for use in adoptive therapies, with or without T cell engineering. PMID:26635796

  4. NKG2D receptor regulates human effector T-cell cytokine production

    PubMed Central

    Barber, Amorette

    2011-01-01

    Although innate immune signals shape the activation of naive T cells, it is unclear how innate signals influence effector T-cell function. This study determined the effects of stimulating the NKG2D receptor in conjunction with the TCR on human effector CD8+ T cells. Stimulation of CD8+ T cells through CD3 and NKG2D simultaneously or through a chimeric NKG2D receptor, which consists of NKG2D fused to the intracellular region of CD3ζ, activated β-catenin and increased expression of β-catenin–induced genes, whereas T cells stimulated through the TCR or a combination of the TCR and CD28 did not. Activation by TCR and NKG2D prevented expression and production of anti-inflammatory cytokines IL-10, IL-9, IL-13, and VEGF-α in a β-catenin– and PPARγ- dependent manner. NKG2D stimulation also modulated the cytokine secretion of T cells activated simultaneously through CD3 and CD28. These data indicate that activating CD8+ T cells through the NKG2D receptor along with the TCR modulates signal transduction and the production of anti-inflammatory cytokines. Thus, human effector T cells alter their function depending on which innate receptors are engaged in conjunction with the TCR complex. PMID:21518928

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

    Cancer.gov

    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

  6. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

    PubMed Central

    Fraietta, Joseph A.; Beckwith, Kyle A.; Patel, Prachi R.; Ruella, Marco; Zheng, Zhaohui; Barrett, David M.; Lacey, Simon F.; Melenhorst, Jan Joseph; McGettigan, Shannon E.; Cook, Danielle R.; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B.; Cogdill, Alexandria P.; Gill, Saar; Porter, David L.; Woyach, Jennifer A.; Long, Meixiao; Johnson, Amy J.; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L.; June, Carl H.; Byrd, John C.

    2016-01-01

    Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. PMID:26813675

  7. Different Levels of T-Cell Receptor Triggering Induce Distinct Functions in Hepatitis B and Hepatitis C Virus-Specific Human CD4+ T-Cell Clones

    PubMed Central

    Diepolder, Helmut M.; Gruener, Norbert H.; Gerlach, J. Tilman; Jung, Maria-Christina; Wierenga, Eddy A.; Pape, Gerd R.

    2001-01-01

    CD4+ T cells play a major role in the host defense against viruses and intracellular microbes. During the natural course of such an infection, specific CD4+ T cells are exposed to a wide range of antigen concentrations depending on the body compartment and the stage of disease. While epitope variants trigger only subsets of T-cell effector functions, the response of virus-specific CD4+ T cells to various concentrations of the wild-type antigen has not been systematically studied. We stimulated hepatitis B virus core- and hepatitis C virus NS3-specific CD4+ T-cell clones which had been isolated from patients with acute hepatitis during viral clearance with a wide range of specific antigen concentrations and determined the phenotypic changes and the induction of T-cell effector functions in relation to T-cell receptor internalization. A low antigen concentration induced the expression of T-cell activation markers and adhesion molecules in CD4+ T-cell clones in the absence of cytokine secretion and proliferation. The expression of CD25, HLA-DR, CD69, and intercellular cell adhesion molecule 1 increased as soon as T-cell receptor internalization became detectable. A 30- to 100-fold-higher antigen concentration, corresponding to the internalization of 20 to 30% of T-cell receptor molecules, however, was required for the induction of proliferation as well as for gamma interferon and interleukin-4 secretion. These data indicate that virus-specific CD4+ T cells can respond to specific antigen in a graded manner depending on the antigen concentration, which may have implications for a coordinate regulation of specific CD4+ T-cell responses. PMID:11483723

  8. Improving Therapy of Chronic Lymphocytic Leukemia (CLL) with Chimeric Antigen Receptor (CAR) T Cells

    PubMed Central

    Fraietta, Joseph A.; Schwab, Robert D.; Maus, Marcela V.

    2016-01-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically-engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and re-directing the immune system against cancer. This review will briefly summarize T cell therapies in development for CLL disease. We discuss the role of T cell function and phenotype, T cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

  9. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    PubMed Central

    Cartellieri, Marc; Bachmann, Michael; Feldmann, Anja; Bippes, Claudia; Stamova, Slava; Wehner, Rebekka; Temme, Achim; Schmitz, Marc

    2010-01-01

    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. PMID:20467460

  10. Chimeric antigen receptor T-cell neuropsychiatric toxicity in acute lymphoblastic leukemia.

    PubMed

    Prudent, Vasthie; Breitbart, William S

    2017-01-04

    Chimeric antigen receptor T cells are used in the treatment of B-cell leukemias. Common chimeric antigen receptor T-cell toxicities can range from mild flu-like symptoms, such as fever and myalgia, to a more striking neuropsychiatric toxicity that can present as discrete neurological symptoms and delirium. We report here two cases of chimeric antigen receptor T-cell neuropsychiatric toxicity, one who presented as a mild delirium and aphasia that resolved without intervention, and one who presented with delirium, seizures, and respiratory insufficiency requiring intensive treatment. The current literature on the treatment and proposed mechanisms of this clinically challenging chimeric antigen receptor T-cell complication is also presented.

  11. Regulator T cells: specific for antigen and/or antigen receptors?

    PubMed

    Rubin, B; de Durana, Y Diaz; Li, N; Sercarz, E E

    2003-05-01

    Adaptive immune responses are regulated by many different molecular and cellular effectors. Regulator T cells are coming to their rights again, and these T cells seem to have ordinary alpha/beta T-cell receptors (TCRs) and to develop in the thymus. Autoimmune responses are tightly regulated by such regulatory T cells, a phenomenon which is beneficial to the host in autoimmune situations. However, the regulation of autoimmune responses to tumour cells is harmful to the host, as this regulation delays the defence against the outgrowth of neoplastic cells. In the present review, we discuss whether regulatory T cells are specific for antigen and/or for antigen receptors. Our interest in these phenomena comes from the findings that T cells produce many more TCR-alpha and TCR-beta chains than are necessary for surface membrane expression of TCR-alphabeta heterodimers with CD3 complexes. Excess TCR chains are degraded by the proteasomes, and TCR peptides thus become available to the assembly pathway of major histocompatibility complex class I molecules. Consequently, do T cells express two different identification markers on the cell membrane, the TCR-alphabeta clonotype for recognition by B-cell receptors and clonotypic TCR-alphabeta peptides for recognition by T cells?

  12. Chimeric Antigen Receptor T Cells against CD19 for Multiple Myeloma

    PubMed Central

    Garfall, Alfred L.; Maus, Marcela V.; Hwang, Wei-Ting; Lacey, Simon F.; Mahnke, Yolanda D.; Melenhorst, J. Joseph; Zheng, Zhaohui; Vogl, Dan T.; Cohen, Adam D.; Weiss, Brendan M.; Dengel, Karen; Kerr, Naseem D.S.; Bagg, Adam; Levine, Bruce L.; June, Carl H.; Stadtmauer, Edward A.

    2015-01-01

    SUMMARY A patient with refractory multiple myeloma received an infusion of CTL019 cells, a cellular therapy consisting of autologous T cells transduced with an anti-CD19 chimeric antigen receptor, after myeloablative chemotherapy (melphalan, 140 mg per square meter of body-surface area) and autologous stem-cell transplantation. Four years earlier, autologous transplantation with a higher melphalan dose (200 mg per square meter) had induced only a partial, transient response. Autologous transplantation followed by treatment with CTL019 cells led to a complete response with no evidence of progression and no measurable serum or urine monoclonal protein at the most recent evaluation, 12 months after treatment. This response was achieved despite the absence of CD19 expression in 99.95% of the patient’s neoplastic plasma cells. (Funded by Novartis and others; ClinicalTrials.gov number, NCT02135406.) PMID:26352815

  13. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

    PubMed Central

    1996-01-01

    The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck. PMID:8642247

  14. Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street.

    PubMed

    Barrett, David M; Grupp, Stephan A; June, Carl H

    2015-08-01

    The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT.

  15. Interleukin-1 Receptors Are Differentially Expressed in Normal and Psoriatic T Cells

    PubMed Central

    Kovács-Sólyom, Ferenc; Prihoda, Judit; Kui, Róbert; Kemény, Lajos; Gyulai, Rolland

    2014-01-01

    This study was carried out to examine the possible role of interleukin-1 (IL-1) in the functional insufficiency of regulatory T cells in psoriasis, by comparing the expression of IL-1 receptors on healthy control and psoriatic T cells. Patients with moderate-to-severe chronic plaque psoriasis and healthy volunteers, matched in age and sex, were selected for all experiments. CD4+CD25− effector and CD4+CD25+CD127low regulatory T cells were separated and used for the experiments. Expression of the mRNA of IL-1 receptors (IL-1R1, IL-1R2, and sIL-1R2) was determined by quantitative real-time RT-PCR. Cell surface IL-1 receptor expression was assessed by flow cytometry. Relative expression of the signal transmitting IL-1 receptor type 1 (IL-1R1) mRNA is higher in resting psoriatic effector and regulatory T cells, and activation induces higher IL-1R1 protein expression in psoriatic T cells than in healthy cells. Psoriatic regulatory and effector T cells express increased mRNA levels of the decoy IL-1 receptors (IL-1R2 and sIL-1R2) upon activation compared to healthy counterparts. Psoriatic T cells release slightly more sIL-1R2 into their surrounding than healthy T cells. In conclusion, changes in the expression of IL-1 receptors in psoriatic regulatory and effector T cells could contribute to the pathogenesis of psoriasis. PMID:24665164

  16. Altered expression of chemokine receptor CXCR5 on T cells of myasthenia gravis patients.

    PubMed

    Saito, Ryuji; Onodera, Hiroshi; Tago, Hideaki; Suzuki, Yasushi; Shimizu, Masayuki; Matsumura, Yuji; Kondo, Takashi; Itoyama, Yasuto

    2005-12-30

    Myasthenia gravis (MG) is characterized by the T cell-dependent production of anti-acetylcholine receptor (AChR) antibodies. The chemokine receptor CXCR5 regulates lymphocyte migration and is expressed on a subset of CD4+ T cells named follicular helper T cells (T(FH)), the key modulators of antibody production by B cells. We studied the frequency of CXCR5-positive lymphocytes in the peripheral blood of MG patients before and after therapy (thymectomy plus glucocorticoid). Before therapy, the MG patients showed a significantly higher frequency of CXCR5+ CD4+ T cells in the peripheral blood compared with the control group, while no significant difference in the percentages of CXCR5+ CD4+ T cells was observed between the patients of the hyperplasia group and those of the thymoma group. The CXCR5+ CD4+ T cell frequency correlated with the disease severity. The CXCR5+ CD4+ T cell frequency of MG patients positive for other autoantibodies together with anti-AChR antibodies was significantly higher than in those having only anti-AChR antibodies. After therapy, the CXCR5+ CD4+ T cell percentage decreased gradually to the control level with a significant inverse correlation between the CXCR5+ CD4+ T cell frequency and duration after the initiation of MG therapy. The CXCR5+ CD4+ T cell populations in the hyperplastic thymuses and thymomas were not significantly different from those in the control thymuses. These results suggest that CXCR5+ CD4+ T cells play an important role in the disease activity of MG and that some MG patients have a systemic abnormality in T cell-dependent antibody production.

  17. Disruption of PTH Receptor 1 in T Cells Protects against PTH-Induced Bone Loss

    PubMed Central

    Tawfeek, Hesham; Bedi, Brahmchetna; Li, Jau-Yi; Adams, Jonathan; Kobayashi, Tatsuya; Weitzmann, M. Neale; Kronenberg, Henry M.; Pacifici, Roberto

    2010-01-01

    Background Hyperparathyroidism in humans and continuous parathyroid hormone (cPTH) treatment in mice cause bone loss by regulating the production of RANKL and OPG by stromal cells (SCs) and osteoblasts (OBs). Recently, it has been reported that T cells are required for cPTH to induce bone loss as the binding of the T cell costimulatory molecule CD40L to SC receptor CD40 augments SC sensitivity to cPTH. However it is unknown whether direct PTH stimulation of T cells is required for cPTH to induce bone loss, and whether T cells contribute to the bone catabolic activity of PTH with mechanisms other than induction of CD40 signaling in SCs. Methodology/Principal Findings Here we show that silencing of PTH receptor 1 (PPR) in T cells blocks the bone loss and the osteoclastic expansion induced by cPTH, thus demonstrating that PPR signaling in T cells is central for PTH-induced reduction of bone mass. Mechanistic studies revealed that PTH activation of the T cell PPR stimulates T cell production of the osteoclastogenic cytokine tumor necrosis factor α (TNF). Attesting to the relevance of this effect, disruption of T cell TNF production prevents PTH-induced bone loss. We also show that a novel mechanism by which TNF mediates PTH induced osteoclast formation is upregulation of CD40 expression in SCs, which increases their RANKL/OPG production ratio. Conclusions/Significance These findings demonstrate that PPR signaling in T cells plays an essential role in PTH induced bone loss by promoting T cell production of TNF. A previously unknown effect of TNF is to increase SC expression of CD40, which in turn increases SC osteoclastogenic activity by upregulating their RANKL/OPG production ratio. PPR-dependent stimulation of TNF production by T cells and the resulting TNF regulation of CD40 signaling in SCs are potential new therapeutic targets for the bone loss of hyperparathyroidism. PMID:20808842

  18. Selective manipulation of the human T-cell receptor repertoire expressed by thymocytes in organ culture.

    PubMed Central

    Merkenschlager, M; Fisher, A G

    1992-01-01

    A recently described organ culture system for human thymocytes is shown to support the generation of a diverse T-cell receptor repertoire in vitro: thymocytes of the alpha beta lineage, including representatives of the V beta families 5.2/5.3, 6.7, and 8, accounted for the majority of T-cell receptor-positive cells throughout a 3-week culture period. Thymocytes bearing gamma delta receptors were also identified, particularly among the CD4 CD8 double-negative subset. The T-cell receptor repertoire expressed in organ culture responded to experimental manipulation with staphylococcal enterotoxins. Staphylococcal enterotoxin D (a powerful activator of human peripheral T cells expressing V beta 5.2/5.3 receptors) caused a marked reduction of V beta 5.2/5.3 expression, as determined with the V beta-specific antibody 42/1C1. Evidence is presented that this loss of V beta 5.2/5.3 expression resulted from the selective deletion of activated thymocytes by apoptosis, in concert with T-cell receptor modulation. These effects of staphylococcal enterotoxin D were specific (since staphylococcal enterotoxin E did not influence V beta 5.2/5.3 expression) and V beta-selective (since expression of V beta 6.7 remained unaffected by staphylococcal enterotoxin D). On the basis of these observations, we suggest that thymic organ culture provides a powerful approach to study the generation of the human T-cell repertoire. Images PMID:1584760

  19. Interleukin-21 Receptor Gene Induction in Human T Cells Is Mediated by T-Cell Receptor-Induced Sp1 Activity

    PubMed Central

    Wu, Zheng; Kim, Hyoung-Pyo; Xue, Hai-Hui; Liu, Hong; Zhao, Keji; Leonard, Warren J.

    2005-01-01

    Interleukin-21 (IL-21) plays important roles in regulating the immune response. IL-21 receptor (IL-21R) mRNA is expressed at a low level in human resting T cells but is rapidly induced by mitogenic stimulation. We now investigate the basis for IL21R gene regulation in T cells. We found that the −80 to −20 region critically regulates IL-21R promoter activity and corresponds to a major DNase I-hypersensitive site. Electrophoretic mobility shift assays, DNA affinity chromatography followed by mass spectrometry, and chromatin immunoprecipitation assays revealed that Sp1 binds to this region in vitro and in vivo. Moreover, mutation of the Sp1 motif markedly reduced IL-21R promoter activity, and Sp1 small interfering RNAs effectively diminished IL-21R expression in activated T cells. Interestingly, upon T-cell receptor (TCR) stimulation, T cells increased IL-21R expression and Sp1 protein levels while decreasing Sp1 phosphorylation. Moreover, phosphatase inhibitors that increased phosphorylation of Sp1 diminished IL-21R transcription. These data indicate that TCR-induced IL-21R expression is driven by TCR-mediated augmentation of Sp1 protein levels and may partly depend on the dephosphorylation of Sp1. PMID:16260592

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

    PubMed Central

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

    2015-01-01

    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. PMID:25753381

  1. Accumulation of cytolytic CD8{sup +} T cells in B16-melanoma and proliferation of mature T cells in TIS21-knockout mice after T cell receptor stimulation

    SciTech Connect

    Ryu, Min Sook; Woo, Min-Yeong; Kwon, Daeho; Hong, Allen E.; Song, Kye Yong; Park, Sun; Lim, In Kyoung

    2014-10-01

    In vivo and in vitro effects of TIS21 gene on the mature T cell activation and antitumor activities were explored by employing MO5 melanoma orthograft and splenocytes isolated from the TIS21-knockout (KO) mice. Proliferation and survival of mature T cells were significantly increased in the KO than the wild type (WT) cells, indicating that TIS21 inhibits the rate of mature T cell proliferation and its survival. In MO5 melanoma orthograft model, the KO mice recruited much more CD8{sup +} T cells into the tumors at around day 14 after tumor cell injection along with reduced tumor volumes compared with the WT. The increased frequency of granzyme B{sup +} CD8{sup +} T cells in splenocytes of the KO mice compared with the WT may account for antitumor-immunity of TIS21 gene in the melanoma orthograft. In contrast, reduced frequencies of CD107a{sup +} CD8{sup +} T cells in the splenocytes of KO mice may affect the loss of CD8{sup +} T cell infiltration in the orthograft at around day 19. These results indicate that TIS21 exhibits antiproliferative and proapoptotic effects in mature T cells, and differentially affects the frequencies of granzyme B{sup +} CD8{sup +} T-cells and CD107a{sup +} CD8{sup +} T-cells, thus transiently regulating in vivo anti-tumor immunity. - Highlights: • Constitutive expression of TIS21 in splenocytes and upregulation by TCR stimulation. • Proliferation of mature T-cells in spleen of TIS21KO mice after TCR stimulation. • Inhibition of cell death in mature T-cells of TIS21KO mice compared with the wild type. • Inhibition of melanoma growth in TIS21KO mice and CD8{sup +} T cell infiltration in tumor. • Reduction of CD 107{sup +}CD8{sup +} T cells, but increased granzyme B{sup +} CD8{sup +} T cells in TIS21KO mice.

  2. Receptor Pre-Clustering and T cell Responses: Insights into Molecular Mechanisms.

    PubMed

    Castro, Mario; van Santen, Hisse M; Férez, María; Alarcón, Balbino; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    T cell activation, initiated by T cell receptor (TCR) mediated recognition of pathogen-derived peptides presented by major histocompatibility complex class I or II molecules (pMHC), shows exquisite specificity and sensitivity, even though the TCR-pMHC binding interaction is of low affinity. Recent experimental work suggests that TCR pre-clustering may be a mechanism via which T cells can achieve such high sensitivity. The unresolved stoichiometry of the TCR makes TCR-pMHC binding and TCR triggering, an open question. We formulate a mathematical model to characterize the pre-clustering of T cell receptors (TCRs) on the surface of T cells, motivated by the experimentally observed distribution of TCR clusters on the surface of naive and memory T cells. We extend a recently introduced stochastic criterion to compute the timescales of T cell responses, assuming that ligand-induced cross-linked TCR is the minimum signaling unit. We derive an approximate formula for the mean time to signal initiation. Our results show that pre-clustering reduces the mean activation time. However, additional mechanisms favoring the existence of clusters are required to explain the difference between naive and memory T cell responses. We discuss the biological implications of our results, and both the compatibility and complementarity of our approach with other existing mathematical models.

  3. Receptor Pre-Clustering and T cell Responses: Insights into Molecular Mechanisms

    PubMed Central

    Castro, Mario; van Santen, Hisse M.; Férez, María; Alarcón, Balbino; Lythe, Grant; Molina-París, Carmen

    2014-01-01

    T cell activation, initiated by T cell receptor (TCR) mediated recognition of pathogen-derived peptides presented by major histocompatibility complex class I or II molecules (pMHC), shows exquisite specificity and sensitivity, even though the TCR–pMHC binding interaction is of low affinity. Recent experimental work suggests that TCR pre-clustering may be a mechanism via which T cells can achieve such high sensitivity. The unresolved stoichiometry of the TCR makes TCR–pMHC binding and TCR triggering, an open question. We formulate a mathematical model to characterize the pre-clustering of T cell receptors (TCRs) on the surface of T cells, motivated by the experimentally observed distribution of TCR clusters on the surface of naive and memory T cells. We extend a recently introduced stochastic criterion to compute the timescales of T cell responses, assuming that ligand-induced cross-linked TCR is the minimum signaling unit. We derive an approximate formula for the mean time to signal initiation. Our results show that pre-clustering reduces the mean activation time. However, additional mechanisms favoring the existence of clusters are required to explain the difference between naive and memory T cell responses. We discuss the biological implications of our results, and both the compatibility and complementarity of our approach with other existing mathematical models. PMID:24817867

  4. Role of monocyte fucose-receptors in T-cell fibronectin activity.

    PubMed Central

    Donson, J; Mandy, K; Feng, Z H; Mandy, S; Brown, E J; Godfrey, H P

    1991-01-01

    T-cell fibronectin (FN) is a lymphokine produced by antigen- and mitogen-activated T cells that agglutinates human monocytes at femtomolar concentrations. This extreme degree of activity derives from co-operative interactions between multiple FN domains and multiple monocyte integrin protein receptors. T-cell FN, like other FN, is a glycoprotein. The role interactions between T-cell FN carbohydrate and lectin-like monocyte surface receptors play in mediating T-cell FN activity was studied by determining the ability of monosaccharides to inhibit T-cell FN activity. L-Fucose and L-rhamnose significantly inhibited T-cell FN-mediated monocyte agglutination at concentrations as low as 0.01 mM; D-glucose, D- or L-galactose, D- or L-mannose and D-fucose were not inhibitory at 10-100 mM. This inhibition appeared to be due to interference with the binding of T-cell FN fucose residues to monocyte fucose receptors since: (i) treatment of T-cell FN with alpha-L-fucosidase abolished its agglutinating activity for human monocytes, while treatment with beta-D-galactosidase or with alpha-L-fucosidase in the presence of L-fucose had no effect; (ii) treatment of monocytes with alpha-L-fucosidase did not affect their response to T-cell FN; and (iii) L-fucose or L-rhamnose did not alter the expression of monocyte integrin FN receptors under conditions where T-cell FN-mediated monocyte agglutination was completely inhibited. In vivo, 1 mumol intracutaneous L-fucose inhibited expression of delayed hypersensitivity by 30% (P much less than 0.001); similar doses of L-rhamnose inhibited responses by 10% (P less than 0.02). These data implicate a fucose receptor in monocyte response to T-cell FN, and suggest that T-cell FN is only one of the mediators involved in initiating delayed hypersensitivity reactions in vivo. PMID:1769694

  5. IL-7 receptor blockade following T cell depletion promotes long-term allograft survival

    PubMed Central

    Mai, Hoa-Le; Boeffard, Françoise; Longis, Julie; Danger, Richard; Martinet, Bernard; Haspot, Fabienne; Vanhove, Bernard; Brouard, Sophie; Soulillou, Jean-Paul

    2014-01-01

    T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti–IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4– and anti-CD8–mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation. PMID:24569454

  6. T Cell Receptor CDR3 Sequence but Not Recognition Characteristics Distinguish Autoreactive Effector and Foxp3+ Regulatory T Cells

    PubMed Central

    Liu, Xin; Nguyen, Phuong; Liu, Wei; Cheng, Cheng; Steeves, Meredith; Obenauer, John C.; Ma, Jing; Geiger, Terrence L.

    2010-01-01

    SUMMARY The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)+ regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRα retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation. PMID:20005134

  7. Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling.

    PubMed

    Zhang, Sai; Laouar, Amale; Denzin, Lisa K; Sant'Angelo, Derek B

    2015-07-16

    The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge.

  8. Zbtb16 (PLZF) is stably suppressed and not inducible in non-innate T cells via T cell receptor-mediated signaling

    PubMed Central

    Zhang, Sai; Laouar, Amale; Denzin, Lisa K.; Sant’Angelo, Derek B.

    2015-01-01

    The transcription factor PLZF (promyelocytic leukemia zinc finger; zbtb16) is essential for nearly all of the unique characteristics of NKT cells including their rapid and potent response to antigen. In the immune system, zbtb16 expression is only found in innate cells. Conventional T cells that ectopically express PLZF spontaneously acquire an activated, effector phenotype. Activation induced expression of lineage defining transcription factors such as T-bet, FoxP3, RORγt, GATA3 and others is essential for naïve T cell differentiation into effector T cells. In this study, we used sensitive genetic-based approaches to assess the induction of PLZF expression in non-innate T cells by T cell receptor (TCR)-mediated activation. Surprisingly, we found that PLZF was stably repressed in non-innate T cells and that TCR-mediated signaling was not sufficient to induce PLZF in conventional T cells. The inactivated state of PLZF was stably maintained in mature T cells, even under inflammatory conditions imposed by bacterial infection. Collectively, our data show that, in contrast to multiple recent reports, PLZF expression is highly specific to innate T cells and cannot be induced in conventional T cells via TCR-mediated activation or inflammatory challenge. PMID:26178856

  9. Molecular basis of cross-reactivity among allergen-specific human T cells: T-cell receptor V alpha gene usage and epitope structure.

    PubMed Central

    Mohapatra, S S; Mohapatra, S; Yang, M; Ansari, A A; Parronchi, P; Maggi, E; Romagnani, S

    1994-01-01

    Cross-reactivities between the major grass pollen allergens, at the level of T-cell recognition was examined employing several Lolium perenne I (Lol p I)-specific human T-cell clones. Nine of these Lol p I-specific T-cell clones exhibited cross-recognition of the recombinant Poa pratensis IX (Poa p IX) allergen, rKBG7.2, indicating that these two major antigens of a grass pollen share T-cell epitopes. Furthermore, proliferative responses of two other T-cell clones demonstrated that individual allergens of diverse grass pollens also possess common T-cell epitopes. Examination of the T-cell receptor (TcR) V alpha genes of these T-cell clones indicated that these cloned cells utilized distinct J alpha genes and that nine out of 10 clones possessed V alpha 13 gene. Furthermore, sequence comparisons of several allergenic molecules indicated that this cross-reactivity may be due to the presence of epitope(s) with structure(s) similar to the major T-cell epitope of Poa p IX allergens. Taken together, these results suggest for the first time that the major grass pollen allergens share cross-reacting T-cell epitope(s), and that this cross-reactivity is due to the structural homologies among allergens and restricted usage of TcR V alpha genes. PMID:7510663

  10. Triggering of toll-like receptor signaling pathways in T cells contributes to the anti-tumor efficacy of T cell responses.

    PubMed

    Salem, Mohamed Labib

    2011-06-30

    Traditionally, expression of toll-like receptors (TLRs) has been associated with innate immune cells in particular professional antigen presenting cells and natural killer cells. This led to the concept that the adjuvant effects of ligation of TLR in a host occur mainly in innate immune cells. However, this concept has been challenged by recent studies including ours demonstrating that T cells express appreciated levels of different TLRs, which can serve as costimulatory co-receptors during polyclonal and antigen-specific stimulation of T cells. Because T cells express low levels of TLRs as compared to innate immune cells, increasing the expression levels of TLRs in T cells can significantly maximize their responses to the costimulatory effects of TLR ligation. This review article focuses on the potential role of TLR expression in T cells in their responses to vaccination regimen containing TLR agonists and how it can be modulated to optimize anti-tumor immunity.

  11. Role of Prolactin in the Recovered T-Cell Development of Early Partially Decapitated Chicken Embryo

    PubMed Central

    Moreno, J.; Varas, A.; Vicente, A.

    1998-01-01

    Although different experimental approaches have suggested certain regulation of the mammalian immune system by the neuroendocrine system, the precise factors involved in the process are largely unknown. In previous reports, we demonstrated important changes in the thymic development of chickens deprived of the major neuroendocrine centers by the removal of embryonic prosencephalon at 33-38 hr of incubation (DCx embryos) (Herradón et al., 1991; Moreno et al., 1995). In these embryos, there was a stopping of T-cell maturation that resulted in an accumulation of the most immature T-cell subsets (CD4-CD8- cells and CD4-CD81o cells) and, accordingly, in decreased numbers of DP (CD4+CD8+) thymocytes and mature CD3+TcRαβ + cells, but not CD3+TcRγδ lymphocytes. In the present work, we restore the thymic histology as well as the percentage of distinct T-cell subsets of DCx embryos by supplying recombinant chicken prolactin, grafting of embryonic pituitary gland, or making cephalic chick-quail chimeras. The recovery was not, however, whole and the percentage of CD3+TcRαβ thymocytes did not reach the normal values observed in 17-day-old control Sham-DCx embryos. The results are discussed on the basis of a key role for prolactin in chicken T-cell maturation. This hormone could regulate the transition of DN (CD4-CD8-) thymocytes to the DP (CD4+CD8+) cell compartment through its capacity for inducing IL-2 receptor expression on the former. PMID:9851358

  12. On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments

    PubMed Central

    Toor, Amir A.; Toor, Abdullah A.; Rahmani, Mohamed; Manjili, Masoud H.

    2016-01-01

    The human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor α (TRA) and β (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5′ to the 3′ end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in gene-segment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices. PMID:26763333

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

    NASA Astrophysics Data System (ADS)

    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).

  14. T cell receptor recognition of CD1b presenting a mycobacterial glycolipid

    PubMed Central

    Gras, Stephanie; Van Rhijn, Ildiko; Shahine, Adam; Cheng, Tan-Yun; Bhati, Mugdha; Tan, Li Lynn; Halim, Hanim; Tuttle, Kathryn D.; Gapin, Laurent; Le Nours, Jérôme; Moody, D. Branch; Rossjohn, Jamie

    2016-01-01

    CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αβ T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and β-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid. PMID:27807341

  15. KLF2 deficiency in T cells results in unrestrained cytokine production and bystander chemokine receptor upregulation

    PubMed Central

    Weinreich, Michael A.; Takada, Kensuke; Skon, Cara; Reiner, Steven L.; Jameson, Stephen C.; Hogquist, Kristin A.

    2009-01-01

    SUMMARY The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid binding receptor, S1P1, and the selectin, CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirm the upregulation of the chemokine receptor CXCR3 on KLF2 deficient T cells. However, we show that this is a cell nonautonomous effect, as revealed by CXCR3 upregulation on WT bystander cells in mixed bone marrow chimeras with KLF2 deficient cells. Furthermore, we show that KLF2 deficient T cells overproduce IL-4, leading to the upregulation of CXCR3 through an IL-4 receptor and eomesodermin dependent pathway. Consistent with the increased IL-4 production, we find high levels of serum IgE in mice with T cell specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P1 and CD62L, and restrains spontaneous cytokine production in naive T cells. PMID:19592277

  16. Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

    PubMed Central

    Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

    2015-01-01

    Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

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

    PubMed

    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

    2016-07-08

    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.

  18. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction.

    PubMed

    Nika, Konstantina; Soldani, Cristiana; Salek, Mogjiborahman; Paster, Wolfgang; Gray, Adrian; Etzensperger, Ruth; Fugger, Lars; Polzella, Paolo; Cerundolo, Vincenzo; Dushek, Omer; Höfer, Thomas; Viola, Antonella; Acuto, Oreste

    2010-06-25

    T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to approximately 40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-zeta phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors.

  19. An essential role for IL-2 receptor in regulatory T cell function

    PubMed Central

    Levine, Andrew G; Fan, Xiying; Klein, Ulf; Zheng, Ye; Gasteiger, Georg; Feng, Yongqiang; Fontenot, Jason D.; Rudensky, Alexander Y.

    2016-01-01

    Regulatory T (Treg) cells, expressing abundant amounts of the IL-2 receptor (IL-2R), are reliant on IL-2 produced by activated T cells. This feature implied a key role for a simple network based on IL-2 consumption by Treg cells in their suppressor function. However, congenital deficiency in IL-2R results in reduced expression of the Treg cell lineage specification factor Foxp3, confounding experimental efforts to understand the role of IL-2R expression and signaling in Treg suppressor function. Using genetic gain and loss of function approaches, we demonstrate that IL-2 capture is dispensable for control of CD4+ T cells, but is important for limiting CD8+ T cell activation, and that IL-2R dependent STAT5 transcription factor activation plays an essential role in Treg cell suppressor function separable from T cell receptor signaling. PMID:27595233

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

    PubMed Central

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

    2016-01-01

    Summary CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour‐specific CD4+ T cells occur in low frequency, express relatively low‐affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour‐specific HLA class I‐restricted TCRs prior to adoptive transfer. The lack of help from the co‐receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild‐type and a range of affinity‐enhanced TCRs specific for the HLA A*0201‐restricted NY‐ESO‐1‐ and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement. PMID:27324616

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

    PubMed

    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.

  2. Chimeric antigen receptor T cell therapy in AML: How close are we?

    PubMed

    Gill, Saar

    2016-12-01

    The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML.

  3. TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling1

    PubMed Central

    Xie, Ping; Kraus, Zachary J.; Stunz, Laura L.; Liu, Yan; Bishop, Gail A.

    2011-01-01

    We recently reported that TRAF3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. Here we report the generation and characterization of T cell-specific TRAF3−/− mice, in which the TRAF3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell-lineage did not affect the numbers or proportions of CD4+,CD8+ or double positive or negative thymocytes, or CD4 or CD8 T cell populations in secondary lymphoid organs except that the T cell specific TRAF3−/− mice had a two-fold increase in FoxP3+ T cells.. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3 deficient mice exhibited defective IgG1 responses to a T dependent antigen, and impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon co-stimulation, and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide new insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity. PMID:21084666

  4. Preclinical targeting of aggressive T-cell malignancies using anti-CD5 chimeric antigen receptor.

    PubMed

    Chen, K H; Wada, M; Pinz, K G; Liu, H; Lin, K-W; Jares, A; Firor, A E; Shuai, X; Salman, H; Golightly, M; Lan, F; Senzel, L; Leung, E L; Jiang, X; Ma, Y

    2017-02-10

    The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.Leukemia advance online publication, 10 February 2017; doi:10.1038/leu.2017.8.

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

    PubMed

    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.

  6. Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity

    PubMed Central

    1990-01-01

    gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR. PMID:2109035

  7. T cells engineered with a T cell receptor against the prostate antigen TARP specifically kill HLA-A2+ prostate and breast cancer cells.

    PubMed

    Hillerdal, Victoria; Nilsson, Berith; Carlsson, Björn; Eriksson, Fredrik; Essand, Magnus

    2012-09-25

    To produce genetically engineered T cells directed against prostate and breast cancer cells, we have cloned the T-cell receptor recognizing the HLA-A2-restricted T-cell receptor γ-chain alternate reading-frame protein (TARP)(4-13) epitope. TARP is a protein exclusively expressed in normal prostate epithelium and in adenocarcinomas of the prostate and breast. Peripheral blood T cells transduced with a lentiviral vector encoding the TARP-TCR proliferated well when exposed to peptide-specific stimuli. These cells exerted peptide-specific IFN-γ production and cytotoxic activity. Importantly, HLA-A2(+) prostate and breast cancer cells expressing TARP were also killed, demonstrating that the TARP(4-13) epitope is a physiologically relevant target for T-cell therapy of prostate and breast cancer. In conclusion, we present the cloning of a T cell receptor (TCR) directed against a physiologically relevant HLA-A2 epitope of TARP. To our knowledge this report on engineering of T cells with a TCR directed against an antigen specifically expressed by prostate cells is unique.

  8. Signalling pathways induced by protease-activated receptors and integrins in T cells.

    PubMed

    Bar-Shavit, Rachel; Maoz, Miriam; Yongjun, Yin; Groysman, Maya; Dekel, Idit; Katzav, Shulamit

    2002-01-01

    Recent characterization of the thrombin receptor indicates that it plays a role in T-cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease-activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T-leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP-70 and SLP-76, two key proteins in T-cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin-coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand-signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR-induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

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

    PubMed

    Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

    2017-03-29

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

  10. Ligand-Driven T Cell Receptor Selection in Celiac Disease.

    PubMed

    Singh, Nishant K; Baker, Brian M

    2016-10-04

    Recognition of antigens by T cell receptors (TCRs) underlies cellular immunity. By comparing how different TCRs recognize the key antigens associated with celiac disease, Petersen et al. (2016), in this issue of Structure, show how celiac antigen properties select immunologically distinct yet structurally and physically compatible TCRs, ultimately driving autoimmunity.

  11. Partial Regulatory T Cell Depletion Prior to Schistosomiasis Vaccination Does Not Enhance the Protection

    PubMed Central

    Zhou, Sha; Xu, Zhipeng; Hoellwarth, Jason; Chen, Xiaojun; He, Lei; Zhang, Rongbo; Liu, Feng; Wang, Jun; Su, Chuan

    2012-01-01

    CD4+CD25+ regulatory T cells (Tregs) do not only influence self-antigen specific immune responses, but also dampen the protective effect induced by a number of vaccines. The impact of CD4+CD25+ Tregs on vaccines against schistosomiasis, a neglected tropical disease that is a major public health concern, however, has not been examined. In this study, a DNA vaccine encoding a 26 kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST) was constructed and its potential effects were evaluated by depleting CD25+ cells prior to pVAX1-Sj26GST immunization. This work shows that removal of CD25+ cells prior to immunization with the pVAX1-Sj26GST schistosomiasis DNA vaccine significantly increases the proliferation of splenocytes and IgG levels. However, CD25+ cell-depleted mice immunized with pVAX1-Sj26GST show no improved protection against S. japonicum. Furthermore, depletion of CD25+ cells causes an increase in both pro-inflammatory cytokines (e.g. IFN-γ, GM-CSF and IL-4) and an anti-inflammatory cytokine (e.g. IL-10), with CD4+CD25- T cells being one of the major sources of both IFN-γ and IL-10. These findings indicate that partial CD25+ cell depletion fails to enhance the effectiveness of the schistosome vaccine, possibly due to IL-10 production by CD4+CD25- T cells, or other cell types, after CD25+ cell depletion during vaccination. PMID:22802961

  12. Drug Hypersensitivity: How Drugs Stimulate T Cells via Pharmacological Interaction with Immune Receptors.

    PubMed

    Pichler, Werner J; Adam, Jacqueline; Watkins, Stephen; Wuillemin, Natascha; Yun, James; Yerly, Daniel

    2015-01-01

    Small chemicals like drugs tend to bind to proteins via noncovalent bonds, e.g. hydrogen bonds, salt bridges or electrostatic interactions. Some chemicals interact with other molecules than the actual target ligand, representing so-called 'off-target' activities of drugs. Such interactions are a main cause of adverse side effects to drugs and are normally classified as predictable type A reactions. Detailed analysis of drug-induced immune reactions revealed that off-target activities also affect immune receptors, such as highly polymorphic human leukocyte antigens (HLA) or T cell receptors (TCR). Such drug interactions with immune receptors may lead to T cell stimulation, resulting in clinical symptoms of delayed-type hypersensitivity. They are assigned the 'pharmacological interaction with immune receptors' (p-i) concept. Analysis of p-i has revealed that drugs bind preferentially or exclusively to distinct HLA molecules (p-i HLA) or to distinct TCR (p-i TCR). P-i reactions differ from 'conventional' off-target drug reactions as the outcome is not due to the effect on the drug-modified cells themselves, but is the consequence of reactive T cells. Hence, the complex and diverse clinical manifestations of delayed-type hypersensitivity are caused by the functional heterogeneity of T cells. In the abacavir model of p-i HLA, the drug binding to HLA may result in alteration of the presenting peptides. More importantly, the drug binding to HLA generates a drug-modified HLA, which stimulates T cells directly, like an allo-HLA. In the sulfamethoxazole model of p-i TCR, responsive T cells likely require costimulation for full T cell activation. These findings may explain the similarity of delayed-type hypersensitivity reactions to graft-versus-host disease, and how systemic viral infections increase the risk of delayed-type hypersensitivity reactions.

  13. Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens

    PubMed Central

    Kirschner, Andreas; Thiede, Melanie; Blaeschke, Franziska; Richter, Günther H.S.; Gerke, Julia S.; Baldauf, Michaela C.; Grünewald, Thomas G.P.; Busch, Dirk H.; Burdach, Stefan; Thiel, Uwe

    2016-01-01

    Aim Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells. Methods We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells. Results Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319. Conclusion Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells. PMID:27447745

  14. Characterization of a single-chain T-cell receptor expressed in Escherichia coli.

    PubMed

    Hoo, W F; Lacy, M J; Denzin, L K; Voss, E W; Hardman, K D; Kranz, D M

    1992-05-15

    Despite progress in defining the nature of major histocompatibility complex products that are recognized by the T-cell antigen receptor, the binding properties and structure of the receptor have not been solved. The primary problem has been the difficulty in obtaining sufficient quantities of active receptor. In this report we show that a single-chain T-cell receptor gene can be expressed in Escherichia coli. The protein consists of the variable (V) regions of the alpha and beta chains (V alpha and V beta) encoded by the cytotoxic T-lymphocyte clone 2C (a H-2b anti-H-2d alloreactive cell line) linked by a 25-amino acid flexible peptide. Solubilized extracts that contain the 27-kDa V alpha 3V beta 8 protein are positive in solid-phase immunoassays with the anti-V beta 8 antibody KJ16 and the anti-clonotypic antibody 1B2. Approximately 1% of the protein can be specifically purified on a 1B2-conjugated column. These results indicate that a fraction of the protein is able to fold into a native conformation and that single-chain proteins should be useful not only as immunogens for eliciting anti-T-cell receptor antibodies but in the study of T-cell receptor structure and function.

  15. Yersinia pseudotuberculosis supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling.

    PubMed

    Pasztoi, Maria; Bonifacius, Agnes; Pezoldt, Joern; Kulkarni, Devesha; Niemz, Jana; Yang, Juhao; Teich, René; Hajek, Janina; Pisano, Fabio; Rohde, Manfred; Dersch, Petra; Huehn, Jochen

    2017-04-04

    Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4(+) T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4(+) T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3(+) regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4(+) T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4(+) T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3(+) Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4(+) T cell subsets by altering their TCR downstream signaling.

  16. Review: Current clinical applications of chimeric antigen receptor (CAR) modified T cells.

    PubMed

    Geyer, Mark B; Brentjens, Renier J

    2016-11-01

    The past several years have been marked by extraordinary advances in clinical applications of immunotherapy. In particular, adoptive cellular therapy utilizing chimeric antigen receptor (CAR)-modified T cells targeted to CD19 has demonstrated substantial clinical efficacy in children and adults with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) and durable clinical benefit in a smaller subset of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or B-cell non-Hodgkin lymphoma (B-NHL). Early-phase clinical trials are currently assessing CAR T-cell safety and efficacy in additional malignancies. Here, we discuss clinical results from the largest series to date investigating CD19-targeted CAR T cells in B-ALL, CLL, and B-NHL, including discussion of differences in CAR T-cell design and production and treatment approach, as well as clinical efficacy, nature of severe cytokine release syndrome and neurologic toxicities, and CAR T-cell expansion and persistence. We additionally review the current and forthcoming use of CAR T cells in multiple myeloma and several solid tumors and highlight challenges and opportunities afforded by the current state of CAR T-cell therapies, including strategies to overcome inhibitory aspects of the tumor microenvironment and enhance antitumor efficacy.

  17. Crossreactive T Cells Spotlight the Germline Rules for [alpha beta] T Cell-Receptor Interactions with MHC Molecules

    SciTech Connect

    Dai, Shaodong; Huseby, Eric S.; Rubtsova, Kira; Scott-Browne, James; Crawford, Frances; Macdonald, Whitney A.; Marrack, Philippa; Kappler, John W.

    2008-10-31

    To test whether highly crossreactive {alpha}{beta} T cell receptors (TCRs) produced during limited negative selection best illustrate evolutionarily conserved interactions between TCR and major histocompatibility complex (MHC) molecules, we solved the structures of three TCRs bound to the same MHC II peptide (IA{sup b}-3K). The TCRs had similar affinities for IA{sup b}-3K but varied from noncrossreactive to extremely crossreactive with other peptides and MHCs. Crossreactivity correlated with a shrinking, increasingly hydrophobic TCR-ligand interface, involving fewer TCR amino acids. A few CDR1 and CDR2 amino acids dominated the most crossreactive TCR interface with MHC, including V{beta}8 48Y and 54E and V{alpha}4 29Y, arranged to impose the familiar diagonal orientation of TCR on MHC. These interactions contribute to MHC binding by other TCRs using related V regions, but not usually so dominantly. These data show that crossreactive TCRs can spotlight the evolutionarily conserved features of TCR-MHC interactions and that these interactions impose the diagonal docking of TCRs on MHC.

  18. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.

    PubMed

    Bendle, Gavin M; Linnemann, Carsten; Hooijkaas, Anna I; Bies, Laura; de Witte, Moniek A; Jorritsma, Annelies; Kaiser, Andrew D M; Pouw, Nadine; Debets, Reno; Kieback, Elisa; Uckert, Wolfgang; Song, Ji-Ying; Haanen, John B A G; Schumacher, Ton N M

    2010-05-01

    The transfer of T cell receptor (TCR) genes can be used to induce immune reactivity toward defined antigens to which endogenous T cells are insufficiently reactive. This approach, which is called TCR gene therapy, is being developed to target tumors and pathogens, and its clinical testing has commenced in patients with cancer. In this study we show that lethal cytokine-driven autoimmune pathology can occur in mouse models of TCR gene therapy under conditions that closely mimic the clinical setting. We show that the pairing of introduced and endogenous TCR chains in TCR gene-modified T cells leads to the formation of self-reactive TCRs that are responsible for the observed autoimmunity. Furthermore, we demonstrate that adjustments in the design of gene therapy vectors and target T cell populations can be used to reduce the risk of TCR gene therapy-induced autoimmune pathology.

  19. Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-02-01

    The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

  20. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse

    NASA Astrophysics Data System (ADS)

    Choudhuri, Kaushik; Llodrá, Jaime; Roth, Eric W.; Tsai, Jones; Gordo, Susana; Wucherpfennig, Kai W.; Kam, Lance C.; Stokes, David L.; Dustin, Michael L.

    2014-03-01

    The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These

  1. T cell receptor (TCR) gene transfer with lentiviral vectors allows efficient redirection of tumor specificity in naive and memory T cells without prior stimulation of endogenous TCR.

    PubMed

    Circosta, Paola; Granziero, Luisa; Follenzi, Antonia; Vigna, Elisa; Stella, Stefania; Vallario, Antonella; Elia, Angela Rita; Gammaitoni, Loretta; Vitaggio, Katiuscia; Orso, Francesca; Geuna, Massimo; Sangiolo, Dario; Todorovic, Maja; Giachino, Claudia; Cignetti, Alessandro

    2009-12-01

    We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

  2. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas.

    PubMed

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S; Furness, Andrew J S; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H; McGranahan, Nicholas; Rowan, Andrew J; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L; Larkin, James; Swanton, Charles

    2013-12-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367-16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, -0.218 to 0.465). 3-93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches.

  3. Expression of T cell antigen receptor during differentiation

    SciTech Connect

    Allison, J.P.; Lanier, L.L.; Guyden, J.; Richie, E.R.

    1986-03-01

    The authors have used flow cytometry with monoclonal antibodies, radioimmuneprecipitation with a rabbit antiserum to common epitopes of the TCR, and Northern and Southern blot analysis with cloned TCR genes to study antigen receptor (TCR) expression by normal murine and human thymocytes and by primary murine thymomas. L3T4-,Lyt2- murine thymomas corresponding to the earliest stage of thymic differentiation, were found to have rearranged TCR beta genes, and to express low levels of beta transcript, but lacked alpha gene transcript and failed to express TCR on the cell surface. L3T4+,Lyt2+ thymomas were variable, but the majority were found to contain significant levels of both alpha and beta transcripts and to express TCR at the cell surface. Similarly, alpha and beta transcripts and TCR protein were detected in sorted L3T4+,Lyt2+ murine thymocytes. Using three color fluorescence, the authors determined that app. 70% of human T4+T8+ thymocytes also expressed T3, a component of the TCR complex. These data indicate that in mouse and man expression of TCR occurs in the immature, or cortical, thymic population.

  4. T-cell receptor-CD4 physical association in a murine T-cell hybridoma: induction by antigen receptor ligation.

    PubMed Central

    Mittler, R S; Goldman, S J; Spitalny, G L; Burakoff, S J

    1989-01-01

    By employing flow cytometric analysis and fluorescence resonance energy transfer (FRET), we examined the physical relationship between the T-cell receptor-CD3 complex (Ti-CD3) and the CD4 molecule on helper T cells. Through the use of an L3T4-negative murine T-cell hybridoma infectant expressing the human CD4 gene and having antigen specificity for HLA-DR, we show that binding of the Ti-CD3 complex with an anti-CD3 monoclonal antibody induces its redistribution proximal to cell-surface CD4. FRET efficiency was 9.4% on cells labeled with rhodaminated anti-CD3 and fluoresceinated anti-CD4. FRET was found to be temperature dependent, since similarly treated cells held at 4 degrees C displayed a FRET efficiency of less than 1%. Energy transfer was evident within 3 min after warming cells to 37 degrees C. Energy transfer was not detected between Ti-CD3 and the abundantly expressed leukocyte common antigen (CD45). Of greater significance was our observation that hybridomas infected with a truncated CD4 gene lacking the cytoplasmic domain failed to transfer energy despite the fact that CD4 was expressed on the cell surface at levels equivalent to or greater than the wild type. These studies suggest that after crosslinking of the Ti-CD3 on CD4+ T cells, a physical association occurs between the antigen receptor complex and CD4 and that the association is dependent upon the presence of the cytoplasmic domain of CD4. PMID:2530583

  5. CD8 Co-receptor promotes susceptibility of CD8+ T cells to transforming growth factor-β (TGF-β)-mediated suppression

    PubMed Central

    Zloza, Andrew; Jagoda, Michael C.; Lyons, Gretchen E.; Graves, Michael C.; Kohlhapp, Frederick J.; O’Sullivan, Jeremy A.; Lacek, Andrew T.; Nishimura, Michael I.

    2015-01-01

    CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-β have been studied individually, the influence of CD8 co-receptor on TGF-β function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-β-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αβCD8 molecule, we demonstrate that cells expressing full-length αβCD8 were highly susceptible, αβCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-β. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-β suppression. Resistance was not associated with TGF-β receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-β. Based on the important role which TGF-β-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies. PMID:21193909

  6. T Cell Receptor Activation of NF-κB in Effector T Cells: Visualizing Signaling Events Within and Beyond the Cytoplasmic Domain of the Immunological Synapse.

    PubMed

    Traver, Maria K; Paul, Suman; Schaefer, Brian C

    2017-01-01

    The T cell receptor (TCR) to NF-κB signaling pathway plays a critical role in regulation of proliferation and effector T cell differentiation and function. In naïve T cells, data suggest that most or all key cytoplasmic NF-κB signaling occurs in a TCR-proximal manner at the immunological synapse (IS). However, the subcellular organization of cytoplasmic NF-κB-activating complexes in effector T cells is more complex, involving signaling molecules and regulatory mechanisms beyond those operative in naïve cells. Additionally, in effector T cells, much signaling occurs at cytoplasmic locations distant from the IS. Visualization of these cytoplasmic signaling complexes has provided key insights into the complex and dynamic regulation of NF-κB signal transduction in effector T cells. In this chapter, we provide in-depth protocols for activating and preparing effector T cells for fluorescence imaging, as well as a discussion of the effective application of distinct imaging methodologies, including confocal and super-resolution microscopy and imaging flow cytometry.

  7. Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor.

    PubMed

    van den Berg, Joost H; Gomez-Eerland, Raquel; van de Wiel, Bart; Hulshoff, Lenie; van den Broek, Daan; Bins, Adriaan; Tan, Hanno L; Harper, Jane V; Hassan, Namir J; Jakobsen, Bent K; Jorritsma, Annelies; Blank, Christian U; Schumacher, Ton N M; Haanen, John B A G

    2015-09-01

    Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26-35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed.

  8. Case Report of a Fatal Serious Adverse Event Upon Administration of T Cells Transduced With a MART-1-specific T-cell Receptor

    PubMed Central

    van den Berg, Joost H; Gomez-Eerland, Raquel; van de Wiel, Bart; Hulshoff, Lenie; van den Broek, Daan; Bins, Adriaan; Tan, Hanno L; Harper, Jane V; Hassan, Namir J; Jakobsen, Bent K; Jorritsma, Annelies; Blank, Christian U; Schumacher, Ton N M; Haanen, John B A G

    2015-01-01

    Here, we describe a fatal serious adverse event observed in a patient infused with autologous T-cell receptor (TCR) transduced T cells. This TCR, originally obtained from a melanoma patient, recognizes the well-described HLA-A*0201 restricted 26–35 epitope of MART-1, and was not affinity enhanced. Patient 1 with metastatic melanoma experienced a cerebral hemorrhage, epileptic seizures, and a witnessed cardiac arrest 6 days after cell infusion. Three days later, the patient died from multiple organ failure and irreversible neurologic damage. After T-cell infusion, levels of IL-6, IFN-γ, C-reactive protein (CRP), and procalcitonin increased to extreme levels, indicative of a cytokine release syndrome or T-cell-mediated inflammatory response. Infused T cells could be recovered from blood, broncho-alveolar lavage, ascites, and after autopsy from tumor sites and heart tissue. High levels of NT-proBNP indicate semi-acute heart failure. No cross reactivity of the modified T cells toward a beating cardiomyocyte culture was observed. Together, these observations suggest that high levels of inflammatory cytokines alone or in combination with semi-acute heart failure and epileptic seizure may have contributed substantially to the occurrence of the acute and lethal event. Protocol modifications to limit the risk of T-cell activation-induced toxicity are discussed. PMID:25896248

  9. C-C chemokine receptor type-4 transduction of T cells enhances interaction with dendritic cells, tumor infiltration and therapeutic efficacy of adoptive T cell transfer.

    PubMed

    Rapp, Moritz; Grassmann, Simon; Chaloupka, Michael; Layritz, Patrick; Kruger, Stephan; Ormanns, Steffen; Rataj, Felicitas; Janssen, Klaus-Peter; Endres, Stefan; Anz, David; Kobold, Sebastian

    2016-03-01

    T cell infiltration at the tumor site has been identified as a major predictor for the efficacy of adoptive T cell therapy. The chemokine C-C motif ligand 22 (CCL22) is highly expressed by immune cells in murine and human pancreatic cancer. Expression of its corresponding receptor, C-C chemokine receptor type 4 (CCR4), is restricted to regulatory T cells (Treg). We show that transduction of cytotoxic T cells (CTL) with CCR4 enhances their immigration into a pancreatic cancer model. Further, we show that binding of CCR4 with CCL22 strengthens the binding of T cell LFA-1 to dendritic cell (DC) ICAM-1 and increases CTL activation. In vivo, in a model of subcutaneous pancreatic cancer, treatment of tumor-bearing mice with CCR4-transduced CTL led to the eradication of established tumors in 40% of the mice. In conclusion, CCR4 overexpression in CTL is a promising therapeutic strategy to enhance the efficacy of adoptive T cell transfer (ACT).

  10. T cell receptor-dependent activation of mTOR signaling in T cells is mediated by Carma1 and MALT1, but not Bcl10.

    PubMed

    Hamilton, Kristia S; Phong, Binh; Corey, Catherine; Cheng, Jing; Gorentla, Balachandra; Zhong, Xiaoping; Shiva, Sruti; Kane, Lawrence P

    2014-06-10

    Signaling to the mechanistic target of rapamycin (mTOR) regulates diverse cellular processes, including protein translation, cellular proliferation, metabolism, and autophagy. Most models place Akt upstream of the mTOR complex, mTORC1; however, in T cells, Akt may not be necessary for mTORC1 activation. We found that the adaptor protein Carma1 [caspase recruitment domain (CARD)-containing membrane-associated protein 1] and at least one of its associated proteins, the paracaspase MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), were required for optimal activation of mTOR in T cells in response to stimulation of the T cell receptor (TCR) and the co-receptor CD28. However, Bcl10, which binds to Carma1 and MALT1 to form a complex that mediates signals from the TCR to the transcription factor NF-κB (nuclear factor κB), was not required. The catalytic activity of MALT1 was required for the proliferation of stimulated CD4+ T cells, but not for early TCR-dependent activation events. Consistent with an effect on mTOR, MALT1 activity was required for the increased metabolic flux in activated CD4+ T cells. Together, our data suggest that Carma1 and MALT1 play previously unappreciated roles in the activation of mTOR signaling in T cells after engagement of the TCR.

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

    PubMed Central

    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.

    2016-01-01

    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 is able to 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. While 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 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 read-out to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection. PMID:27030598

  12. Lymphocyte toxicity and T cell receptor excision circles in workers exposed to benzene.

    PubMed

    Lan, Qing; Zhang, Luoping; Hakim, Fran; Shen, Min; Memon, Sarfraz; Li, Guilan; Vermeulen, Roel; Smith, Martyn T; Rappaport, Stephen M; Hayes, Richard; Linet, Martha; Yin, Songnian; Rothman, Nathaniel; Rabkin, Charles S

    2005-05-30

    We have previously reported that benzene decreases peripheral white blood cell and platelet counts and specifically lowers subsets of several blood cell types, including CD4+-T cells, B cells, NK cells, and granulocytes. Diminished thymus function has been implicated as a mechanism for CD4+-T cell loss in other conditions such as AIDS by assays of T cell receptor excision circles (TRECs), a marker of naive T cells that have recently emigrated from the thymus. To evaluate alteration of thymic function as a mechanism for benzene's effects on CD4+-T cell counts, we measured total TREC levels in 45 benzene-exposed workers and 45 unexposed controls. There was no significant difference in TREC levels per 10(6) peripheral blood leukocytes in the benzene-exposed workers compared to the controls. Although our study does not rule out counterbalancing alterations of TREC levels in specific T cell subsets, benzene's lymphotoxicity does not appear to be mediated through diminished thymus function.

  13. Antitumor Effects of Chimeric Receptor Engineered Human T Cells Directed to Tumor Stroma

    PubMed Central

    Kakarla, Sunitha; Chow, Kevin KH; Mata, Melinda; Shaffer, Donald R; Song, Xiao-Tong; Wu, Meng-Fen; Liu, Hao; Wang, Lisa L; Rowley, David R; Pfizenmaier, Klaus; Gottschalk, Stephen

    2013-01-01

    Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors. PMID:23732988

  14. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

    PubMed

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen

    2009-12-18

    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  15. Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T.

    PubMed

    Marmor, M D; Benatar, T; Ratcliffe, M J

    1993-03-01

    Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest

  16. Evaluation of bovine thymic function by measurement of signal joint T-cell receptor excision circles.

    PubMed

    Hisazumi, Rinnosuke; Kayumi, Miya; Zhang, Weidong; Kikukawa, Ryuji; Nasu, Tetuo; Yasuda, Masahiro

    2016-01-01

    A signal joint T-cell receptor excision circle (sjTREC) is a circular DNA produced by T-cell receptor α gene rearrangement in the thymus. Measurements of sjTREC values have been used to evaluate thymic function. We recently established a quantitative PCR (QPCR) assay of bovine sjTREC. In the present study, we used this QPCR assay to measure the sjTREC value in bovine peripheral blood mononuclear cells and we then evaluated the relationships between sjTREC values and peripheral blood T-cell number, growth stage, gender, and meteorological season. The sjTREC value was highest at the neonatal stage, and its value subsequently decreased with age. On the other hand, the peripheral T-cell number increased with age. The sjTREC value in calves up to 50-days old was significantly higher for males than for females, suggesting that thymic function might differ by gender. In addition, the sjTREC value and the peripheral T-cell number were significantly higher in calves in the summer season than in calves in the winter season. These data suggest that bovine thymic function is highly variable and varies according to the growth stage, gender, and environmental factors such as air temperature or the UV index.

  17. Coevolution of T-cell receptors with MHC and non-MHC ligands

    PubMed Central

    Castro, Caitlin C.; Luoma, Adrienne M.; Adams, Erin J.

    2015-01-01

    Summary The structure and amino acid diversity of the T-cell receptor (TCR), similar in nature to that of Fab portions of antibodies, would suggest these proteins have a nearly infinite capacity to recognize antigen. Yet all currently defined native T cells expressing an α and β chain in their TCR can only sense antigen when presented in the context of a major histocompatibility complex (MHC) molecule. This MHC molecule can be one of many that exist in vertebrates, presenting small peptide fragments, lipid molecules, or small molecule metabolites. Here we review the pattern of TCR recognition of MHC molecules throughout a broad sampling of species and T-cell lineages and also touch upon T cells that do not appear to require MHC presentation for their surveillance function. We review the diversity of MHC molecules and information on the corresponding T-cell lineages identified in divergent species. We also discuss TCRs with structural domains unlike that of conventional TCRs of mouse and human. By presenting this broad view of TCR sequence, structure, domain organization, and function, we seek to explore how this receptor has evolved across time and been selected for alternative antigen-recognition capabilities in divergent lineages. PMID:26284470

  18. T cell receptor transgenic lymphocytes infiltrating murine tumors are not induced to express foxp3

    PubMed Central

    2011-01-01

    Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor β (TGFβ). B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment. PMID:22112546

  19. Cooperative B7-1/2 (CD80/CD86) and B7-DC Costimulation of CD4+ T Cells Independent of the PD-1 Receptor

    PubMed Central

    Shin, Tahiro; Kennedy, Gene; Gorski, Kevin; Tsuchiya, Haruo; Koseki, Haruhiko; Azuma, Miyuki; Yagita, Hideo; Chen, Lieping; Powell, Jonathan; Pardoll, Drew; Housseau, Franck

    2003-01-01

    B7-DC is a recently discovered member of the B7 family that binds to PD-1 and is selectively expressed by dendritic cells (DCs). It has been shown to either costimulate or inhibit T cell responses. To assess the role of B7-DC in DC–T cell interactions, DCs from B7-DC knockout (KO) mice were generated and compared with DCs from wild-type (WT) and B7–1/B7–2 double KO mice. B7–1/B7–2–deficient DCs, while strongly diminished in their ability to stimulate naive CD4+ T cells, nonetheless retain partial activity. DCs from B7-DC KO mice are diminished in their ability to activate CD4+ T cells, demonstrating that DC-expressed B7-DC serves a predominantly stimulatory rather than inhibitory function in the initiation of T cell responses. B7-DC costimulates expression of CD40L with faster kinetics than B7–1 and displays potent synergy with B7–1 and B7–2 for T cell proliferation and cytokine production, indicating that these B7 family members work in concert to stimulate T cells. Finally, costimulation with B7-DC alone or in conjunction with B7–1 is PD-1 independent, indicating that B7-DC costimulates T cells via a second receptor. PMID:12847135

  20. SAP-Dependent and -Independent Regulation of Innate T Cell Development Involving SLAMF Receptors.

    PubMed

    De Calisto, Jaime; Wang, Ninghai; Wang, Guoxing; Yigit, Burcu; Engel, Pablo; Terhorst, Cox

    2014-01-01

    Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) plays an essential role in the immune system mediating the function of several members of the SLAM family (SLAMF) of receptors, whose expression is essential for T, NK, and B-cell responses. Additionally, the expression of SAP in double-positive thymocytes is mandatory for natural killer T (NKT) cells and, in mouse, for innate CD8(+) T cell development. To date, only two members of the SLAMF of receptors, Slamf1 and Slamf6, have been shown to positively cooperate during NKT cell differentiation in mouse. However, it is less clear whether other members of this family may also participate in the development of these innate T cells. Here, we show that Slamf[1 + 6](-/-) and Slamf[1 + 5 + 6](-/-) B6 mice have ~70% reduction of NKT cells compared to wild-type B6 mice. Unexpectedly, the proportion of innate CD8(+) T cells slightly increased in the Slamf[1 + 5 + 6](-/-) , but not in the Slamf[1 + 6](-/-) strain, suggesting that Slamf5 may function as a negative regulator of innate CD8(+) T cell development. Accordingly, Slamf5(-/-) B6 mice showed an exclusive expansion of innate CD8(+) T cells, but not NKT cells. Interestingly, the SAP-independent Slamf7(-/-) strain showed an expansion of both splenic innate CD8(+) T cells and thymic NKT cells. On the other hand, and similar to what was recently shown in Slamf3(-/-) BALB/c mice, the proportions of thymic promyelocytic leukemia zinc finger (PLZF(hi)) NKT cells and innate CD8(+) T cells significantly increased in the SAP-independent Slamf8(-/-) BALB/c strain. In summary, these results show that NKT and innate CD8(+) T cell development can be regulated in a SAP-dependent and -independent fashion by SLAMF receptors, in which Slamf1, Slamf6, and Slamf8 affect development of NKT cells, and that Slamf5, Slamf7, and Slamf8 affect the development of innate CD8(+) T cells.

  1. The Pathogen Recognition Receptor NOD2 Regulates Human FOXP3+ T Cell Survival

    PubMed Central

    Rahman, Meher K.; Midtling, Emilie H.; Svingen, Phyllis A.; Xiong, Yuning; Bell, Michael P.; Tung, Jeanne; Smyrk, Tom; Egan, Larry J.; Faubion, William A.

    2013-01-01

    The expression of pathogen recognition receptors in human FOXP3+ T regulatory cells is established, yet the function of these receptors is currently obscure. In the process of studying the function of both peripheral and lamina propria FOXP3+ lymphocytes in patients with the human inflammatory bowel disease Crohn’s disease, we observed a clear deficiency in the quantity of FOXP3+ lymphocytes in patients with disease-associated polymorphisms in the pathogen recognition receptor gene NOD2. Subsequently, we determined that the NOD2 ligand, muramyl dipeptide (MDP), activates NF-κB in primary human FOXP3+ T cells. This activation is functionally relevant, as MDP-stimulated human FOXP3+ T cells are protected from death receptor Fas-mediated apoptosis. Importantly, apoptosis protection was not evident in MDP-stimulated FOXP3+ T cells isolated from a patient with the disease-associated polymorphism. Thus, we propose that one function of pathogen recognition receptors in human T regulatory cells is the protection against death receptor-mediated apoptosis in a Fas ligand-rich environment, such as that of the inflamed intestinal subepithelial space. PMID:20483763

  2. T-cell receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy.

    PubMed

    Noy, Roy; Eppel, Malka; Haus-Cohen, Maya; Klechevsky, Einav; Mekler, Orian; Michaeli, Yaeil; Denkberg, Galit; Reiter, Yoram

    2005-06-01

    Major histocompatibility complex class I molecules play a central role in the immune response against a variety of cells that have undergone malignant transformation by shaping the T-cell repertoire and presenting peptide antigens from endogeneous antigens to CD8+ cytotoxic T-cells. Diseased tumor or virus-infected cells are present on class I major histocompatibility complex molecule peptides that are derived from tumor-associated antigens or viral-derived proteins. Due to their unique specificity, such major histocompatibility complex-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific major histocompatibility complex-peptide complexes that are involved in the immune response against cancer or viral infections would allow for a specific immunotherapeutic treatment of these diseases. It has recently been demonstrated that antibodies with the antigen-specific, major histocompatibility complex-restricted specificity of T-cells can be generated by taking advantage of the selection power of phage display technology. In addition to their tumor targeting capabilities, antibodies that mimic the fine specificity of T-cell receptors can serve as valuable research reagents that enable study of human class I peptide-major histocompatibility complex ligand presentation, as well as T-cell receptor peptide-major histocompatibility complex interactions. T-cell receptor-like antibody molecules may prove to be useful tools for studying major histocompatibility complex class I antigen presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases and autoimmune disorders.

  3. Genomic organization of the mouse T-cell receptor beta-chain gene family.

    PubMed Central

    Lai, E; Barth, R K; Hood, L

    1987-01-01

    We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments. Images PMID:3035555

  4. Pappalysin-1 T cell receptor transgenic allo-restricted T cells kill Ewing sarcoma in vitro and in vivo

    PubMed Central

    Kirschner, Andreas; Thiede, Melanie; Alba Rubio, Rebeca; Richter, Günther H. S.; Kirchner, Thomas; Busch, Dirk H.; Burdach, Stefan; Thiel, Uwe

    2017-01-01

    ABSTRACT Pregnancy-associated plasma protein-A (PAPPA), also known as pappalysin, is a member of the insulin-like growth factor (IGF) family. PAPPA acts as a protease, cleaving IGF inhibitors, i.e., IGF binding proteins (IGFBPs), thereby setting free IGFs. The insulin/IGF-axis is involved in cancer in general and in Ewing sarcoma (ES) in particular. ES is a highly malignant bone tumor characterized by early metastatic spread. PAPPA is associated with various cancers. It is overexpressed and required for proliferation in ES. PAPPA also stimulates normal bone growth. We isolated HLA-A*02:01+/peptide-restricted T cells from A*02:01− healthy donors directed against PAPPA, generated by priming with A*02:01+ PAPPA peptide loaded dendritic cells. After TCR identification, retrovirally TCR transduced CD8+ T cells were assessed for their in vitro specificity and in vivo efficacy in human ES bearing Rag2−/−γc−/− mice. Engraftment in mice and tumor infiltration of TCR transgenic T cells in the mice was evaluated. The TCR transgenic T cell clone PAPPA-2G6 demonstrated specific reactivity toward HLA-A*02:01+/PAPPA+ ES cell lines. We furthermore detected circulating TCR transgenic T cells in the blood in Rag2−/−γc−/− mice and in vivo engraftment in bone marrow. Tumor growth in mice with xenografted ES was significantly reduced after treatment with PAPPA-2G6 TCR transgenic T cells in contrast to controls. Tumors of treated mice revealed tumor-infiltrating PAPPA-2G6 TCR transgenic T cells. In summary, we demonstrate that PAPPA is a first-rate target for TCR-based immunotherapy of ES. PMID:28344885

  5. A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

    PubMed Central

    Prosser, H M; Wotton, D; Gegonne, A; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1992-01-01

    The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1. Images PMID:1409722

  6. Surface expression of functional T cell receptor chains formed by interlocus recombination on human T lymphocytes

    PubMed Central

    1994-01-01

    Structural diversity of lymphocyte antigen receptors (the immunoglobulin [Ig] of B cells and the alpha/beta or gamma/delta T cell receptor [TCR] of T cells) is generated through somatic rearrangements of V, D, and J gene segments. Classically, these recombination events involve gene segments from the same Ig or TCR locus. However, occurrence of "trans" rearrangements between distinct loci has also been described, although in no instances was the surface expression of the corresponding protein under normal physiological conditions demonstrated. Here we show that hybrid TCR genes generated by trans rearrangement between V gamma and (D) J beta elements are translated into functional antigen receptor chains, paired with TCR alpha chains. Like classical alpha/beta T cells, cells expressing these hybrid TCR chains express either CD4 or CD8 coreceptors and are frequently alloreactive. These results have several implications in terms of T cell repertoire selection and relationships between TCR structure and specificity. First, they suggest that TCR alloreactivity is determined by the repertoire selection processes operating during lymphocyte development rather than by structural features specific to V alpha V beta regions. Second, they suggest the existence of close structural relationships between gamma/delta and alpha/beta TCR and more particularly, between V gamma and V beta regions. Finally, since a significant fraction of PBL (at least 1/10(4)) expressed hybrid TCR chains on their surface, these observations indicate that trans rearrangements significantly contribute to the combinatorial diversification of the peripheral immune repertoire. PMID:7964454

  7. Quantifying signaling-induced reorientation of T cell receptors during immunological synapse formation

    PubMed Central

    Moss, William C.; Irvine, Darrell J.; Davis, Mark M.; Krummel, Matthew F.

    2002-01-01

    Productive T cell recognition of antigen-presenting cells (APCs) is normally accompanied by the formation of a cell–cell contact called the “immunological synapse.” Our understanding of the steps leading up to this formation has been limited by the absence of tools for analyzing 3D surfaces and surface distributions as they change over time. Here we use a 3D fluorescence quantitation method to show that T cell receptors are recruited in bulk within the first minute after the onset of activation and with velocities ranging from 0.04 to 0.1 μm/s; a speed significantly greater than unrestricted diffusion. Our method reveals a second feature of this reorientation: a conformational change as the T cell pushes more total membrane into the interface creating a larger contact area for additional receptors. Analysis of individual T cell receptor velocities using a single-particle tracking method confirms our velocity measurement. This method should permit the quantitation of other dynamic membrane events and the associated movement of cell-surface molecules. PMID:12415110

  8. T-Cell Receptor (TCR) Clonotype-Specific Differences in Inhibitory Activity of HIV-1 Cytotoxic T-Cell Clones Is Not Mediated by TCR Alone.

    PubMed

    Flerin, Nina C; Chen, Huabiao; Glover, Tynisha D; Lamothe, Pedro A; Zheng, Jian Hua; Fang, Justin W; Ndhlovu, Zaza M; Newell, Evan W; Davis, Mark M; Walker, Bruce D; Goldstein, Harris

    2017-03-15

    Functional analysis of T-cell responses in HIV-infected individuals has indicated that virus-specific CD8(+) T cells with superior antiviral efficacy are well represented in HIV-1 controllers but are rare or absent in HIV-1 progressors. To define the role of individual T-cell receptor (TCR) clonotypes in differential antiviral CD8(+) T-cell function, we performed detailed functional and mass cytometric cluster analysis of multiple CD8(+) T-cell clones recognizing the identical HLA-B*2705-restricted HIV-1 epitope KK10 (KRWIILGLNK). Effective and ineffective CD8(+) T-cell clones segregated based on responses to HIV-1-infected and peptide-loaded target cells. Following cognate peptide stimulation, effective HIV-specific clones displayed significantly more rapid TCR signal propagation, more efficient initial lytic granule release, and more sustained nonlytic cytokine and chemokine secretion than ineffective clones. To evaluate the TCR clonotype contribution to CD8(+) T-cell function, we cloned the TCR α and β chain genes from one effective and two ineffective CD8(+) T-cell clones from an elite controller into TCR-expressing lentivectors. We show that Jurkat/MA cells and primary CD8(+) T cells transduced with lentivirus expressing TCR from one of the ineffective clones exhibited a level of activation by cognate peptide and inhibition of in vitro HIV-1 infection, respectively, that were comparable to those of the effective clonotype. Taken together, these data suggest that the potent antiviral capacity of some HIV-specific CD8(+) T cells is a consequence of factors in addition to TCR sequence that modulate functionality and contribute to the increased antiviral capacity of HIV-specific CD8(+) T cells in elite controllers to inhibit HIV infection.IMPORTANCE The greater ex vivo antiviral inhibitory activity of CD8(+) T cells from elite controllers than from HIV-1 progressors supports the crucial role of effective HIV-specific CD8(+) T cells in controlling HIV-1

  9. Dominant and shared T cell receptor beta chain variable regions of T cells inducing synovial hyperplasia in rheumatoid arthritis.

    PubMed

    Mima, T; Ohshima, S; Sasai, M; Nishioka, K; Shimizu, M; Murata, N; Yasunami, R; Matsuno, H; Suemura, M; Kishimoto, T; Saeki, Y

    1999-09-16

    Previously, we demonstrated the presence of at least two distinct subpopulations of patients with rheumatoid arthritis (RA) employing a cell-transfer experiment using severe combined immunodeficient (SCID) mice. One group of patients, whose T cells derived from the rheumatoid joints, induced synovial hyperplasia (SH) in the SCID mice (the positive group). The other group did not display the induction of SH (the negative group). TCR/Vbeta gene usage analysis indicated that some dominant T cell subpopulations were oligoclonally expanding only in the rheumatoid joints, and not in the periphery of the patients of the positive group. Moreover, these T cell subpopulations were not seen in the joints of patients in the negative group or in non-RA patients. In addition, the preferential uses of certain TCR/Vbetas (Vbeta8, Vbeta12, Vbeta13, and Vbeta14) genes were demonstrated in these T cells. In this study, to investigate whether these T cells are driven by a certain antigen(s), the third complementarity determining regions (CDR3s) of TCR/Vbeta, especially Vbeta8 and Vbeta14 PCR products, were cloned and sequenced. As a result, a dominant CDR3 sequence, CASS-PRERAT-YEQ, was found in Vbeta14+ T cells from the rheumatoid joint of a patient (Patient 1) of the positive group with a Vbeta14 skew. The identical CDR3 sequence also predominated in Vbeta14+ T cells from the rheumatoid joint of another patient (Patient 7) of the positive group with a Vbeta14 skew. In addition, in the patients (Patients 4, 7, 8) of the positive group with a Vbeta8 skew, other dominant CDR3 sequences, CASS-ENS-YEQ and CASS-LTEP-DTQ, were found as in the case of Vbeta14. However, no identical CDR3 sequences were detected dominantly in the joints of the patients in the negative group or in non-RA patients. A Vbeta14+ T cell clone (TCL), named G3, with the identical CDR3 sequence, CASS-PRERAT-YEQ, was isolated successfully from Patient 1, and cell transfer of G3 with autologous irradiated peripheral

  10. Transduction of human T cells with a novel T-cell receptor confers anti-HCV reactivity.

    PubMed

    Zhang, Yi; Liu, Yeuying; Moxley, Kelly M; Golden-Mason, Lucy; Hughes, Michael G; Liu, Tongxin; Heemskerk, Mirjam H M; Rosen, Hugo R; Nishimura, Michael I

    2010-07-29

    Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world's population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073-1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4(+) and CD8(+) T cells recognized the HCV NS3:1073-1081 peptide-loaded targets and HCV(+) hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-gamma, IL-2, and TNF-alpha) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8(-) Jurkat cells and CD4(+) PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

  11. Aryl hydrocarbon receptor deficiency in T cells suppresses the development of collagen-induced arthritis

    PubMed Central

    Nakahama, Taisuke; Kimura, Akihiro; Nguyen, Nam Trung; Chinen, Ichino; Hanieh, Hamza; Nohara, Keiko; Fujii-Kuriyama, Yoshiaki; Kishimoto, Tadamitsu

    2011-01-01

    The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis have not been elucidated. Here, we show that Ahr deficiency ameliorated collagen-induced arthritis, a mouse model of RA. Collagen-immunized Ahr KO mice showed decreased serum levels of such proinflammatory cytokines as IL-1β and IL-6. The Th17 and Th1 cell populations in lymph nodes from these mice decreased and increased, respectively, whereas the percentage of regulatory T cells was unchanged. Interestingly, a lack of Ahr specifically in T cells significantly suppressed collagen-induced arthritis development, whereas Ahr deficiency in macrophages had no effect. These finding indicate that the development of experimental autoimmune arthritis depends on the presence of Ahr in T cells, and that Th1/Th17 balance may be particularly important for this process. PMID:21825138

  12. Identification of putative human T cell receptor delta complementary DNA clones

    SciTech Connect

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  13. Glucocorticoid receptor in T cells mediates protection from autoimmunity in pregnancy

    PubMed Central

    Engler, Jan Broder; Kursawe, Nina; Solano, María Emilia; Patas, Kostas; Wehrmann, Sabine; Heckmann, Nina; Lühder, Fred; Reichardt, Holger M.; Arck, Petra Clara; Gold, Stefan M.

    2017-01-01

    Pregnancy is one of the strongest inducers of immunological tolerance. Disease activity of many autoimmune diseases including multiple sclerosis (MS) is temporarily suppressed by pregnancy, but little is known about the underlying molecular mechanisms. Here, we investigated the endocrine regulation of conventional and regulatory T cells (Tregs) during reproduction. In vitro, we found the pregnancy hormone progesterone to robustly increase Treg frequencies via promiscuous binding to the glucocorticoid receptor (GR) in T cells. In vivo, T-cell–specific GR deletion in pregnant animals undergoing experimental autoimmune encephalomyelitis (EAE), the animal model of MS, resulted in a reduced Treg increase and a selective loss of pregnancy-induced protection, whereas reproductive success was unaffected. Our data imply that steroid hormones can shift the immunological balance in favor of Tregs via differential engagement of the GR in T cells. This newly defined mechanism confers protection from autoimmunity during pregnancy and represents a potential target for future therapy. PMID:28049829

  14. Inhibition of T cell receptor signaling by cholesterol sulfate, a naturally occurring derivative of membrane cholesterol

    PubMed Central

    Wang, Feng; Beck-García, Katharina; Zorzin, Carina; Schamel, Wolfgang W. A.; Davis, Mark M.

    2016-01-01

    Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor–CD3 complex (TCR). Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. Biochemical studies show that CS disrupted TCR multimers, apparently by displacing cholesterol, known to bind TCRβ. Moreover, CS-deficient mice displayed a heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation. PMID:27213689

  15. Rebalancing immune specificity and function in cancer by T-cell receptor gene therapy

    PubMed Central

    Udyavar, Akshata; Geiger, Terrence L.

    2010-01-01

    Adoptive immunotherapy with tumor-specific T lymphocytes has demonstrated clinical benefit in some cancers, particularly melanoma. Yet isolating and expanding tumor-specific cells from patients is challenging, and there is limited ability to control T cell affinity and response characteristics. T cell receptor (TCR) gene therapy, in which T lymphocytes for immunotherapy are redirected using introduced rearranged TCR, has emerged as an important alternative. Successful TCR gene therapy requires consideration of a number of issues, including TCR specificity and affinity, optimal gene therapy constructs, types of T cells administered, and the survival and activity of the modified cells. In this review, we highlight the rationale for and experience with, as well as new approaches to enhance TCR gene therapy. PMID:20680493

  16. Use of anti-idiotypic antibodies to identify a receptor for the T-cell I-J determinant.

    PubMed Central

    Zupko, K; Waltenbaugh, C; Diamond, B

    1985-01-01

    In order to identify the molecule(s) interacting with the I-J determinant on suppressor T cells, we have generated two anti-idiotypic sera: one to monoclonal anti-I-Jd antibody and one to monoclonal anti-I-Jk antibody. These antisera specifically block suppressor T-cell function in a genetically restricted manner and have no effect on helper T-cell activation. Both recognize a marker on primary monocytes and B cells but not on T cells. A myeloma cell line bearing this marker has been identified. Therefore, these antisera may recognize a molecule on cells interacting with suppressor T cells that is involved in mediating suppressor T-cell activity. The relationship between the T-cell I-J determinant and the molecule identified by the anti-idiotype may be similar to the relationship between the receptor on helper T cells and Ia molecules. Images PMID:2932739

  17. A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

    PubMed

    Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

    2006-09-01

    Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

  18. A Natural Variant of the T Cell Receptor-Signaling Molecule Vav1 Reduces Both Effector T Cell Functions and Susceptibility to Neuroinflammation

    PubMed Central

    Kassem, Sahar; Bernard, Isabelle; Dejean, Anne S.; Liblau, Roland; Fournié, Gilbert J.; Colacios, Céline

    2016-01-01

    The guanine nucleotide exchange factor Vav1 is essential for transducing T cell antigen receptor signals and therefore plays an important role in T cell development and activation. Our previous genetic studies identified a locus on rat chromosome 9 that controls the susceptibility to neuroinflammation and contains a non-synonymous polymorphism in the major candidate gene Vav1. To formally demonstrate the causal implication of this polymorphism, we generated a knock-in mouse bearing this polymorphism (Vav1R63W). Using this model, we show that Vav1R63W mice display reduced susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by MOG35-55 peptide immunization. This is associated with a lower production of effector cytokines (IFN-γ, IL-17 and GM-CSF) by autoreactive CD4 T cells. Despite increased proportion of Foxp3+ regulatory T cells in Vav1R63W mice, we show that this lowered cytokine production is intrinsic to effector CD4 T cells and that Treg depletion has no impact on EAE development. Finally, we provide a mechanism for the above phenotype by showing that the Vav1R63W variant has normal enzymatic activity but reduced adaptor functions. Together, these data highlight the importance of Vav1 adaptor functions in the production of inflammatory cytokines by effector T cells and in the susceptibility to neuroinflammation. PMID:27438086

  19. Epigallocatechin-3-gallate inhibits expression of receptors for T cell regulatory cytokines and their downstream signaling in mouse CD4+ T cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously showed a suppressive effect of epigallocatechin-3-gallate (EGCG) on T cell cycling and expansion as well as a paradoxical effect on IL-2 levels (up-regulating) and IL-2 receptor (IL-2R)alpha expression (down-regulating). Thus, in the current study we tested the hypothesis that EGCG aff...

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

    PubMed

    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.

  1. Insights into the Relationship between Toll Like Receptors and Gamma Delta T Cell Responses

    PubMed Central

    Dar, Asif Amin; Patil, Rushikesh Sudam; Chiplunkar, Shubhada Vivek

    2014-01-01

    The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, tumor progression and responses to therapy. The composition and characteristics of the tumor microenvironment vary widely and are important in determining the anti-tumor immune response. Successful immunization requires activation of both innate and adaptive immunity. Generally, immune system is compromised in patients with cancer due to immune suppression, loss of tumor antigen expression and dysfunction of antigen presenting cells (APC). Thus, therapeutic immunization leading to cancer regression remains a significant challenge. Certain cells of the immune system, including dendritic cells (DCs) and gamma delta (γδ) T cells are capable of driving potent anti-tumor responses. The property of MHC-unrestricted cytotoxicity, high potential of cytokine release, tissue tropism and early activation in infections and malignant disease makes γδ T cells as an emerging candidate for immunotherapy. Various strategies are being developed to enhance anti-tumor immune responses of γδ T cells and DCs one of them is the use of novel adjuvants like toll like receptors (TLR) agonists, which enhance γδ T cell function directly or through DC activation, which has ability to prime γδ T cells. TLR agonists are being used clinically either alone or in combination with tumor antigens and has shown initial success in both enhancing immune responses and eliciting anti-tumor activity. TLR activated γδ T cells and DCs nurture each other’s activation. This provides a potent base for first line of defense and manipulation of the adaptive response against pathogens and cancer. The available data provides a strong rationale for initiating combinatorial therapy for the treatment of diseases and this review will summarize the application of adjuvants (TLRs) for boosting immune response of γδ T cells to treat cancer and infectious diseases and their use in combinatorial therapy

  2. Biological characterization and partial purification of an idiotype and antigen specific T cell lymphokine

    SciTech Connect

    Bowen, M.E.

    1987-01-01

    An idiotype (Id) and antigen-specific T cell lymphokine has been partially purified and characterized biologically. This lymphokine appears to be derived from the T helper/sub 2/ (Th/sub 2/) lymphocyte and plays a key role in the optimal expression of the cross-reactive idiotype (CRI/sup +/-TMA) associated with both anti-phenyltrimethylammonium (TMA) and anti-trinitrophenyl (TNP) antibodies. An apparent molecular weight of 30-35 Kd was determined using molecular sieve chromatography. Upon SDS-polyacrylamide gel electrophoresis (SDS-PAGE) however, the biological activity migrated to 68 Kd as well as 35 Kd. Equivalent amounts of activity are found in both SDS-PAGE fractions. The Th/sub 2/F has two isoelectric points, 5.7 and 6.3, although 99% of the activity is found at pH 6.3. The Id-enhancing factor is an acid stable and heat labile protein. As in the case for the expression of serum CRI-TMA, the production of the Th/sub 2/F is linked to the allotype (Igh-1/sup e/) of the heavy chain locus. Using Con A Sns from various genetically distinct strains of mice, it has been shown that the production of the Th/sub 2/F is allotype-linked, and works across major histocompatibility (MHC) barriers. Isolation of Th/sub 2/F has been carried out using a combination of affinity chromatography and gel filtration. The partially purified material has been /sup 125/I labeled and analyzed by SDS-PAGE and flat bed isoelectric focusing. Two radiolabeled proteins which could be the Th/sub 2/F were identified.

  3. Changing the peptide specificity of a human T cell receptor by directed evolution

    PubMed Central

    Smith, Sheena N.; Wang, Yuhang; Baylon, Javier L.; Singh, Nishant K.; Baker, Brian M.; Tajkhorshid, Emad; Kranz, David M.

    2014-01-01

    Binding of a T cell receptor (TCR) to a peptide/major histocompatibility complex is the key interaction involved in antigen specificity of T cells. The recognition involves up to six complementarity determining regions (CDR) of the TCR. Efforts to examine the structural basis of these interactions and to exploit them in adoptive T cell therapies has required the isolation of specific T cell clones and their clonotypic TCRs. Here we describe a strategy using in vitro, directed evolution of a single TCR to change its peptide specificity, thereby avoiding the need to isolate T cell clones. The human TCR A6, which recognizes the viral peptide Tax/HLA-A2, was converted to TCR variants that recognized the cancer peptide MART1/HLA-A2. Mutational studies and molecular dynamics simulations identified CDR residues that were predicted to be important in the specificity switch. Thus, in vitro engineering strategies alone can be used to discover TCRs with desired specificities. PMID:25376839

  4. T cell receptor peptide therapy for autoimmune encephalomyelitis: stronger immunization is necessary for effective vaccination.

    PubMed

    Matsumoto, Y; Tsuchida, M; Hanawa, H; Abo, T

    1994-02-01

    Although T cell receptor (TCR) peptide therapy was initially reported to be a very effective method for prevention of the development of experimental autoimmune encephalomyelitis (EAE), it was recently demonstrated that the same peptide immunization led to enhanced and chronic EAE in some cases. In the present study, we examined the effect of the TCR peptide (V beta 8.2-39-59) vaccination on the development of EAE by employing several immunization protocols. We found that TCR peptide vaccination effectively prevented EAE development only when the peptide was injected with Mycobacterium tuberculosis-enriched CFA in the vicinity of the challenge site. Under such conditions, a sufficient number of peptide-reactive T cells were generated. Flow cytometry and immunohistochemical analyses using anti-peptide antibody and anti-V beta 8.2 mAb revealed that despite the presence of V beta 8.2+ cells, very few peptide-positive T cells appeared in the lymphoid organs throughout the course of EAE. These findings imply that antibodies that are generated after immunization with V beta 8P are hardly accessible to their specific epitopes in the native protein. Insufficient generation of both T cells and antibodies against V beta 8.2-positive T cells may be attributable to the outcome of the therapy. To establish effective TCR peptide immunotherapy, these disadvantages should be overcome by using other TCR sequences and/or by employing a more suitable adjuvant.

  5. Those other mammals: the immunoglobulins and T cell receptors of marsupials and monotremes.

    PubMed

    Miller, Robert D

    2010-02-01

    This review summarizes analyses of marsupial and monotreme immunoglobulin and T cell receptor genetics and expression published over the past decade. Analyses of recently completed whole genome sequences from the opossum and the platypus have yielded insight into the evolution of the common antigen receptor systems, as well as discovery of novel receptors that appear to have been lost in eutherian mammals. These species are also useful for investigation of the development of the immune system in organisms notable for giving birth to highly altricial young, as well as the evolution of maternal immunity through comparison of oviparous and viviparous mammals.

  6. Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.

    PubMed

    Freeman, J Douglas; Warren, René L; Webb, John R; Nelson, Brad H; Holt, Robert A

    2009-10-01

    T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

  7. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

    PubMed Central

    Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

    2016-01-01

    The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

  8. An altered repertoire of T cell receptor V gene expression by rheumatoid synovial fluid T lymphocytes.

    PubMed Central

    Lunardi, C; Marguerie, C; So, A K

    1992-01-01

    The pattern of T cell receptor V gene expression by lymphocytes from rheumatoid synovial fluid and paired peripheral blood samples was compared using a polymerase chain reaction (PCR)-based assay. Eight rheumatoid arthritis (RA) patients who had varying durations of disease (from 2 to 20 years) were studied. In all patients there was evidence of a different pattern of V gene expression between the two compartments. Significantly increased expression of at least one V alpha or V beta gene family by synovial fluid T cells was observed in all the patients studied. Three different V alpha (V alpha 10, 15 and 18) and three V beta (V beta 4, 5 and 13) families were commonly elevated. Sequencing of synovial V beta transcripts demonstrated that the basis of increased expression of selected V gene families in the synovial fluid was due to the presence of dominant clonotypes within those families, which constituted up to 53% of the sequences isolated from one particular synovial V gene family. There were considerable differences in the NDJ sequences found in synovial and peripheral blood T cell receptor (TCR) transcripts of the same V beta gene family. These data suggest that the TCR repertoire in the two compartments differs, and that antigen-driven expansion of particular synovial T cell populations is a component of rheumatoid synovitis, and is present in all stages of the disease. PMID:1458680

  9. Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.

    PubMed

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.

  10. Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

    PubMed

    Thommen, Daniela S; Schreiner, Jens; Müller, Philipp; Herzig, Petra; Roller, Andreas; Belousov, Anton; Umana, Pablo; Pisa, Pavel; Klein, Christian; Bacac, Marina; Fischer, Ozana S; Moersig, Wolfgang; Savic Prince, Spasenija; Levitsky, Victor; Karanikas, Vaios; Lardinois, Didier; Zippelius, Alfred

    2015-12-01

    Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

  11. VCP, the mammalian homolog of cdc48, is tyrosine phosphorylated in response to T cell antigen receptor activation.

    PubMed Central

    Egerton, M; Ashe, O R; Chen, D; Druker, B J; Burgess, W H; Samelson, L E

    1992-01-01

    Activation of T cells through the T cell antigen receptor (TCR) results in the rapid tyrosine phosphorylation of a number of cellular proteins, one of the earliest being a 100 kDa protein. We have sought to identify this 100 kDa substrate by partially purifying the protein by antiphosphotyrosine (APT) affinity purification, in order to obtain amino acid sequence data and, using this information, to isolate the cDNA clone encoding the molecule. We report here that the amino acid sequence data showed pp100 to be the murine equivalent of porcine valosin containing protein (VCP), a finding confirmed from the cloning and sequencing of the murine pp100 cDNA. Sequence analysis has shown VCP to be a member of a family of ATP binding, homo-oligomeric proteins, and the mammalian homolog of Saccharomyces cerevisiae cdc48p, a protein essential to the completion of mitosis in yeast. We also provide proof that both endogenous and expressed murine VCP are tyrosine phosphorylated in response to T cell activation. Thus we have identified a novel component of the TCR mediated tyrosine kinase activation pathway that may provide a link between TCR ligation and cell cycle control. Images PMID:1382975

  12. Treatment of solid tumors with chimeric antigen receptor-engineered T cells: current status and future prospects.

    PubMed

    Di, Shengmeng; Li, Zonghai

    2016-04-01

    Chimeric antigen receptors (CARs) are artificial recombinant receptors that generally combine the antigen-recognition domain of a monoclonal antibody with T cell activation domains. Recent years have seen great success in clinical trials employing CD19-specific CAR-T cell therapy for B cell leukemia. Nevertheless, solid tumors remain a major challenge for CAR-T cell therapy. This review summarizes the preclinical and clinical studies on the treatment of solid tumors with CAR-T cells. The major hurdles for the success of CAR-T and the novel strategies to address these hurdles have also been described and discussed.

  13. T-cell receptor gamma--delta lymphocytes and Eimeria vermiformis infection.

    PubMed Central

    Rose, M E; Hesketh, P; Rothwell, L; Gramzinski, R A

    1996-01-01

    The role of T-cell receptor gamma--delta T lymphocytes in coccidiosis was examined by determining the course of infection with Eimeria vermiformis in BALB/c mice depleted of gamma--delta lymphocytes by treatment with GL3 monoclonal antibody. The replication of the parasite in primary infections was not greatly, or consistently, affected by this treatment, and there was no correlation between the extent of depletion of small intestinal intraepithelial lymphocytes and the number of oocysts produced. The resistance of immunized mice to challenge was not compromised by depletion of intraintestinal epithelial lymphocytes when their depletion was effected at the time of primary infection and/or administration of the challenge inoculum. Thus, T-cell receptor gamma--delta T lymphocytes do not appear to be crucial to the establishment, or the control, of primary infection with E. vermiformis and are not principal mediators of the solid immunity to challenge that this infection induces. PMID:8890252

  14. Ultra-deep T cell receptor sequencing reveals the complexity and intratumour heterogeneity of T cell clones in renal cell carcinomas

    PubMed Central

    Gerlinger, Marco; Quezada, Sergio A; Peggs, Karl S; Furness, Andrew JS; Fisher, Rosalie; Marafioti, Teresa; Shende, Vishvesh H; McGranahan, Nicholas; Rowan, Andrew J; Hazell, Steven; Hamm, David; Robins, Harlan S; Pickering, Lisa; Gore, Martin; Nicol, David L; Larkin, James; Swanton, Charles

    2013-01-01

    The recognition of cancer cells by T cells can impact upon prognosis and be exploited for immunotherapeutic approaches. This recognition depends on the specific interaction between antigens displayed on the surface of cancer cells and the T cell receptor (TCR), which is generated by somatic rearrangements of TCR α- and β-chains (TCRb). Our aim was to assess whether ultra-deep sequencing of the rearranged TCRb in DNA extracted from unfractionated clear cell renal cell carcinoma (ccRCC) samples can provide insights into the clonality and heterogeneity of intratumoural T cells in ccRCCs, a tumour type that can display extensive genetic intratumour heterogeneity (ITH). For this purpose, DNA was extracted from two to four tumour regions from each of four primary ccRCCs and was analysed by ultra-deep TCR sequencing. In parallel, tumour infiltration by CD4, CD8 and Foxp3 regulatory T cells was evaluated by immunohistochemistry and correlated with TCR-sequencing data. A polyclonal T cell repertoire with 367–16 289 (median 2394) unique TCRb sequences was identified per tumour region. The frequencies of the 100 most abundant T cell clones/tumour were poorly correlated between most regions (Pearson correlation coefficient, –0.218 to 0.465). 3–93% of these T cell clones were not detectable across all regions. Thus, the clonal composition of T cell populations can be heterogeneous across different regions of the same ccRCC. T cell ITH was higher in tumours pretreated with an mTOR inhibitor, which could suggest that therapy can influence adaptive tumour immunity. These data show that ultra-deep TCR-sequencing technology can be applied directly to DNA extracted from unfractionated tumour samples, allowing novel insights into the clonality of T cell populations in cancers. These were polyclonal and displayed ITH in ccRCC. TCRb sequencing may shed light on mechanisms of cancer immunity and the efficacy of immunotherapy approaches. Copyright © 2013 Pathological Society of

  15. Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones.

    PubMed Central

    De Magistris, M T; Di Tommaso, A; Domenighini, M; Censini, S; Tagliabue, A; Oksenberg, J R; Steinman, L; Judd, A K; O'Sullivan, D; Rappuoli, R

    1992-01-01

    The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines. Images PMID:1313575

  16. Interleukin-21 (IL-21) synergizes with IL-2 to enhance T-cell receptor-induced human T-cell proliferation and counteracts IL-2/transforming growth factor-β-induced regulatory T-cell development

    PubMed Central

    Battaglia, Alessandra; Buzzonetti, Alexia; Baranello, Cinzia; Fanelli, Mara; Fossati, Marco; Catzola, Valentina; Scambia, Giovanni; Fattorossi, Andrea

    2013-01-01

    Interleukin-2 (IL-2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL-2 facilitates regulatory T (Treg) cell development. IL-21 is a type I cytokine acting as a potent T-cell co-mitogen but less efficient than IL-2 in sustaining T-cell proliferation. Using various in vitro models for T-cell receptor (TCR)-dependent human T-cell proliferation, we found that IL-21 synergized with IL-2 to make CD4+ and CD8+ T cells attain a level of expansion that was impossible to obtain with IL-2 alone. Synergy was mostly evident in naive CD4+ cells. IL-2 and tumour-released transforming growth factor-β (TGF-β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin-21 hampered Treg cell expansion induced by IL-2/TGF-β combination in naive CD4+ cells by facilitating non-Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL-21 did not modulate the conversion of naive activated CD4+ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down-modulation of IL-2/TGF-β-induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL-21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4+ and CD8+ T cells. Present data provide proof-of-concept for evaluating a combinatorial approach that would reduce the IL-2 needed to sustain T-cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T-cell response. PMID:23278180

  17. Circulating regulatory anti–T cell receptor antibodies in patients with myasthenia gravis

    PubMed Central

    Jambou, Florence; Zhang, Wei; Menestrier, Monique; Klingel-Schmitt, Isabelle; Michel, Olivier; Caillat-Zucman, Sophie; Aissaoui, Abderrahim; Landemarre, Ludovic; Berrih-Aknin, Sonia; Cohen-Kaminsky, Sylvia

    2003-01-01

    Serum anti–T cell receptor (TCR) Ab’s are involved in immune regulation directed against pathogenic T cells in experimental models of autoimmune diseases. Our identification of a dominant T cell population expressing the Vβ5.1 TCR gene (TCRBV5-1), which is responsible for the production of pathogenic anti-acetylcholine receptor (AChR) autoantibodies in HLA-DR3 patients with early-onset myasthenia gravis (EOMG), prompted us to explore the occurrence, reactivity, and regulatory role of anti-TCR Ab’s in EOMG patients and disease controls with clearly defined other autoantibodies. In the absence of prior vaccination against the TCR, EOMG patients had elevated anti-Vβ5.1 Ab’s of the IgG class. This increase was restricted largely to EOMG cases with HLA-DR3 and with less severe disease, and it predicted clinical improvement in follow-up studies. EOMG patient sera containing anti-TCR Ab’s bound specifically the native TCR on intact Vβ5.1-expressing cells and specifically inhibited the proliferation and IFN-γ production of purified Vβ5.1-expressing cells to alloantigens in mixed lymphocyte reaction and the proliferation of a Vβ5.1-expressing T cell clone to an AChR peptide, indicating a regulatory function for these Ab’s. This evidence of spontaneously active anti-Vβ5.1 Ab’s in EOMG patients suggests dynamic protective immune regulation directed against the excess of pathogenic Vβ5.1-expressing T cells. Though not sufficient to prevent a chronic, exacerbated autoimmune process, it might be boosted using a TCR peptide as vaccine. PMID:12865414

  18. Stimulatory and Inhibitory Killer Immunoglobulin-Like Receptor Molecules are Expressed and Functional on Lupus T Cells1

    PubMed Central

    Basu, Dhiman; Liu, Ying; Wu, Ailing; Yarlagadda, Sushma; Gorelik, Gabriela J.; Kaplan, Mariana J.; Hewagama, Anura; Hinderer, Robert C.; Strickland, Faith M.; Richardson, Bruce C.

    2009-01-01

    T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell immunoglobulin–like receptor (KIR3) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Antibodies to the stimulatory molecule KIR2DL4 triggered IFN-γ release by lupus T cells, and production was proportional to disease activity. Similarly, crosslinking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and suggest that antibodies to inhibitory KIR may be a treatment for this disease. PMID:19675166

  19. Stimulatory and inhibitory killer Ig-like receptor molecules are expressed and functional on lupus T cells.

    PubMed

    Basu, Dhiman; Liu, Ying; Wu, Ailing; Yarlagadda, Sushma; Gorelik, Gabriela J; Kaplan, Mariana J; Hewagama, Anura; Hinderer, Robert C; Strickland, Faith M; Richardson, Bruce C

    2009-09-01

    T cells from lupus patients have hypomethylated DNA and overexpress genes normally suppressed by DNA methylation that contribute to disease pathogenesis. We found that stimulatory and inhibitory killer cell Ig-like receptor (KIR) genes are aberrantly overexpressed on experimentally demethylated T cells. We therefore asked if lupus T cells also overexpress KIR, and if the proteins are functional. T cells from lupus patients were found to overexpress KIR genes, and expression was proportional to disease activity. Abs to the stimulatory molecule KIR2DL4 triggered IFN-gamma release by lupus T cells, and production was proportional to disease activity. Similarly, cross-linking the inhibitory molecule KIR3DL1 prevented the autoreactive macrophage killing that characterizes lupus T cells. These results indicate that aberrant T cell KIR expression may contribute to IFN overproduction and macrophage killing in human lupus, and they suggest that Abs to inhibitory KIR may be a treatment for this disease.

  20. Immunotherapy of HCC metastases with autologous T cell receptor redirected T cells, targeting HBsAg in a liver transplant patient.

    PubMed

    Qasim, Waseem; Brunetto, Maurizia; Gehring, Adam J; Xue, Shao-An; Schurich, Anna; Khakpoor, Atefeh; Zhan, Hong; Ciccorossi, Pietro; Gilmour, Kimberly; Cavallone, Daniela; Moriconi, Francesco; Farzhenah, Farzin; Mazzoni, Alessandro; Chan, Lucas; Morris, Emma; Thrasher, Adrian; Maini, Mala K; Bonino, Ferruccio; Stauss, Hans; Bertoletti, Antonio

    2015-02-01

    HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.

  1. Proliferation of thymocytes in relation to T-cell receptor beta-chain expression.

    PubMed

    Parkin, I G; Owen, J J; Jenkinson, E J

    1988-05-01

    During proliferation and differentiation of maturing thymocytes, T-cell receptor beta-chain products are first expressed in the cytoplasm. Only subsequently are they expressed on the cell surface, presumably as part of the alpha beta/CD3 receptor complex. This study uses double immunofluorescence labelling to identify these cytoplasmic and surface phases separately in relationship to cell-cycle parameters. The use of a mitotic arrest agent and tritiated thymidine autoradiography both show that cells with cytoplasmic beta-chains are in cell cycle, whereas cells with surface beta-chains are cycling slowly, if at all.

  2. Antigen-receptor gene-modified T cells for treatment of glioma.

    PubMed

    Ikeda, Hiroaki; Shiku, Hiroshi

    2012-01-01

    Immunological effector cells and molecules have been shown to access intracranial tumor sites despite the existence of blood brain barrier (BBB) or immunosuppressive mechanisms associated with brain tumors. Recent progress in T-cell biology and tumor immunology made possible to develop strategies of tumor-associated antigen-specific immunotherapeutic approaches such as vaccination with defined antigens and adoptive T-cell therapy with antigen-specific T cells including gene-modified T cells for the treatment of patients with brain tumors. An array of recent reports on the trials of active and passive immunotherapy for patients with brain tumors have documented safety and some preliminary clinical efficacy, although the ultimate judgment for clinical benefits awaits rigorous evaluation in trials of later phases. Nevertheless, treatment with lymphocytes that are engineered to express tumor-specific receptor genes is a promising immunotherapy against glioma, based on the significant efficacy reported in the trials for patients with other types of malignancy. Overcoming the relative difficulty to apply immunotherapeutic approach to intracranial region, current advances in the understanding of human tumor immunology and the gene-therapy methodology will address the development of effective immunotherapy of brain tumors.

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

    PubMed

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

    2016-03-01

    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.

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

    PubMed Central

    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

  5. Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire

    PubMed Central

    Sims, Jennifer S.; Grinshpun, Boris; Feng, Yaping; Ung, Timothy H.; Neira, Justin A.; Samanamud, Jorge L.; Canoll, Peter; Shen, Yufeng; Sims, Peter A.; Bruce, Jeffrey N.

    2016-01-01

    Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a “signature” set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy. PMID:27261081

  6. First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

    PubMed

    Borroto, Aldo; Reyes-Garau, Diana; Jiménez, M Angeles; Carrasco, Esther; Moreno, Beatriz; Martínez-Pasamar, Sara; Cortés, José R; Perona, Almudena; Abia, David; Blanco, Soledad; Fuentes, Manuel; Arellano, Irene; Lobo, Juan; Heidarieh, Haleh; Rueda, Javier; Esteve, Pilar; Cibrián, Danay; Martinez-Riaño, Ana; Mendoza, Pilar; Prieto, Cristina; Calleja, Enrique; Oeste, Clara L; Orfao, Alberto; Fresno, Manuel; Sánchez-Madrid, Francisco; Alcamí, Antonio; Bovolenta, Paola; Martín, Pilar; Villoslada, Pablo; Morreale, Antonio; Messeguer, Angel; Alarcon, Balbino

    2016-12-21

    Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

  7. T-Cell Receptor Gene Therapy of Established Tumors in a Murine Melanoma Model

    PubMed Central

    Abad, John D.; Wrzensinski, Claudia; Overwijk, Willem; De Witte, Moniek A.; Jorritsma, Annelies; Hsu, Gary; Gattinoni, Luca; Cohen, Cyrille J.; Paulos, Chrystal M.; Palmer, Douglas C.; Haanen, John B. A. G.; Schumacher, Ton N. M.; Rosenberg, Steven A.; Restifo, Nicholas P.; Morgan, Richard A.

    2008-01-01

    Summary Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-γ secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies. PMID:18157006

  8. T-cell receptor gene therapy of established tumors in a murine melanoma model.

    PubMed

    Abad, John D; Wrzensinski, Claudia; Overwijk, Willem; De Witte, Moniek A; Jorritsma, Annelies; Hsu, Cary; Gattinoni, Luca; Cohen, Cyrille J; Paulos, Chrystal M; Palmer, Douglas C; Haanen, John B A G; Schumacher, Ton N M; Rosenberg, Steven A; Restifo, Nicholas P; Morgan, Richard A

    2008-01-01

    Adoptive cell transfer therapy using tumor-infiltrating lymphocytes for patients with metastatic melanoma has demonstrated significant objective response rates. One major limitation of these current therapies is the frequent inability to isolate tumor-reactive lymphocytes for treatment. Genetic engineering of peripheral blood lymphocytes with retroviral vectors encoding tumor antigen-specific T-cell receptors (TCRs) bypasses this restriction. To evaluate the efficacy of TCR gene therapy, a murine treatment model was developed. A retroviral vector was constructed encoding the pmel-1 TCR genes targeting the B16 melanoma antigen, gp100. Transduction of C57BL/6 lymphocytes resulted in efficient pmel-1 TCR expression. Lymphocytes transduced with this retrovirus specifically recognized gp100-pulsed target cells as measured by interferon-gamma secretion assays. Upon transfer into B16 tumor-bearing mice, the genetically engineered lymphocytes significantly slowed tumor development. The effectiveness of tumor treatment was directly correlated with the number of TCR-engineered T cells administered. These results demonstrated that TCR gene therapy targeting a native tumor antigen significantly delayed the growth of established tumors. When C57BL/6 lymphocytes were added to antigen-reactive pmel-1 T cells, a reduction in the ability of pmel-1 T cell to treat B16 melanomas was seen, suggesting that untransduced cells may be deleterious to TCR gene therapy. This model may be a powerful tool for evaluating future TCR gene transfer-based strategies.

  9. Expression and function of a variant T cell receptor complex lacking CD3-gamma

    PubMed Central

    1991-01-01

    A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface- radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3- epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma. PMID:1713248

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

    PubMed Central

    Lawrence, Christina; Pelikan, Richard C.; Moore, Jacen S.; Pan, Zijian; Radfar, Lida; Lewis, David M.; Grundahl, Kiely M.; Kelly, Jennifer A.; Wiley, Graham B.; 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

  11. New role for the (pro)renin receptor in T-cell development.

    PubMed

    Geisberger, Sabrina; Maschke, Ulrike; Gebhardt, Matthias; Kleinewietfeld, Markus; Manzel, Arndt; Linker, Ralf A; Chidgey, Ann; Dechend, Ralf; Nguyen, Genevieve; Daumke, Oliver; Muller, Dominik N; Wright, Mark D; Binger, Katrina J

    2015-07-23

    The (pro)renin receptor (PRR) was originally thought to be important for regulating blood pressure via the renin-angiotensin system. However, it is now emerging that PRR has instead a generic role in cellular development. Here, we have specifically deleted PRR from T cells. T-cell-specific PRR-knockout mice had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4(+) and CD8(+) thymocytes, and a large increase in double-negative (DN) precursors. Gene expression analysis on sorted DN3 thymocytes indicated that PRR-deficient thymocytes have perturbations in key cellular pathways essential at the DN3 stage, including transcription and translation. Further characterization of DN T-cell progenitors leads us to propose that PRR deletion affects thymocyte survival and development at multiple stages; from DN3 through to DN4, double-positive, and single-positive CD4 and CD8. Our study thus identifies a new role for PRR in T-cell development.

  12. ɣδ T cell receptor ligands and modes of antigen recognition

    PubMed Central

    Champagne, Eric

    2011-01-01

    T lymphocytes expressing the γδ-type of T cell receptors for antigens contribute to all aspects of immune responses, including defenses against viruses, bacteria, parasites and tumors, allergy and autoimmunity. Multiple subsets have been individualized in humans as well as in mice and they appear to recognize in a TCR-dependent manner antigens as diverse as small non-peptidic molecules, soluble or membrane-anchored polypeptides and molecules related to MHC antigens on cell surfaces, implying diverse modes of antigen recognition. We review here the γδ TCR ligands which have been identified along the years and their characteristics, with emphasis on a few systems which have been extensively studied such as human γδ T cells responding to phosphoantigens or murine γδ T cells activated by allogeneic MHC antigens. We discuss a speculative model of antigen recognition involving simultaneous TCR recognition of MHC-like and non-MHC ligands which could fit with most available data and shares many similarities with the classical model of MHC-restricted antigen recognition for peptides or lipids by T cells subsets with αβ-type TCRs. PMID:21298486

  13. T-cell receptor analysis in Omenn's syndrome: evidence for defects in gene rearrangement and assembly.

    PubMed

    Brooks, E G; Filipovich, A H; Padgett, J W; Mamlock, R; Goldblum, R M

    1999-01-01

    Patients with Omenn's syndrome have a form of severe immune deficiency that is associated with pathological features of graft-versus-host disease, except for the lack of foreign engraftment. It has been hypothesized that the disease's unique clinical features are mediated by an expanded population of autologous self-reactive T cells of limited clonality. In the current study, an investigation of the T-cell receptor (TCR) repertoire was undertaken to identify defects in T-cell rearrangement and development. The TCR repertoire in this group of patients was exquisitely restricted in the number of different TCR clonotypes, and some of these clonotypes seemed to have similar recognition motifs in the antigen-binding region, indicating antigen-driven proliferation of T lymphocytes. The TCRs from some patients lacked N- or P-nucleotide insertions and used proximal variable and joining gene segments, suggesting abnormal intrathymic T-cell development. Finally, abnormal assembly of gene segments and truncated rearrangements within nonproductive alleles suggested abnormalities in TCR rearrangement mechanisms. Overall, the findings suggest that inefficient and/or abnormal generation of TCRs may be a consistent feature of this disease.

  14. Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

    PubMed Central

    Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

    2016-01-01

    The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

  15. T cell receptor restriction of diabetogenic autoimmune NOD T cells

    PubMed Central

    Simone, E.; Daniel, D.; Schloot, N.; Gottlieb, P.; Babu, S.; Kawasaki, E.; Wegmann, D.; Eisenbarth, G. S.

    1997-01-01

    Restricted use of T cell receptor (TCR) gene segments is characteristic of several induced autoimmune disease models. TCR sequences have previously been unavailable for pathogenic T cells which react with a defined autoantigen in a spontaneous autoimmune disease. The majority of T cell clones, derived from islets of NOD mice which spontaneously develop type I diabetes, react with insulin peptide B-(9–23). We have sequenced the α and β chains of TCRs from these B-(9–23)-reactive T cell clones. No TCR β chain restriction was found. In contrast, the clones (10 of 13) used Vα13 coupled with one of two homologous Jα segments (Jα45 or Jα34 in 8 of 13 clones). Furthermore, 9 of 10 of the Vα13 segments are a novel NOD sequence that we have tentatively termed Vα13.3. This dramatic α chain restriction, similar to the β chain restriction of other autoimmune models, provides a target for diagnostics and immunomodulatory therapy. PMID:9122227

  16. Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

    PubMed

    Molnár, Eszter; Swamy, Mahima; Holzer, Martin; Beck-García, Katharina; Worch, Remigiusz; Thiele, Christoph; Guigas, Gernot; Boye, Kristian; Luescher, Immanuel F; Schwille, Petra; Schubert, Rolf; Schamel, Wolfgang W A

    2012-12-14

    The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRβ chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRβ chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

  17. A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

    PubMed Central

    Cartellieri, Marc; Koristka, Stefanie; Arndt, Claudia; Feldmann, Anja; Stamova, Slava; von Bonin, Malte; Töpfer, Katrin; Krüger, Thomas; Geib, Mathias; Michalk, Irene; Temme, Achim; Bornhäuser, Martin; Lindemann, Dirk; Ehninger, Gerhard; Bachmann, Michael P.

    2014-01-01

    Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes. PMID:24699869

  18. Statistical inference of the generation probability of T-cell receptors from sequence repertoires.

    PubMed

    Murugan, Anand; Mora, Thierry; Walczak, Aleksandra M; Callan, Curtis G

    2012-10-02

    Stochastic rearrangement of germline V-, D-, and J-genes to create variable coding sequence for certain cell surface receptors is at the origin of immune system diversity. This process, known as "VDJ recombination", is implemented via a series of stochastic molecular events involving gene choices and random nucleotide insertions between, and deletions from, genes. We use large sequence repertoires of the variable CDR3 region of human CD4+ T-cell receptor beta chains to infer the statistical properties of these basic biochemical events. Because any given CDR3 sequence can be produced in multiple ways, the probability distribution of hidden recombination events cannot be inferred directly from the observed sequences; we therefore develop a maximum likelihood inference method to achieve this end. To separate the properties of the molecular rearrangement mechanism from the effects of selection, we focus on nonproductive CDR3 sequences in T-cell DNA. We infer the joint distribution of the various generative events that occur when a new T-cell receptor gene is created. We find a rich picture of correlation (and absence thereof), providing insight into the molecular mechanisms involved. The generative event statistics are consistent between individuals, suggesting a universal biochemical process. Our probabilistic model predicts the generation probability of any specific CDR3 sequence by the primitive recombination process, allowing us to quantify the potential diversity of the T-cell repertoire and to understand why some sequences are shared between individuals. We argue that the use of formal statistical inference methods, of the kind presented in this paper, will be essential for quantitative understanding of the generation and evolution of diversity in the adaptive immune system.

  19. Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer.

    PubMed

    Feng, Kaichao; Guo, Yelei; Dai, Hanren; Wang, Yao; Li, Xiang; Jia, Hejin; Han, Weidong

    2016-05-01

    The successes achieved by chimeric antigen receptor-modified T (CAR-T) cells in hematological malignancies raised the possibility of their use in non-small lung cancer (NSCLC). In this phase I clinical study (NCT01869166), patients with epidermal growth factor receptor (EGFR)-positive (>50% expression), relapsed/refractory NSCLC received escalating doses of EGFR-targeted CAR-T cell infusions. The EGFR-targeted CAR-T cells were generated from peripheral blood after a 10 to 13-day in vitro expansion. Serum cytokines in peripheral blood and copy numbers of CAR-EGFR transgene in peripheral blood and in tissue biopsy were monitored periodically. Clinical responses were evaluated with RECIST1.1 and immune- related response criteria, and adverse events were graded with CTCAE 4.0. The EGFR-targeted CAR-T cell infusions were well-tolerated without severe toxicity. Of 11 evaluable patients, two patients obtained partial response and five had stable disease for two to eight months. The median dose of transfused CAR(+) T cells was 0.97×10(7) cells kg(-1) (interquartile range (IQR), 0.45 to 1.09×10(7) cells kg(-1)). Pathological eradication of EGFR positive tumor cells after EGFR-targeted CAR-T cell treatment can be observed in tumor biopsies, along with the CAR-EGFR gene detected in tumor-infiltrating T cells in all four biopsied patients. The EGFR-targeted CAR-T cell therapy is safe and feasible for EGFR-positive advanced relapsed/refractory NSCLC.

  20. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

    PubMed Central

    Witkover, Aviva; Tanaka, Yuetsu; Fields, Paul; Bangham, Charles R. M.

    2016-01-01

    There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. PMID:27893842

  1. T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells.

    PubMed

    Rowan, Aileen G; Witkover, Aviva; Melamed, Anat; Tanaka, Yuetsu; Cook, Lucy B M; Fields, Paul; Taylor, Graham P; Bangham, Charles R M

    2016-11-01

    There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease.

  2. Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4

    PubMed Central

    Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M.; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L.; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A.; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L.; Burgdorf, Sven

    2016-01-01

    The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8+ T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality. PMID:27601670

  3. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

    PubMed Central

    Baixauli, Francesc; Martín-Cófreces, Noa B; Morlino, Giulia; Carrasco, Yolanda R; Calabia-Linares, Carmen; Veiga, Esteban; Serrador, Juan M; Sánchez-Madrid, Francisco

    2011-01-01

    During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse. PMID:21326213

  4. The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse.

    PubMed

    Baixauli, Francesc; Martín-Cófreces, Noa B; Morlino, Giulia; Carrasco, Yolanda R; Calabia-Linares, Carmen; Veiga, Esteban; Serrador, Juan M; Sánchez-Madrid, Francisco

    2011-04-06

    During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

  5. Toll-Like Receptor 3 Ligand Dampens Liver Inflammation by Stimulating Vα14 Invariant Natural Killer T Cells to Negatively Regulate γδT Cells

    PubMed Central

    Gardner, Tommy R.; Chen, Qingling; Jin, Yijun; Ajuebor, Maureen N.

    2010-01-01

    Vα14 invariant natural killer T (Vα14iNKT) cells are at the interface between the innate and adaptive immune responses and are thus critical for providing full engagement of host defense. We investigated the role of polyriboinosinic:polycytidylic acid (poly I:C), a replication-competent viral double-stranded RNA mimic and a specific agonist that recognizes the cellular sensor Toll-like receptor 3 (TLR3), in regulating Vα14iNKT cell activation. We established for the first time that hepatic Vα14iNKT cells up-regulate TLR3 extracellularly after poly I:C treatment. Notably, activation of TLR3-expressing hepatic Vα14iNKT cells by a TLR3 ligand was suppressed by TLR3 deficiency. Our studies also revealed that Vα14iNKT cell activation in response to poly I:C administration uniquely suppressed the accumulation and activation of intrahepatic γδT cells (but not natural killer cells) by inducing apoptosis. Furthermore, we established that activated hepatic Vα14iNKT cells (via cytokines and possibly reactive oxygen species) influenced the frequency and absolute number of intrahepatic γδT cells, as evidenced by increased hepatic γδT cell accumulation in Vα14iNKT cell-deficient mice after poly I:C treatment relative to wild-type mice. Thus, hepatic Vα14iNKT cells and intrahepatic γδT cells are functionally linked on application of TLR3 agonist. Overall, our results demonstrate a novel and previously unrecognized anti-inflammatory role for activated hepatic Vα14iNKT cells in negatively regulating intrahepatic γδT cell accumulation (probably through TLR3 signaling) and thereby preventing potentially harmful activation of intrahepatic γδT cells. PMID:20167870

  6. A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

    PubMed Central

    Nicholson, Lindsay B.; Murtaza, Anwar; Hafler, Brian P.; Sette, Alessandro; Kuchroo, Vijay K.

    1997-01-01

    Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ. PMID:9256473

  7. A fast and robust method to clone and functionally validate T-cell receptors.

    PubMed

    Birkholz, Katrin; Hofmann, Christian; Hoyer, Stefanie; Schulz, Birgit; Harrer, Thomas; Kämpgen, Eckhart; Schuler, Gerold; Dörrie, Jan; Schaft, Niels

    2009-07-31

    Sequencing, cloning and functional testing of T-cell-receptor (TCR) alpha- and beta-chains from T-cell clones is often required in immunotherapy and in immunological research. However, the determination of the TCR chains by a simple PCR is not possible, since, in contrast to the 3' constant domain and untranslated region (UTR), no conserved sequences are present in the 5' region. Furthermore, subsequent functional testing of cloned TCRs requires laborious cell culture experiments, often involving primary human material and time-consuming viral transduction strategies. Here we present a universal PCR-based protocol, adapted from the capswitch technology, that allows for amplification of the TCR alpha- and beta-chain mRNAs without knowledge of the TCR variable domain subtype by attaching a designed sequence to the mRNA's 5' end. Two different MelanA/HLA-A2-specific and one HIVgag/HLA-A2-specific TCR were cloned that way, and were functionally tested by a newly developed easy, fast, and low-cost method: we electroporated Jurkat T cells simultaneously with TCR-encoding RNA and an NFAT-reporter construct, and measured the activation status of the cells upon specific stimulation. The results of this assay correlated with the cytokine release, functional avidity, proliferative activity, and the ability to recognize MelanA/HLA-A2-presenting tumor cells of bulk T cells electroporated with RNA encoding the same TCR. Together these two protocols represent a rapid and low-cost tool for the identification and functional testing of TCRs of T-cell clones, which can then be applied in immunotherapy or immunological research.

  8. Evolution of T-Cell Receptor Gamma and Delta Constant Region and Other T-Cell-Related Proteins in the Human-Rodent-Artiodactyl Triplet

    PubMed Central

    Ciccarese, S.; Lanave, C.; Saccone, C.

    1997-01-01

    In this paper we report a detailed comparative and evolutionary analysis of the sequences of constant T-cell receptor (Tcr) Cγδ genes of artiodactyls compared to the homologous sequences of rodents and primates. Because of the frequency and physiological distribution of γδ T-cells in different animals, rodents and humans are defined as ``γδ low'' species and ruminants as ``γδ high'' species. Such a characteristic seems to be due to an adaptive role of γδ T-cell function. By analyzing the ruminant gene phylogeny of Tcr Cγ we were able to estimate the distance between cattle and sheep at 18 million years ago, a time that is in agreement with other nonmolecular estimates. For Tcr Cγδ genes a peculiar phylogenetic relationship was found, with human and mouse clustering together and leaving artiodactyls apart. By using appropriate outgroups, the same phylogenetic pattern was obtained with other T-cell related sequences: namely, Tcr Cα chain, CD3 γ and δ invariant subunits, Interleukin-2, Interleukin-2 receptor α chain and Interleukin-1β with the exception of Tcr Cβ chain and Interleukin-1α. In contrast, the analysis of all other T-cell nonrelated genes available in primary databases reveals a different tree, where primates and artiodactyls are sister taxa and rodents are apart in accordance with the current view of mammalian phylogeny. These data are relevant to important evolutionary issues. They show how misleading a phylogeny based on a single or on a few homologous genes may be. In addition they demonstrate that genes with correlated functions may evolve in a lineage specific manner probably in relation to environmental conditions. PMID:9071594

  9. A natural anti-T-cell receptor monoclonal antibody protects against experimental autoimmune encephalomyelitis.

    PubMed

    Fernández-Malavé, Edgar; Stark-Aroeira, Luiz

    2011-05-01

    The therapeutic potential of natural anti-T-cell receptor (TCR) antibodies is largely unknown. We investigated whether passive administration of C1-19, a novel natural anti-TCRVβ8 monoclonal antibody, could interfere with the development of EAE. Treatment with C1-19 prevented myelin basic protein (MBP)-induced EAE in Vβ8-sufficient B10.PL but not in Vβ8-deficient SJL mice. Furthermore, C1-19 reduced disease severity when administrated shortly after disease onset. These protective effects of C1-19 correlated with a Th2 bias of the cytokine response, in the absence of T-cell deletion or anergy. Together, these findings indicate that natural anti-TCR antibodies could function as therapeutic tools in autoimmune inflammatory diseases.

  10. End-binding protein 1 controls signal propagation from the T cell receptor

    PubMed Central

    Martín-Cófreces, Noa B; Baixauli, Francesc; López, María J; Gil, Diana; Monjas, Alicia; Alarcón, Balbino; Sánchez-Madrid, Francisco

    2012-01-01

    The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome. PMID:22922463

  11. In vitro membrane reconstitution of the T cell receptor proximal signaling network

    PubMed Central

    Hui, Enfu; Vale, Ronald D.

    2014-01-01

    T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45, and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over a ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that coclustering TCR-Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs. PMID:24463463

  12. Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids

    PubMed Central

    2014-01-01

    Background Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR. Methods Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student’s t-test was employed for statistical evaluation. Results We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk

  13. Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains.

    PubMed

    Beck-García, Katharina; Beck-García, Esmeralda; Bohler, Sheila; Zorzin, Carina; Sezgin, Erdinc; Levental, Ilya; Alarcón, Balbino; Schamel, Wolfgang W A

    2015-04-01

    In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

  14. cap alpha. -chain locus of the T-cell antigen receptor is involved in the t(10; 14) chromosome translocation of T-cell acute lymphocytic leukemia

    SciTech Connect

    Kagan, J.; Finan, J.; Letofsky, J.; Besa, E.C.; Nowell, P.C.; Croce, C.M.

    1987-07-01

    Human leukemic T cells carrying a t(10;14)(q24;q11) chromosome translocation were fused with mouse leukemic T cells, and the hybrids were examined for genetic markers of human chromosomes 10 and 14. Hybrids containing the human 10q+ chromosome had the human genes for terminal deoxynucleotidyltransferase that has been mapped at 10q23-q25 and for C/sub ..cap alpha../ (the constant region of TCRA (the ..cap alpha..-chain locus of the T-cell antigen receptor gene)), but not for V/sub ..cap alpha../ (the variable region of TCRA). Hybrids containing the human 14q- chromosome retained the V/sub ..cap alpha../genes. Thus the 14q11 breakpoint in the t(10;14) chromosome translocation directly involves TCRA, splitting the locus in a region between the V/sub ..cap alpha../ and the C/sub ..cap alpha../ genes. These results suggest that the translocation of the C/sub ..cap alpha../ locus to a putative cellular protooncogene located proximal to the breakpoint at 10q24, for which the authors propose the name TCL3, results in its deregulation, leading to T-cell leukemia. Since hybrids with the 10q+ chromosome also retained the human terminal deoxynucleotidyltransferase gene, it is further concluded that the terminal deoxynucleotidyltransferase locus is proximal to the TCL3 gene, at band 10q23-q24.

  15. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity

    PubMed Central

    Attaf, Meriem; Holland, Stephan J.; Bartok, Istvan; Dyson, Julian

    2016-01-01

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface. PMID:27775030

  16. αβ T cell receptor germline CDR regions moderate contact with MHC ligands and regulate peptide cross-reactivity.

    PubMed

    Attaf, Meriem; Holland, Stephan J; Bartok, Istvan; Dyson, Julian

    2016-10-24

    αβ T cells respond to peptide epitopes presented by major histocompatibility complex (MHC) molecules. The role of T cell receptor (TCR) germline complementarity determining regions (CDR1 and 2) in MHC restriction is not well understood. Here, we examine T cell development, MHC restriction and antigen recognition where germline CDR loop structure has been modified by multiple glycine/alanine substitutions. Surprisingly, loss of germline structure increases TCR engagement with MHC ligands leading to excessive loss of immature thymocytes. MHC restriction is, however, strictly maintained. The peripheral T cell repertoire is affected similarly, exhibiting elevated cross-reactivity to foreign peptides. Our findings are consistent with germline TCR structure optimising T cell cross-reactivity and immunity by moderating engagement with MHC ligands. This strategy may operate alongside co-receptor imposed MHC restriction, freeing germline TCR structure to adopt this novel role in the TCR-MHC interface.

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

    PubMed Central

    Guerrero, Alan D; Moyes, Judy S; Cooper, Laurence JN

    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 cells to target B-cell malignancies 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. PMID:25189715

  18. Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

    DTIC Science & Technology

    2013-05-01

    Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel...of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody 5b. GRANT NUMBER W81XWH-12-1-0184 5c. PROGRAM ELEMENT NUMBER 6...tumors using a brain selective cell line, 231-BR, derived from human breast cancer . Therefore, the experimental model to be used must be immune

  19. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells

    PubMed Central

    Pato, A; Eisenberg, G; Machlenkin, A; Margalit, A; Cafri, G; Frankenburg, S; Merims, S; Peretz, T; Lotem, M; Gross, G

    2015-01-01

    Adoptive T cell therapy of cancer employs a large number of ex-vivo-propagated T cells which recognize their targets either by virtue of their endogenous T cell receptor (TCR) or via genetic reprogramming. However, both cell-extrinsic and intrinsic mechanisms often diminish the in-vivo potency of these therapeutic T cells, limiting their clinical efficacy and broader use. Direct activation of human T cells by Toll-like receptor (TLR) ligands induces T cell survival and proliferation, boosts the production of proinflammatory cytokines and augments resistance to regulatory T cell (Treg) suppression. Removal of the TLR ligand-binding region results in constitutive signalling triggered by the remaining cytosolic Toll/interleukin-1 receptor (TIR) domain. The use of such TIR domains therefore offers an ideal means for equipping anti-tumour T cells with the arsenal of functional attributes required for improving current clinical protocols. Here we show that constitutively active (ca)TLR-4 can be expressed efficiently in human T cells using mRNA electroporation. The mere expression of caTLR-4 mRNA in polyclonal CD8 and CD4 T cells induced the production of interferon (IFN)-γ, triggered the surface expression of CD25, CD69 and 4-1BB and up-regulated a panel of cytokines and chemokines. In tumour-infiltrating lymphocytes prepared from melanoma patients, caTLR-4 induced robust IFN-γ secretion in all samples tested. Furthermore, caTLR-4 enhanced the anti-melanoma cytolytic activity of tumour-infiltrating lymphocytes and augmented the secretion of IFN-γ, tumour necrosis factor (TNF)-α and granulocyte–macrophage colony-stimulating factor (GM-CSF) for at least 4 days post-transfection. Our results demonstrate that caTLR-4 is capable of exerting multiple T cell-enhancing effects and can potentially be used as a genetic adjuvant in adoptive cell therapy. PMID:26212048

  20. Bisphenol A modulates the metabolic regulator oestrogen-related receptor-α in T-cells.

    PubMed

    Cipelli, Riccardo; Harries, Lorna; Okuda, Katsuhiro; Yoshihara, Shin'ichi; Melzer, David; Galloway, Tamara

    2014-01-01

    Bisphenol A (BPA) is a widely used plastics constituent that has been associated with endocrine, immune and metabolic effects. Evidence for how BPA exerts significant biological effects at chronic low levels of exposure has remained elusive. In adult men, exposure to BPA has been associated with higher expression of two nuclear receptors, oestrogen receptor-β (ERβ) and oestrogen-related-receptor-α (ERRα), in peripheral white blood cells in vivo. In this study, we explore the expression of ESR2 (ERβ) and ESRRA (ERRα) in human leukaemic T-cell lymphoblasts (Jurkat cells) exposed to BPA in vitro. We show that exposure to BPA led to enhanced expression of ESRRA within 6 h of exposure (mean±s.e.m.: 1.43±0.08-fold increase compared with the control, P<0.05). After 72 h, expression of ESRRA remained significantly enhanced at concentrations of BPA ≥1 nM. Oxidative metabolism of BPA by rat liver S9 fractions yields the potent oestrogenic metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). Exposure of cells to 1-100 nM MBP increased the expression of both ESRRA (significantly induced, P<0.05, at 1, 10, 100 nM) and ESR2 (1.32±0.07-fold increase at 100 nM exposure, P<0.01). ERRα is a major control point for oxidative metabolism in many cell types, including T-cells. Following exposure to both BPA and MBP, we found that cells showed a decrease in cell proliferation rate. Taken together, these results confirm the bioactivity of BPA against putative T-cell targets in vitro at concentrations relevant to general human exposure.

  1. Expression of T cell receptors by thymocytes: in situ staining and biochemical analysis.

    PubMed Central

    Cristanti, A; Colantoni, A; Snodgrass, R; von Boehmer, H

    1986-01-01

    We have examined the in situ expression of T cell receptor (TCR) V beta 8 protein in murine thymus during ontogeny using the monoclonal antibody F23.1. Positive cells were first detected at day 15 of gestation (0.6%). By day 16 the frequency of positive cells increased dramatically (4.18%). From day 16 to day 17 positive cells doubled (8.17%). The first clusters of F23.1 positive cells were seen at day 17. In the cortex, positive cells decreased from 14% in the newborn mice to 9.8% in 8-week-old mice, whereas in the medulla the frequency remained unchanged at 20%. The antibody F23.1, as well as an antiserum raised against the constant region of the beta chain, immunoprecipitated receptor dimers from highly purified Lyt2+, L3T4+ thymocytes and from two thymic lymphomas of cortical phenotype which express full size alpha and beta mRNA. The receptor dimer could not be precipitated from Lyt2-, L3T4- thymocytes. The results are discussed with regard to intrathymic T cell repertoire selection. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2431899

  2. Domain-swapped T cell receptors improve the safety of TCR gene therapy.

    PubMed

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-11-08

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.

  3. Influence of immunologic status on age prediction using signal joint T cell receptor excision circles.

    PubMed

    Cho, Sohee; Seo, Hee Jin; Lee, Ji Hyun; Kim, Moon Young; Lee, Soong Deok

    2017-02-01

    Age estimation based on quantifying signal joint T cell receptor excision circle (sjTREC) in T cells has been established to be a promising approach in forensic practice and demonstrated in different ethnic groups. Considering that the homeostasis of T cells carrying sjTRECs is closely related to the immunologic status of a person, it is important to investigate the influence of various immunologic statuses on the age estimation model. In this study, quantification of sjTREC contents was performed for groups of people with various immune system statuses, and the result showed less correlation with chronological age (r (2) = 0.424) than in the healthy group (r (2) = 0.648). The simulation model indicated that this influence could increase the range of prediction in the age estimation model, and the mean absolute deviation (MAD) between chronological age and predicted age. Through this study, it was demonstrated that immunologic status is a factor that affects the accuracy of age prediction using sjTREC quantification.

  4. Cloning, expression and interaction of human T-cell receptors with the bacterial superantigen SSA.

    PubMed

    De Marzí, Mauricio C; Fernández, Marisa M; Sundberg, Eric J; Molinero, Luciana; Zwirner, Norberto W; Llera, Andrea S; Mariuzza, Roy A; Malchiodi, Emilio L

    2004-10-01

    Superantigens (SAgs) are a class of disease-causing and immunostimulatory proteins of bacterial or viral origin that activate a large number of T-cells through interaction with the Vbeta domain of T-cell receptors (TCRs). In this study, recombinant TCR beta chains were constructed with human variable domains Vbeta5.2, Vbeta1 and Vbeta2.1, expressed as inclusion bodies, refolded and purified. The Streptococcus pyogenes SAg SSA-1 was cloned and expressed as a soluble periplasmic protein. SSA-1 was obtained both as a monomer and a dimer that has an intermolecular disulfide bond. We analyzed the biological activity of the recombinant SAgs by proliferation assays. The results suggest that SSA dimerization occludes the TCR interaction site. Naturally occurring SSA dimerization was also observed in supernatants of S. pyogenes isolates. An SSA mutant [SSA(C26S)] was produced to eliminate the Cys responsible for dimerization. Affinity assays using a resonant biosensor showed that both the mutant and monomeric wild type SSA have affinity for human Vbeta5.2 and Vbeta1 with Kd of 9-11 microm with a fast kass and a moderately fast kdiss. In spite of the reported stimulation of Vbeta2.1 bearing T-cells by SSA, we observed no measurable interaction.

  5. Chimeric antigen receptors for the adoptive T cell therapy of hematologic malignancies.

    PubMed

    Davila, Marco L; Bouhassira, Diana C G; Park, Jae H; Curran, Kevin J; Smith, Eric L; Pegram, Hollie J; Brentjens, Renier

    2014-04-01

    The genetic modification of autologous T cells with chimeric antigen receptors (CARs) represents a breakthrough for gene engineering as a cancer therapy for hematologic malignancies. By targeting the CD19 antigen, we have demonstrated robust and rapid anti-leukemia activity in patients with heavily pre-treated and chemotherapy-refractory B cell acute lymphoblastic leukemia (B-ALL). We demonstrated rapid induction of deep molecular remissions in adults, which has been recently confirmed in a case report involving a child with B-ALL. In contrast to the results when treating B-ALL, outcomes have been more modest in patients with chronic lymphocytic leukemia (CLL) or other non-hodgkin's lymphoma (NHL). We review the clinical trial experience targeting B-ALL and CLL and speculate on the possible reasons for the different outcomes and propose potential optimization to CAR T cell therapy when targeting CLL or other indolent NHL. Lastly, we discuss the pre-clinical development and potential for clinical translation for using CAR T cells against multiple myeloma and acute myeloid leukemia. We highlight the potential risks and benefits by targeting these poor outcome hematologic malignancies.

  6. Public T cell receptors confer high-avidity CD4 responses to HIV controllers

    PubMed Central

    Galperin, Moran; Lambotte, Olivier; Gras, Stéphanie; Lim, Annick; Mukhopadhyay, Madhura; Campbell, Kristy-Anne; Lemercier, Brigitte; Claireaux, Mathieu; Hendou, Samia; Lechat, Pierre; de Truchis, Pierre; Boufassa, Faroudy; Rossjohn, Jamie; Delfraissy, Jean-François; Arenzana-Seisdedos, Fernando; Chakrabarti, Lisa A.

    2016-01-01

    The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure. PMID:27111229

  7. Domain-swapped T cell receptors improve the safety of TCR gene therapy

    PubMed Central

    Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

    2016-01-01

    T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

  8. Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.

    PubMed

    MacDonald, Katherine G; Hoeppli, Romy E; Huang, Qing; Gillies, Jana; Luciani, Dan S; Orban, Paul C; Broady, Raewyn; Levings, Megan K

    2016-04-01

    Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

  9. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    PubMed Central

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2013-01-01

    Summary Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CART cells. Our review then describes our own and other investigators’ work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. PMID:24329793

  10. Lymphoma Remissions Caused by Anti-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels.

    PubMed

    Kochenderfer, James N; Somerville, Robert P T; Lu, Tangying; Shi, Victoria; Bot, Adrian; Rossi, John; Xue, Allen; Goff, Stephanie L; Yang, James C; Sherry, Richard M; Klebanoff, Christopher A; Kammula, Udai S; Sherman, Marika; Perez, Arianne; Yuan, Constance M; Feldman, Tatyana; Friedberg, Jonathan W; Roschewski, Mark J; Feldman, Steven A; McIntyre, Lori; Toomey, Mary Ann; Rosenberg, Steven A

    2017-03-14

    Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum interleukin-15 (IL-15). Patients who achieved a remission had a median peak blood CAR(+) cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR(+) cell level of 15/μL ( P = .027). High serum IL-15 levels were associated with high peak blood CAR(+) cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-15 levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

  11. Downregulation of T cell receptor expression by CD8(+) lymphocytes in kidney allografts.

    PubMed Central

    Mannon, R B; Kotzin, B L; Nataraj, C; Ferri, K; Roper, E; Kurlander, R J; Coffman, T M

    1998-01-01

    Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses. PMID:9616223

  12. Localization of a site on bacterial superantigens that determines T cell receptor beta chain specificity

    PubMed Central

    1993-01-01

    A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCR) beta chain (V beta). The V beta specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the V beta specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human V beta 5- or V beta 8-bearing T cells, or mouse V beta 3 or V beta 11. We also found that residues in the NH2 terminus were required for wild-type levels of V beta-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR V beta contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the V beta specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining V beta specificity resembles current models being proposed for virally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level. PMID:7678849

  13. Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope

    PubMed Central

    1996-01-01

    Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535- 551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO

  14. NFAT5 induction by the pre-T-cell receptor serves as a selective survival signal in T-lymphocyte development.

    PubMed

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-10-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre-T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre-T-cell receptor.

  15. NFAT5 induction by the pre–T-cell receptor serves as a selective survival signal in T-lymphocyte development

    PubMed Central

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-01-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre–T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre–T-cell receptor. PMID:24043824

  16. Postthymic expansion in human CD4 naive T cells defined by expression of functional high-affinity IL-2 receptors.

    PubMed

    Pekalski, Marcin L; Ferreira, Ricardo C; Coulson, Richard M R; Cutler, Antony J; Guo, Hui; Smyth, Deborah J; Downes, Kate; Dendrou, Calliope A; Castro Dopico, Xaquin; Esposito, Laura; Coleman, Gillian; Stevens, Helen E; Nutland, Sarah; Walker, Neil M; Guy, Catherine; Dunger, David B; Wallace, Chris; Tree, Timothy I M; Todd, John A; Wicker, Linda S

    2013-03-15

    As the thymus involutes with age, the maintenance of peripheral naive T cells in humans becomes strongly dependent on peripheral cell division. However, mechanisms that orchestrate homeostatic division remain unclear. In this study we present evidence that the frequency of naive CD4 T cells that express CD25 (IL-2 receptor α-chain) increases with age on subsets of both CD31(+) and CD31(-) naive CD4 T cells. Analyses of TCR excision circles from sorted subsets indicate that CD25(+) naive CD4 T cells have undergone more rounds of homeostatic proliferation than their CD25(-) counterparts in both the CD31(+) and CD31(-) subsets, indicating that CD25 is a marker of naive CD4 T cells that have preferentially responded to survival signals from self-Ags or cytokines. CD25 expression on CD25(-) naive CD4 T cells can be induced by IL-7 in vitro in the absence of TCR activation. Although CD25(+) naive T cells respond to lower concentrations of IL-2 as compared with their CD25(-) counterparts, IL-2 responsiveness is further increased in CD31(-) naive T cells by their expression of the signaling IL-2 receptor β-chain CD122, forming with common γ-chain functional high-affinity IL-2 receptors. CD25 plays a role during activation: CD25(+) naive T cells stimulated in an APC-dependent manner were shown to produce increased levels of IL-2 as compared with their CD25(-) counterparts. This study establishes CD25(+) naive CD4 T cells, which are further delineated by CD31 expression, as a major functionally distinct immune cell subset in humans that warrants further characterization in health and disease.

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

    PubMed Central

    Deniger, Drew C.; Yu, Jianqiang; Huls, M. Helen; Figliola, Matthew J.; Mi, Tiejuan; Maiti, Sourindra N.; Widhopf, George F.; Hurton, Lenka V.; Thokala, Radhika; Singh, Harjeet; Olivares, Simon; Champlin, Richard E.; Wierda, William G.; Kipps, Thomas J.; Cooper, Laurence J. N.

    2015-01-01

    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. PMID:26030772

  18. T cells deficient in inositol 1,4,5-trisphosphate receptor are resistant to apoptosis.

    PubMed Central

    Jayaraman, T; Marks, A R

    1997-01-01

    The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) calcium release channel is present on the endoplasmic reticulum of most cell types. T lymphocytes which have been made deficient in IP3R1 lack detectable IP3-induced intracellular calcium release and exhibit defective signaling via the T-cell receptor (TCR) (T. Jayaraman, E. Ondriasova, K. Ondrias, D. Harnick, and A. R. Marks, Proc. Natl. Acad. Sci. USA 92:6007-6011, 1995). We now show that IP3R1-deficient T cells are resistant to apoptosis induced by dexamethasone, TCR stimulation, ionizing radiation, and Fas. Resistance to TCR-mediated apoptosis in IP3R1-deficient cells is reversed by pharmacologically raising cytoplasmic calcium levels. TCR-mediated apoptosis can be induced in calcium-free media, indicating that extracellular calcium influx is not required. These findings suggest that intracellular calcium release via the IP3R1 is a critical mediator of apoptosis. PMID:9154798

  19. Molecular characterization of the feline T-cell receptor γ alternate reading frame protein (TARP) ortholog.

    PubMed

    Weiss, Alexander Th A; von Deetzen, Marie-Charlotte; Hecht, Werner; Reinacher, Manfred; Gruber, Achim D

    2012-12-01

    T-cell receptor γ alternate reading frame protein (TARP) is expressed by human prostate epithelial, prostate cancer, and mammary cancer cells, but is not found in normal mammary tissue. To date, this protein has only been described in humans. Additionally, no animal model has been established to investigate the potential merits of TARP as tumor marker or a target for adoptive tumor immunotherapy. In this study conducted to characterize feline T-cell receptor γ sequences, constructs very similar to human TARP transcripts were obtained by RACE from the spleen and prostate gland of cats. Transcription of TARP in normal, hyperplastic, and neoplastic feline mammary tissues was evaluated by conventional RT-PCR. In felines similarly to the situation reported in humans, a C-region encoding two open reading frames is spliced to a J-region gene. In contrast to humans, the feline J-region gene was found to be a pseudogene containing a deletion within its recombination signal sequence. Our findings demonstrated that the feline TARP ortholog is transcribed in the prostate gland and mammary tumors but not normal mammary tissues as is the case with human TARP.

  20. Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

    PubMed

    Fujiwara, Hiroshi

    2014-12-15

    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.

  1. Structurally divergent human T cell receptor. gamma. proteins encoded by distinct C. gamma. genes

    SciTech Connect

    Krangel, M.S.; Band, H.; Hata, S.; McLean, J.; Brenner, M.B.

    1987-07-03

    The human T cell receptor (TCR) ..gamma.. polypeptide occurs in structurally distinct forms on certain peripheral blood T lymphocytes. Complementary DNA clones representing the transcripts of functionally rearranged TCR ..gamma.. genes in these cells have been analyzed. The expression of a disulfide-linked and a nondisulfide-linked form of TCR ..gamma.. correlates with the use of the C..gamma..1 and C..gamma..2 constant-region gene segments, respectively. Variability in TCR ..gamma.. polypeptide and disulfide linkage is determined by the number of copies and the sequence of a repeated segment of the constant region. Thus, C..gamma..1 and C..gamma..2 are used to generate structurally distinct, yet functional, T3-associated receptor complexes on peripheral blood lymphocytes. Tryptic peptide mapping suggests that the T3-associated TCR ..gamma.. and delta peptides in the nondisulfide-linked form are distinct.

  2. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1.

    PubMed

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-05-04

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

  3. Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells

    PubMed Central

    Maher, John

    2012-01-01

    Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche in the treatment of both solid and haematological malignancies. PMID:23304553

  4. Purification and partial sequence analysis of human T-cell growth factor.

    PubMed Central

    Robb, R J; Kutny, R M; Chowdhry, V

    1983-01-01

    A murine monoclonal antibody directed against human T-cell growth factor (TCGF) from the JURKAT cell line was used for affinity column purification of the factor. Bound TCGF was eluted nearly quantitatively at low pH, and the recovered factor appeared homogeneous by two-dimensional gel electrophoresis. The molecule is markedly hydrophobic, with a high content of leucine. A single NH2-terminal sequence of 36 residues was obtained by automated Edman degradation, further supporting the homogeneity of the material. Thus, significant quantities of purified TCGF have been prepared in a single step, making possible detailed analysis of its molecular structure and biological role. Images PMID:6604277

  5. Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire

    PubMed Central

    Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

    2016-01-01

    Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells. PMID:27064277

  6. Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells*

    PubMed Central

    Ruperez, Patricia; Gago-Martinez, Ana; Burlingame, A. L.; Oses-Prieto, Juan A.

    2012-01-01

    Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on analysis of phosphotyrosine residues, and little is known about the role of serine/threonine phosphorylation in early activation of the T cell receptor (TCR). Therefore, we performed a quantitative mass spectrometry-based analysis of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, isobaric tag for the relative and absolute quantitation methodology, and strong cation exchange separation, we were able to identify 2814 phosphopeptides. These unique sites were employed to investigate the site-specific phosphorylation dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive changes. We found that upon 5 min of stimulation of the TCR, specific serine and threonine kinase motifs are overrepresented in the set of responsive phosphorylation sites. These phosphorylation events targeted proteins with many different activities and are present in different subcellular locations. Many of these proteins are involved in intracellular signaling cascades related mainly to cytoskeletal reorganization and regulation of small GTPase-mediated signal transduction, probably involved in the formation of the immune synapse. PMID:22499768

  7. T Cell Receptor-dependent Tyrosine Phosphorylation of β2-Chimaerin Modulates Its Rac-GAP Function in T Cells*S⃞

    PubMed Central

    Siliceo, María; Mérida, Isabel

    2009-01-01

    The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation. PMID:19201754

  8. Toll-Like Receptor 3 Signalling Up-Regulates Expression of the HIV Co-Receptor G-Protein Coupled Receptor 15 on Human CD4+ T Cells

    PubMed Central

    Kiene, Miriam; Rethi, Bence; Jansson, Marianne; Dillon, Stephanie; Lee, Eric; Lantto, Rebecka; Wilson, Cara; Pöhlmann, Stefan; Chiodi, Francesca

    2014-01-01

    Background Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. Results Here, we show that GPR15 expression in CD4+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF) and was more prominent on gut-homing compared to lymph node-homing CD4+ T cells. Conclusion These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4+ T cells to HIV infection and target cell availability in the gut in some infected individuals. PMID:24558379

  9. Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene.

    PubMed

    Taylor, N; Bacon, K B; Smith, S; Jahn, T; Kadlecek, T A; Uribe, L; Kohn, D B; Gelfand, E W; Weiss, A; Weinberg, K

    1996-11-01

    A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

  10. A novel regulatory pathway for autoimmune disease: binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance.

    PubMed

    Vandenbark, Arthur A; Meza-Romero, Roberto; Benedek, Gil; Andrew, Shayne; Huan, Jianya; Chou, Yuan K; Buenafe, Abigail C; Dahan, Rony; Reiter, Yoram; Mooney, Jeffery L; Offner, Halina; Burrows, Gregory G

    2013-02-01

    Treatment with partial (p)MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.

  11. Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4.

    PubMed

    Kalia, Vandana; Penny, Laura Anne; Yuzefpolskiy, Yevgeniy; Baumann, Florian Martin; Sarkar, Surojit

    2015-06-16

    Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation.

  12. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

    PubMed Central

    Van Caeneghem, Yasmine; De Munter, Stijn; Tieppo, Paola; Goetgeluk, Glenn; Weening, Karin; Verstichel, Greet; Bonte, Sarah; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Debets, Reno; Vermijlen, David; Abken, Hinrich; Vandekerckhove, Bart

    2017-01-01

    ABSTRACT Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD.

  13. Infiltrating T cells promote prostate cancer metastasis via modulation of FGF11→miRNA-541→androgen receptor (AR)→MMP9 signaling

    PubMed Central

    Hu, Shuai; Li, Lei; Yeh, Shuyuan; Cui, Yun; Li, Xin; Chang, Hong-Chiang; Jin, Jie; Chang, Chawnshang

    2014-01-01

    Early clinical studies suggested infiltrating T cells might be associated with poor outcomes in prostate cancer (PCa) patients. The detailed mechanisms how T cells contribute to PCa progression, however, remained unclear. Here, we found PCa cells have a better capacity to recruit more CD4(+) T cells than the surrounding normal prostate cells via secreting more chemokines-CXCL9. The consequences of more recruited CD4(+) T cells to PCa might then lead to enhance PCa cell invasion. Mechanism dissection revealed that infiltrating CD4(+) T cells might function through the modulation of FGF11→miRNA-541 signals to suppress PCa androgen receptor (AR) signals. The suppressed AR signals might then alter the MMP9 signals to promote the PCa cell invasion. Importantly, suppressed AR signals via AR-siRNA or anti-androgen Enzalutamidein PCa cells also enhanced the recruitment of T cells and the consequences of this positive feed back regulation could then enhance the PCa cell invasion. Targeting these newly identified signals viaFGF11-siRNA, miRNA-541 inhibitor or MMP9 inhibitor all led to partially reverse the enhanced PCa cell invasion. Results from in vivo mouse models also confirmed the in vitro cell lines in co-culture studies. Together, these results concluded that infiltrating CD4(+) T cells could promote PCa metastasis via modulation of FGF11→miRNA-541→AR→MMP9 signaling. Targeting these newly identified signals may provide us a new potential therapeutic approach to better battle PCa metastasis. PMID:25135278

  14. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  15. Minicircle-Based Engineering of Chimeric Antigen Receptor (CAR) T Cells.

    PubMed

    Hudecek, Michael; Gogishvili, Tea; Monjezi, Razieh; Wegner, Julia; Shankar, Ram; Kruesemann, Christa; Miskey, Csaba; Ivics, Zoltán; Schmeer, Marco; Schleef, Martin

    2016-01-01

    Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an important goal in vector development was to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle (MC) DNA. The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform enabling a close-to-random profile of genomic integration. In combination, the MC platform greatly enhances SB transposition and transgene integration resulting in higher numbers of stably modified target cells. We have recently developed a strategy for MC-based SB transposition of chimeric antigen receptor (CAR) transgenes that enable improved transposition rates compared to conventional plasmids and rapid manufacturing of therapeutic CAR T cell doses (Monjezi et al. 2016). This advance enables manufacturing CAR T cells in a virus-free process that relies on SB-mediated transposition from MC DNA to accomplish gene-transfer. Advantages of this approach include a strong safety profile due to the nature of the MC itself and the genomic insertion pattern of MC-derived CAR transposons. In addition, stable transposition and high-level CAR transgene expression, as well as easy and reproducible handling, make MCs a preferred vector source for gene-transfer in advanced cellular and gene therapy. In this chapter, we will review our experience in MC-based CAR T cell engineering and discuss our recent advances in MC manufacturing to accelerate both pre-clinical and clinical implementation.

  16. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor.

    PubMed

    Urbanska, Katarzyna; Lanitis, Evripidis; Poussin, Mathilde; Lynn, Rachel C; Gavin, Brian P; Kelderman, Sander; Yu, Jason; Scholler, Nathalie; Powell, Daniel J

    2012-04-01

    Adoptive immunotherapies composed of T cells engineered to express a chimeric antigen receptor (CAR) offer an attractive strategy for treatment of human cancer. However, CARs have a fixed antigen specificity such that only one tumor-associated antigen (TAA) can be targeted, limiting the efficacy that can be achieved because of heterogeneous TAA expression. For this reason, a more generalized and effective application of CAR therapy would benefit from the capability to produce large panels of CARs against many known TAAs. In this study, we show a novel strategy to extend the recognition specificity potential of a bioengineered lymphocyte population, allowing flexible approaches to redirect T cells against various TAAs. Our strategy employs a biotin-binding immune receptor (BBIR) composed of an extracellular-modified avidin linked to an intracellular T-cell signaling domain. BBIR T cells recognized and bound exclusively to cancer cells pretargeted with specific biotinylated molecules. The versatility afforded by BBIRs permitted sequential or simultaneous targeting of a combination of distinct antigens. Together, our findings show that a platform of universal T-cell specificity can significantly extend conventional CAR approaches, permitting the tailored generation of T cells of unlimited antigen specificity for improving the effectiveness of adoptive T-cell immunotherapies for cancer.

  17. ROLE OF CHIMAERINS, A GROUP OF Rac-SPECIFIC GTPase ACTIVATING PROTEINS, IN T-CELL RECEPTOR SIGNALING

    PubMed Central

    Caloca, María José; Delgado, Pilar; Alarcón, Balbino; Bustelo, Xosé R.

    2008-01-01

    Chimaerins are GTPase-activating proteins that inactivate the GTP-hydrolase Rac1 in a diacylglycerol-dependent manner. To date, the study of chimaerins has been done mostly in neuronal cells. Here, we show that α2- and β2-chimaerin are expressed at different levels in T-cells and that they participate in T-cell receptor signaling. In agreement with this, we have observed that α2- and β2-chimaerins translocate to the T-cell/B-cell immune synapse and, using both gain- and loss-of-function approaches, demonstrated that their catalytic activity is important for the inhibition of the T-cell receptor- and Vav1-dependent stimulation of the transcriptional factor NF-AT. Mutagenesis-based approaches have revealed the molecular determinants that contribute to the biological program of chimaerins during T-cell responses. Unexpectedly, we have found that the translocation of chimaerins to the T-cell/B-cell immune synapse does not rely on the canonical binding of diacylglycerol to the C1 region of these GTPase-activating proteins. Taken together, these results identify chimaerins as candidates for the downmodulation of Rac1 in T-lymphocytes and, in addition, uncover a novel regulatory mechanism that mediates their activation in T-cells. PMID:18249095

  18. T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome

    PubMed Central

    Gaballa, Ahmed; Sundin, Mikael; Stikvoort, Arwen; Abumaree, Muhamed; Uzunel, Mehmet; Sairafi, Darius; Uhlin, Michael

    2016-01-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up. PMID:27727179

  19. Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM

    PubMed Central

    Shirasu, Naoto; Yamada, Hiromi; Shibaguchi, Hirotomo; Kuroki, Motomu; Kuroki, Masahide

    2012-01-01

    The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy. PMID:22547929

  20. Human cord blood T-cell receptor alpha beta cell responses to protein antigens of Paracoccidioides brasiliensis yeast forms.

    PubMed Central

    Munk, M E; Kaufmann, S H

    1995-01-01

    Paracoccidioides brasiliensis causes a chronic granulomatous mycosis, prevalent in South America, and cell-mediated immunity represents the principal mode of protection against this fungal infection. We investigated the response of naive cord blood T cells to P. brasiliensis lysates. Our results show: (1) P. brasiliensis stimulates T-cell expansion, interleukin-2 (IL-2) production and differentiation into cytotoxic T cells; (2) T-cell stimulation depends on P. brasiliensis processing and major histocompatibility complex (MHC) class II expression; (3) the responsive T-cell population expresses alpha beta T-cell receptors (TCR) with different V beta gene products, CD4 and CD45RO; (4) the P. brasiliensis components involved in T-cell expansion primarily reside in a high molecular weight (100,000 MW) and a low molecular weight (< 1000 MW) protein fraction. These results indicate that protein antigens of P. brasiliensis stimulate cord blood CD4 alpha beta T cells, independent from in vivo presensitization, and thus question direct correlation of positive in vitro responses with protective immunity in vivo. PMID:7890308

  1. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma.

    PubMed

    Chmielewski, Markus; Hombach, Andreas A; Abken, Hinrich

    2014-01-01

    Adoptive T-cell therapy recently achieved impressive efficacy in early phase trials, in particular in hematologic malignancies, strongly supporting the notion that the immune system can control cancer. A current strategy of favor is based on ex vivo-engineered patient T cells, which are redirected by a chimeric antigen receptor (CAR) and recognize a predefined target by an antibody-derived binding domain. Such CAR T cells can substantially reduce the tumor burden as long as the targeted antigen is present on the cancer cells. However, given the tremendous phenotypic diversity in solid tumor lesions, a reasonable number of cancer cells are not recognized by a given CAR, considerably reducing the therapeutic success. This article reviews a recently described strategy for overcoming this shortcoming of the CAR T-cell therapy by modulating the tumor stroma by a CAR T-cell-secreted transgenic cytokine like interleukin-12 (IL-12). The basic process is that CAR T cells, when activated by their CAR, deposit IL-12 in the targeted tumor lesion, which in turn attracts an innate immune cell response toward those cancer cells that are invisible to CAR T cells. Such TRUCKs, T cells redirected for universal cytokine-mediated killing, exhibited remarkable efficacy against solid tumors with diverse cancer cell phenotypes, suggesting their evaluation in clinical trials.

  2. Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes.

    PubMed

    Kumar, Rashmi; Ferez, María; Swamy, Mahima; Arechaga, Ignacio; Rejas, María Teresa; Valpuesta, Jose M; Schamel, Wolfgang W A; Alarcon, Balbino; van Santen, Hisse M

    2011-09-23

    Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.

  3. Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns

    PubMed Central

    Ross, Jenny O.; Melichar, Heather J.; Au-Yeung, Byron B.; Herzmark, Paul; Weiss, Arthur; Robey, Ellen A.

    2014-01-01

    Positive selection of CD8 T cells in the thymus is thought to be a multistep process lasting 3–4 d; however, the discrete steps involved are poorly understood. Here, we examine phenotypic changes, calcium signaling, and intrathymic migration in a synchronized cohort of MHC class I-specific thymocytes undergoing positive selection in situ. Transient elevations in intracellular calcium concentration ([Ca2+]i) and migratory pauses occurred throughout the first 24 h of positive selection, becoming progressively briefer and accompanied by a gradual shift in basal [Ca2+]i over time. Changes in chemokine-receptor expression and relocalization from the cortex to medulla occurred between 12 and 24 h after the initial encounter with positive-selecting ligands, a time frame at which the majority of thymocytes retain CD4 and CD8 expression and still require T-cell receptor (TCR) signaling to efficiently complete positive selection. Our results identify distinct phases in the positive selection of MHC class I-specific thymocytes that are distinguished by their TCR-signaling pattern and intrathymic location and provide a framework for understanding the multistep process of positive selection in the thymus. PMID:24927565

  4. A Novel Loop Domain in Superantigens Extends Their T Cell Receptor Recognition Site

    SciTech Connect

    Gunther,S.; Varma, A.; Moza, B.; Kasper, K.; Wyatt, A.; Zhu, P.; Nur-ur Rahman, A.; Li, Y.; Mariuzza, R.; et al.

    2007-01-01

    Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the {alpha}3-{beta}8 loop, a unique {approx}15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hV{beta}5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the {alpha}3-{beta}8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR V{beta} domain specificity of SEK and other group V SAGs.

  5. Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

    PubMed Central

    Katayama, C D; Eidelman, F J; Duncan, A; Hooshmand, F; Hedrick, S M

    1995-01-01

    The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition. Images PMID:7534228

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

    PubMed Central

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

    1993-01-01

    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. Images Fig. 1 PMID:8341702

  7. Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae.

    PubMed

    Drbalova, Jitka; Musilova, Petra; Kubickova, Svatava; Sebestova, Hana; Vahala, Jiri; Rubes, Jiri

    2014-01-01

    The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

  8. Different gamma delta T-cell receptors are expressed on thymocytes at different stages of development.

    PubMed Central

    Ito, K; Bonneville, M; Takagaki, Y; Nakanishi, N; Kanagawa, O; Krecko, E G; Tonegawa, S

    1989-01-01

    We have analyzed the structural diversity of the murine gamma delta T-cell receptor (TCR) heterodimer expressed on CD4- CD8- thymocyte populations and on TCR gamma delta-expressing hybridomas derived from thymocytes of fetal, newborn, and adult mice. We found that CD4- CD8- thymocytes derived from mice of different pre- and postnatal age preferentially express a gamma delta TCR encoded by different subsets of gamma and delta gene segments. This age-dependent differential expression of gamma delta TCR on thymocytes seems to be accomplished in part by a specific control of rearranged gamma genes operating at the level of transcription and/or RNA stability. We discuss the implications of these findings with respect to the recognition roles of the gamma delta TCR. Images PMID:2463632

  9. The complete 685-kilobase DNA sequence of the human {Beta} T cell receptor locus

    SciTech Connect

    Rowen, L.; Koop, B.F.; Hood, L.

    1996-06-21

    The human {Beta} T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two types of coding elements-TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments) and eight trypsinogen genes-that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to be present in complementary DNA libraries. 47 refs., 2 figs., 2 tabs.

  10. The outline structure of the T-cell alpha beta receptor.

    PubMed Central

    Chothia, C; Boswell, D R; Lesk, A M

    1988-01-01

    From an analysis of the immunoglobulins of known structure we derive a list of 40 sites crucial for the conserved structure of the variable domains. We show that, with marginal exceptions, the sequences of the T-cell alpha beta receptors contain, at sites homologous to these 40, the same or very similar residues. Thus the V alpha-V beta dimer has a framework structure very close to that of the immunoglobulins. Further comparisons show that parts of the surface of the V alpha-V beta framework are hypervariable. They also show that the loops that form the antigen-binding site are similar in size to those commonly found in the immunoglobulins but have different conformations. Only limited sequence variations occur in the first loop of the antigen-binding site in both V alpha and V beta. This, and their geometrical arrangement, suggest that they mainly interact with the MHC proteins. PMID:3208747

  11. Gene correction in the evolution of the T cell receptor beta chain

    PubMed Central

    1986-01-01

    Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families. PMID:3783089

  12. Molecular evidence for a thymus-independent partial T cell development in a FOXN1-/- athymic human fetus.

    PubMed

    Fusco, Anna; Panico, Luigi; Gorrese, Marisa; Bianchino, Gabriella; Barone, Maria V; Grieco, Vitina; Vitiello, Laura; D'Assante, Roberta; Romano, Rosa; Palamaro, Loredana; Scalia, Giulia; Vecchio, Luigi Del; Pignata, Claudio

    2013-01-01

    The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1(-/-) human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1(-/-) fetus, in which we previously described a total blockage of CD4(+) and partial blockage of CD8(+) cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3(+) and CD8(+), but not of CD4(+) cells, a few of them exhibiting a CD45RA(+) naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process.

  13. Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles

    PubMed Central

    GUAZZI, V; AIUTI, F; MEZZAROMA, I; MAZZETTA, F; ANDOLFI, G; Mortellaro, A; Pierdominici, M; FANTINI, R; MARZIALI, M; AIUTI, A

    2002-01-01

    Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T-cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T-cell subpopulations in 17 CVID patients in comparison to age-matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA−CD62L+/RA+CD62L− were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA-DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T-cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA−DR+ and CD8+CD45RA−CD62L+/RA+CD62L− T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency. PMID:12165093

  14. T Cell Receptor-Independent Basal Signaling via Erk and Abl Kinases Suppresses RAG Gene Expression

    PubMed Central

    Roose, Jeroen P; Diehn, Maximilian; Tomlinson, Michael G; Lin, Joseph; Alizadeh, Ash A; Botstein, David; Brown, Patrick O

    2003-01-01

    Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation. PMID:14624253

  15. Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

    1994-09-01

    An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

  16. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production

    PubMed Central

    Bilal, Mahmood Yousif; Houtman, Jon C. D.

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. PMID:25870599

  17. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production.

    PubMed

    Bilal, Mahmood Yousif; Houtman, Jon C D

    2015-01-01

    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

  18. The Transmembrane Domain of HIV-1 gp41 Inhibits T-Cell Activation by Targeting Multiple T-Cell Receptor Complex Components through Its GxxxG Motif.

    PubMed

    Rotem, Etai; Reuven, Eliran Moshe; Klug, Yoel A; Shai, Yechiel

    2016-02-23

    To successfully infect and persist within its host, HIV-1 utilizes several immunosuppressive motifs within its gp41 envelope glycoprotein to manipulate and evade the immune system. The transmembrane domain (TMD) of gp41 downregulates T-cell receptor (TCR) signaling through a hitherto unknown mechanism. Interactions between TMDs within the membrane milieu have been shown to be typically mediated by particular amino acids, such as interactions between basic and acidic residues and dimerization motifs as GxxxG. The HIV-1 TMD exhibits both a polar arginine (Arg(696)) residue and a GxxxG motif, making them ideal candidates for mediators of TMD-TCR interaction. Using a primary T-cell activation assay and biochemical and biophysical methods, we demonstrate that the gp41 TMD directly interacts with TMDs of the TCR and the CD3 coreceptors (δ, γ, and ε) within the membrane, presumably leading to impairment of complex assembly. Additionally, we reveal that although Arg(696) does not affect TMD immunosuppression, the GxxxG motif is crucial in mediating gp41's TMD interaction with the CD3 coreceptors of the TCR. These findings suggest that compared with other gp41 immunosuppressive motifs, the gp41 TMD has multiple targets within the TCR complex, suggesting less susceptibility to evolutionary pressure and consequently being advantageous for the virus over the host immune response. Furthermore, as the GxxxG motif mediates interactions of the gp41 TMD with multiple receptors, it emerges as an attractive drug target. This multitarget inhibitory mechanism might be a strategy utilized by HIV to interfere with the function of additional host receptors.

  19. T-Cell Receptor/CD28 Engagement When Combined with Prostaglandin E2 Treatment Leads to Potent Activation of Human T-Cell Leukemia Virus Type 1

    PubMed Central

    Dumais, Nancy; Paré, Marie-Ève; Mercier, Simon; Bounou, Salim; Marriot, Susan J.; Barbeau, Benoit; Tremblay, Michel J.

    2003-01-01

    Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E2 (PGE2). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE2-mediated increase in virus transcription was dependent on protein kinase A activation and induction of the cAMP response element binding protein. Experiments with a mutated reporter construct further revealed the importance of the Tax-responsive elements in the HTLV-1 LTR in the observed up regulation of virus gene expression when TCR/CD28 engagement was combined with PGE2 treatment. The protein tyrosine kinases p56lck and the transmembrane tyrosine phosphatase CD45 were all found to be involved in TCR-CD28-PGE2-directed increase in HTLV-1 LTR activity. This study presents new information on the possible mechanisms underlying reactivation of this retrovirus. PMID:14512564

  20. T-cell receptor/CD28 engagement when combined with prostaglandin E2 treatment leads to potent activation of human T-cell leukemia virus type 1.

    PubMed

    Dumais, Nancy; Paré, Marie-Eve; Mercier, Simon; Bounou, Salim; Marriot, Susan J; Barbeau, Benoit; Tremblay, Michel J

    2003-10-01

    Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E(2) (PGE(2)). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE(2)-mediated increase in virus transcription was dependent on protein kinase A activation and induction of the cAMP response element binding protein. Experiments with a mutated reporter construct further revealed the importance of the Tax-responsive elements in the HTLV-1 LTR in the observed up regulation of virus gene expression when TCR/CD28 engagement was combined with PGE(2) treatment. The protein tyrosine kinases p56(lck) and the transmembrane tyrosine phosphatase CD45 were all found to be involved in TCR-CD28-PGE(2)-directed increase in HTLV-1 LTR activity. This study presents new information on the possible mechanisms underlying reactivation of this retrovirus.

  1. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies.

    PubMed

    Hay, Kevin A; Turtle, Cameron J

    2017-03-01

    Adoptive immunotherapy with chimeric antigen receptor-modified (CAR)-T cells is a rapidly growing therapeutic approach to treating patients with refractory cancer, with over 100 clinical trials in various malignancies in progress. The enthusiasm for CAR-T cells has been driven by the clinical success of CD19-targeted CAR-T cell therapy in B-cell acute lymphoblastic leukemia, and the promising data in B-cell non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Despite the success of targeting CD19 with CAR-T cells in early clinical studies, many challenges remain to improve outcomes, reduce toxicity, and determine the appropriate settings for CAR-T cell immunotherapy. Reviewing the lessons learned thus far in CD19 CAR-T cell trials and how some of these challenges may be overcome will help guide the development of CAR-T cell therapy for malignancies of B-cell origin, as well as for other hematopoietic and non-hematopoietic cancers.

  2. CXCR3 chemokine receptor enables local CD8(+) T cell migration for the destruction of virus-infected cells.

    PubMed

    Hickman, Heather D; Reynoso, Glennys V; Ngudiankama, Barbara F; Cush, Stephanie S; Gibbs, James; Bennink, Jack R; Yewdell, Jonathan W

    2015-03-17

    CD8(+) T cells play a critical role in limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8(+) T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and CXCL10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3(-/-) mice exhibited dramatically impaired CD8(+)-T-cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3(-/-) T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8(+) T cells restored viral clearance in Cxcr3(-/-) animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8(+) T cells to locate virus-infected cells and thereby exert anti-viral effector functions.

  3. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4

    SciTech Connect

    Yin, Yiyuan; Wang, Xin Xiang; Mariuzza, Roy A

    2012-07-11

    Adaptive immunity depends on specific recognition by a T-cell receptor (TCR) of an antigenic peptide bound to a major histocompatibility complex (pMHC) molecule on an antigen-presenting cell (APC). In addition, T-cell activation generally requires binding of this same pMHC to a CD4 or CD8 coreceptor. Here, we report the structure of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a myelin-derived self-peptide bound to HLA-DR4, and CD4. The complex resembles a pointed arch in which TCR and CD4 are each tilted ~65° relative to the T-cell membrane. By precluding direct contacts between TCR and CD4, the structure explains how TCR and CD4 on the T cell can simultaneously, yet independently, engage the same pMHC on the APC. The structure, in conjunction with previous mutagenesis data, places TCR-associated CD3εγ and CD3εδ subunits, which transmit activation signals to the T cell, inside the TCR-pMHC-CD4 arch, facing CD4. By establishing anchor points for TCR and CD4 on the T-cell membrane, the complex provides a basis for understanding how the CD4 coreceptor focuses TCR on MHC to guide TCR docking on pMHC during thymic T-cell selection.

  4. Three-dimensional localization of T-cell receptors in relation to microvilli using a combination of superresolution microscopies

    PubMed Central

    Jung, Yunmin; Riven, Inbal; Feigelson, Sara W.; Kartvelishvily, Elena; Tohya, Kazuo; Miyasaka, Masayuki; Alon, Ronen; Haran, Gilad

    2016-01-01

    Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules. PMID:27647916

  5. Chemokine receptor CXCR3 deficiency exacerbates murine autoimmune cholangitis by promoting pathogenic CD8(+) T cell activation.

    PubMed

    Ma, Hong-Di; Ma, Wen-Tao; Liu, Qing-Zhi; Zhao, Zhi-Bin; Liu, Mu-Zi-Ying; Tsuneyama, Koichi; Gao, Jin-Ming; Ridgway, William M; Ansari, Aftab A; Gershwin, M Eric; Fei, Yun-Yun; Lian, Zhe-Xiong

    2017-03-01

    CXC Chemokine Receptor 3 (CXCR3) is functionally pleiotropic and not only plays an important role in chemotaxis, but also participates in T cell differentiation and may play a critical role in inducing and maintaining immune tolerance. These observations are particularly critical for autoimmune cholangitis in which CXCR3 positive T cells are found around the portal areas of both humans and mouse models of primary biliary cholangitis (PBC). Herein, we investigated the role of CXCR3 in the pathogenesis of autoimmune cholangitis. We have taken advantage of a unique CXCR3 knockout dnTGFβRII mouse to focus on the role of CXCR3, both by direct observation of its influence on the natural course of disease, as well as through adoptive transfer studies into Rag-/- mice. We report herein that not only do CXCR3 deficient mice develop an exacerbation of autoimmune cholangitis associated with an expanded effector memory T cell number, but also selective adoptive transfer of CXCR3 deficient CD8(+) T cells induces autoimmune cholangitis. In addition, gene microarray analysis of CXCR3 deficient CD8(+) T cells reveal an intense pro-inflammatory profile. Our data suggests that the altered gene profiles induced by CXCR3 deficiency promotes autoimmune cholangitis through pathogenic CD8(+) T cells. These data have significance for human PBC and other autoimmune liver diseases in which therapeutic intervention might be directed to chemokines and/or their receptors.

  6. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    PubMed Central

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans. PMID:27242780

  7. Essential role for retinoic acid in the promotion of CD4+ T cell effector responses via retinoic acid receptor alpha

    PubMed Central

    Hall, J.A.; Cannons, J.L.; Grainger, J.R.; Santos, L.M. Dos; Hand, T.W.; Naik, S.; Wohlfert, E.A.; Chou, D.B.; Oldenhove, G.; Robinson, M.; Grigg, M.E.; Kastenmayer, R.; Schwartzberg, P.L.; Belkaid, Y.

    2012-01-01

    SUMMARY Vitamin A and its metabolite, retinoic acid (RA), have recently been implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we show that RA is also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) is the critical mediator of these effects. Strikingly, antagonism of RAR signaling and deficiency in RARα(Rara−/−) results in a cell autonomous CD4+ T cell activation defect. Altogether, these findings reveal a fundamental role for the RA/RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses. PMID:21419664

  8. Quantitative T cell repertoire analysis by deep cDNA sequencing of T cell receptor α and β chains using next-generation sequencing (NGS)

    PubMed Central

    Fang, Hua; Yamaguchi, Rui; Liu, Xiao; Daigo, Yataro; Yew, Poh Yin; Tanikawa, Chizu; Matsuda, Koichi; Imoto, Seiya; Miyano, Satoru; Nakamura, Yusuke

    2015-01-01

    Immune responses play a critical role in various disease conditions including cancer and autoimmune diseases. However, to date, there has not been a rapid, sensitive, comprehensive, and quantitative analysis method to examine T-cell or B-cell immune responses. Here, we report a new approach to characterize T cell receptor (TCR) repertoire by sequencing millions of cDNA of TCR α and β chains in combination with a newly-developed algorithm. Using samples from lung cancer patients treated with cancer peptide vaccines as a model, we demonstrate that detailed information of the V-(D)-J combination along with complementary determining region 3 (CDR3) sequences can be determined. We identified extensive abnormal splicing of TCR transcripts in lung cancer samples, indicating the dysfunctional splicing machinery in T lymphocytes by prior chemotherapy. In addition, we found three potentially novel TCR exons that have not been described previously in the reference genome. This newly developed TCR NGS platform can be applied to better understand immune responses in many disease areas including immune disorders, allergies, and organ transplantations. PMID:25964866

  9. Notch1 promotes survival of E2A-deficient T cell lymphomas through pre-T cell receptor-dependent and -independent mechanisms.

    PubMed

    Reschly, Erica J; Spaulding, Christina; Vilimas, Tomas; Graham, W Vallen; Brumbaugh, Rachel L; Aifantis, Iannis; Pear, Warren S; Kee, Barbara L

    2006-05-15

    Loss of E2A transcription factor activity or activation of the intracellular form of Notch1 (ICN) leads to the development of leukemia or lymphoma in humans or mice, respectively. Current models propose that ICN functions by suppressing E2A through a pre-T cell receptor (TCR)-dependent mechanism. Here we show that lymphomas arising in E2A(-/-) mice require the activation of Notch1 for their survival and have accumulated mutations in, or near, the Notch1 PEST domain, resulting in increased stability and signaling. In contrast, lymphomas arising in p53(-/-) mice show the activation of Notch1, but no mutations were identified in ICN. The requirement for Notch1 signaling in E2A(-/-) lymphomas cannot be overcome by ectopic expression of pTalpha; however, pTalpha is required for optimal survival and expansion of these cells. Our findings indicate that the activation of Notch1 is an important "second hit" for the transformation of E2A(-/-) T cell lymphomas and that Notch1 promotes survival through pre-TCR-dependent and -independent mechanisms.

  10. Dopamine Receptor D3 Signaling on CD4+ T Cells Favors Th1- and Th17-Mediated Immunity.

    PubMed

    Contreras, Francisco; Prado, Carolina; González, Hugo; Franz, Dafne; Osorio-Barrios, Francisco; Osorio, Fabiola; Ugalde, Valentina; Lopez, Ernesto; Elgueta, Daniela; Figueroa, Alicia; Lladser, Alvaro; Pacheco, Rodrigo

    2016-05-15

    Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells.

  11. Class II-restricted T cell receptor engineered in vitro for higher affinity retains peptide specificity and function

    PubMed Central

    Weber, K. Scott; Donermeyer, David L.; Allen, Paul M.; Kranz, David M.

    2005-01-01

    The T cell receptor (TCR) αβ heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb/I-Ek), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (Vβ-linker-Vα) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb/I-Ek to isolate a panel of affinity mutants with KD values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation. PMID:16365315

  12. Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

    PubMed

    Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

    2016-11-01

    To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D.

  13. Expression of CD3-associated antigen-binding receptors on suppressor T cells.

    PubMed Central

    Kuchroo, V K; Steele, J K; Billings, P R; Selvaraj, P; Dorf, M E

    1988-01-01

    Three suppressor T (Ts)-cell hybridomas specific for 4-hydroxy-3-nitrophenyl acetyl (NP) hapten were selected for surface expression of cluster determinant 3 (CD3) by using antibody (anti-CD3) or antigen (NP-bovine serum albumin) panning procedures followed by cloning at limiting dilution. The CD3-selected Ts hybridomas showed a 1-2 logarithmic enrichment in suppressor activity when compared to the parent lines; they also specifically bound NP-coupled sheep red blood cells in rosette assays. This antigen-binding ability could be down-modulated by anti-CD3 antibody. Similarly, surface expression of CD3 was specifically down-modulated by preincubation of these hybridomas with antigen. Anti-CD3 monoclonal antibody under reducing conditions coprecipitated a broad band of 38-50 kDa associated with two CD3 (25 and 16 kDa) bands. T-cell receptor, anti-alpha-specific monoclonal antibody also immunoprecipitated a broad band in the 41 to 49-kDa region. The combined results suggest that, like helper and cytotoxic T lymphocytes, Ts cells also bear antigen-specific receptors associated with CD3 molecules. Images PMID:2973609

  14. Constitutively Active Lck Kinase in T Cells Drives Antigen Receptor Signal Transduction

    PubMed Central

    Nika, Konstantina; Soldani, Cristiana; Salek, Mogjiborahman; Paster, Wolfgang; Gray, Adrian; Etzensperger, Ruth; Fugger, Lars; Polzella, Paolo; Cerundolo, Vincenzo; Dushek, Omer; Höfer, Thomas; Viola, Antonella; Acuto, Oreste

    2010-01-01

    Summary T cell antigen receptor (TCR) and coreceptor ligation is thought to initiate signal transduction by inducing activation of the kinase Lck. Here we showed that catalytically active Lck was present in unstimulated naive T cells and thymocytes and was readily detectable in these cells in lymphoid organs. In naive T cells up to ∼40% of total Lck was constitutively activated, part of which was also phosphorylated on the C-terminal inhibitory site. Formation of activated Lck was independent of TCR and coreceptors but required Lck catalytic activity and its maintenance relied on monitoring by the HSP90-CDC37 chaperone complex to avoid degradation. The amount of activated Lck did not change after TCR and coreceptor engagement; however it determined the extent of TCR-ζ phosphorylation. Our findings suggest a dynamic regulation of Lck activity that can be promptly utilized to initiate T cell activation and have implications for signaling by other immune receptors. PMID:20541955

  15. Targeting oncogenic interleukin-7 receptor signalling with N-acetylcysteine in T cell acute lymphoblastic leukaemia.

    PubMed

    Mansour, Marc R; Reed, Casie; Eisenberg, Amy R; Tseng, Jen-Chieh; Twizere, Jean-Claude; Daakour, Sarah; Yoda, Akinori; Rodig, Scott J; Tal, Noa; Shochat, Chen; Berezovskaya, Alla; DeAngelo, Daniel J; Sallan, Stephen E; Weinstock, David M; Izraeli, Shai; Kung, Andrew L; Kentsis, Alex; Look, A Thomas

    2015-01-01

    Activating mutations of the interleukin-7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T-ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand-independent activation of STAT5. We hypothesized that the reducing agent N-acetylcysteine (NAC), a well-tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R-mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R-mutant DND-41 cells as assessed by non-reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND-41 cells, and Ba/F3 cells transformed by an IL7R-mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND-41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T-ALL.

  16. Prostaglandin synthesis in human T cells: its partial inhibition by lectins and anti-CD3 antibodies as a possible step in T cell activation.

    PubMed

    Aussel, C; Mary, D; Fehlmann, M

    1987-05-15

    The human leukemic T cell line Jurkat was used to study arachidonic acid (AA) metabolism. We demonstrated that Jurkat cells are able to convert AA into prostaglandins (PG) and thromboxanes. The presence of tritiated 6-keto-PGF1 alpha, PGE2, PGA2 (B2), and thromboxane B2 in the culture medium was shown either by thin-layer chromatography after a 4-hr incubation period of [3H]AA-prelabeled Jurkat cells or by using specific radioimmuno assays. PG synthesis was inhibited by both indomethacin and niflumic acid, two cyclooxygenase inhibitors. AA metabolism through the cyclooxygenase pathway was followed during T cell activation. T cells were activated by lectins or anti-CD3 monoclonal antibodies (mAb) to trigger the T3-Ti complex and by 12-0-tetradecanoylphorbol 13-acetate (TPA) to mimic IL 1-dependent pathways. Our results show that lectins and anti-CD3 mAb both reduce the amount of PG released by the cells, whereas TPA did not. We confirmed that a combination of TPA and lectins or TPA and anti-CD3 mAb is necessary to obtain full activation of Jurkat cells if this event is monitored by using measurement of IL 2 synthesis. In addition, lectins and anti-CD3 mAb can be replaced by the cyclooxygenase inhibitors indomethacin or niflumic acid. Indeed, a combination of TPA and one of these two drugs induced maximal IL 2 synthesis. These results thus suggest that a reduction in PG synthesis might be a prerequisite to allow the cascade of events involved in T cell activation.

  17. Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

    SciTech Connect

    Li,H.; van Vranken, S.; Zhao, Y.; Li, Z.; Guo, Y.; Eisele, L.; Li, Y.

    2005-01-01

    The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.

  18. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis.

    PubMed

    Wehr, Alexander; Baeck, Christer; Heymann, Felix; Niemietz, Patricia Maria; Hammerich, Linda; Martin, Christian; Zimmermann, Henning W; Pack, Oliver; Gassler, Nikolaus; Hittatiya, Kanishka; Ludwig, Andreas; Luedde, Tom; Trautwein, Christian; Tacke, Frank

    2013-05-15

    Chronic liver injury characteristically results in hepatic inflammation, which represents a prerequisite for organ fibrosis. Although NKT cells are abundantly present in liver and involved in hepatic inflammation, molecular mechanisms of their recruitment in liver fibrosis remained elusive. We hypothesized that chemokine receptor CXCR6 and its ligand CXCL16 control NKT cell migration and functionality in liver fibrosis. In patients with chronic liver diseases (n = 58), CXCR6 and CXCL16 expression was intrahepatically upregulated compared with controls. In murine liver, Cxcl16 was strongly expressed by endothelium and macrophages, whereas lymphocyte populations (NKT, NK, CD4 T, CD8 T cells) expressed CXCR6. Intravital two-photon microscopy imaging of Cxcr6(+/gfp) and Cxcr6(gfp/gfp) mice and chemotaxis studies in vitro revealed that CXCR6 specifically controls hepatic NKT cell accumulation during the early response upon experimental liver damage. Hepatic invariant NKT cells expressed distinct proinflammatory cytokines including IFN-γ and IL-4 upon injury. CXCR6-deficient mice were protected from liver fibrosis progression in two independent experimental models. Macrophage infiltration and protein levels of inflammatory cytokines IFN-γ, TNF-α, and IL-4 were also reduced in fibrotic livers of Cxcr6(-/-) mice, corroborating that hepatic NKT cells provide essential cytokine signals perpetuating hepatic inflammation and fibrogenesis. Adoptive transfer of NKT cells, but not CD4 T cells, isolated from wild type livers restored hepatic fibrosis in Cxcr6(-/-) mice upon experimental steatohepatitis. Our results demonstrate that hepatic NKT cells accumulate CXCR6-dependent early upon injury, thereby accentuating the inflammatory response in the liver and promoting hepatic fibrogenesis. Interfering with CXCR6/CXCL16 might therefore bear therapeutic potential in liver fibrosis.

  19. Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T cells specifically inhibit Ewing sarcoma growth in vitro and in vivo.

    PubMed

    Blaeschke, Franziska; Thiel, Uwe; Kirschner, Andreas; Thiede, Melanie; Rubio, Rebeca Alba; Schirmer, David; Kirchner, Thomas; Richter, Günther H S; Mall, Sabine; Klar, Richard; Riddell, Stanley; Busch, Dirk H; Krackhardt, Angela; Grunewald, Thomas G P; Burdach, Stefan

    2016-07-12

    The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion.In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction.After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2-/-É£c-/- mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells.CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.

  20. Normalization of the peripheral blood T cell receptor V beta repertoire after cultured postnatal human thymic transplantation in DiGeorge syndrome.

    PubMed

    Davis, C M; McLaughlin, T M; Watson, T J; Buckley, R H; Schiff, S E; Hale, L P; Haynes, B F; Markert, M L

    1997-03-01

    Complete DiGeorge syndrome is an immunodeficiency disease characterized by thymic aplasia and the absence of functioning peripheral T cells. A patient with this syndrome was transplanted with cultured postnatal human thymic tissue. Within 5 weeks of transplantation, flow cytometry, T cell receptor V beta sequence analysis, and cell function studies showed the presence of oligoclonal populations of nonfunctional clonally expanded peripheral T cells that were derived from pretransplantation T cells present in the skin. However, at 3 months posttransplantation, a biopsy of the transplanted thymus showed normal intrathymic T cell maturation of host T cells with normal TCR V beta expression on thymocytes. By 9 months postransplantation, peripheral T cell function was restored and the TCR V beta repertoire became polyclonal, coincident with the appearance of normal T cell function. These data suggest that the transplanted thymus was responsible for the establishment of a new T cell repertoire via thymopoiesis in the chimeric thymic graft.

  1. The dynamics of T-cell receptor repertoire diversity following thymus transplantation for DiGeorge anomaly.

    PubMed

    Ciupe, Stanca M; Devlin, Blythe H; Markert, M Louise; Kepler, Thomas B

    2009-06-01

    T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRbeta V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of

  2. Fas-ligand-mediated paracrine T cell regulation by the receptor NKG2D in tumor immunity.

    PubMed

    Groh, Veronika; Smythe, Kimberly; Dai, Zhenpeng; Spies, Thomas

    2006-07-01

    Tumor-associated ligands of the activating NKG2D receptor can effectively stimulate T cell responses at early but not late stages of tumor growth. In late-stage human tumor settings, we observed MIC-driven proliferation of NKG2D(+)CD4(+) T cells that produced the cytokine Fas ligand (FasL) as a result of NKG2D costimulation but were themselves protected from Fas-mediated growth arrest. In contrast, FasL suppressed proliferation of T cells in vitro that did not receive NKG2D costimulation. Similar observations with normal peripheral blood NKG2D(+)CD8(+) T cells demonstrated unrecognized NKG2D-mediated immune functions, whereby FasL release promotes tumor cell death and NKG2D costimulation prolongs T cell survival. These effects, beneficial in conditions of limited NKG2D ligand expression, may be counterweighed when massive expression and shedding of MIC occurs, such as in some late-stage tumors, that causes sustained NKG2D costimulation and population expansion of immunosuppressive T cells.

  3. Peroxisome Proliferator–Activated Receptor-γ Agonists Prevent In Vivo Remodeling of Human Artery Induced by Alloreactive T Cells

    PubMed Central

    Tobiasova, Zuzana; Zhang, Lufeng; Yi, Tai; Qin, Linfeng; Manes, Thomas D.; Kulkarni, Sanjay; Lorber, Marc I.; Rodriguez, Frederick C.; Choi, Je-Min; Tellides, George; Pober, Jordan S.; Kawikova, Ivana; Bothwell, Alfred L.M.

    2012-01-01

    Background Ligands activating the transcription factor peroxisome proliferator–activated receptor-γ (PPARγ) have antiinflammatory effects. Vascular rejection induced by allogeneic T cells can be responsible for acute and chronic graft loss. Studies in rodents suggest that PPARγ agonists may inhibit graft vascular rejection, but human T-cell responses to allogeneic vascular cells differ from those in rodents, and the effects of PPARγ in human transplantation are unknown. Methods and Results We tested the effects of PPARγ agonists on human vascular graft rejection using a model in which human artery is interposed into the abdominal aorta of immunodeficient mice, followed by adoptive transfer of allogeneic (to the artery donor) human peripheral blood mononuclear cells. Interferon-γ–dependent rejection ensues within 4 weeks, characterized by intimal thickening, T-cell infiltrates, and vascular cell activation, a response resembling clinical intimal arteritis. The PPARγ agonists 15-deoxy-prostaglandin-J2, ciglitazone, and pioglitazone reduced intimal expansion, intimal infiltration of CD45RO+ memory T cells, and plasma levels of inflammatory cytokines. The PPARγ antagonist GW9662 reversed the protective effects of PPARγ agonists, confirming the involvement of PPARγ-mediated pathways. In vitro, pioglitazone inhibited both alloantigen-induced proliferation and superantigen-induced transendothelial migration of memory T cells, indicating the potential mechanisms of PPARγ effects. Conclusion Our results suggest that PPARγ agonists inhibit allogeneic human memory T cell responses and may be useful for the treatment of vascular graft rejection. PMID:21690493

  4. At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

    PubMed

    Daniyan, Anthony F O; Brentjens, Renier J

    2016-12-01

    The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic.

  5. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  6. The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex.

    PubMed

    Liu, Baoyu; Chen, Wei; Natarajan, Kannan; Li, Zhenhai; Margulies, David H; Zhu, Cheng

    2015-07-01

    T cells recognize antigens at the two-dimensional (2D) interface with antigen-presenting cells (APCs), which trigger T-cell effector functions. T-cell functional outcomes correlate with 2D kinetics of membrane-embedded T-cell receptors (TCRs) binding to surface-tethered peptide-major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR-pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T-cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR-pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on-rates but had no effect on 2D off-rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on-rate was insensitive to cellular perturbations and the force-dependent off-rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR-pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T-cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.

  7. The phosphatase JKAP/DUSP22 inhibits t-cell receptor signalling and autoimmunity by inactivating Lck

    Technology Transfer Automated Retrieval System (TEKTRAN)

    JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knocko...

  8. P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

    PubMed

    Figliuolo, Vanessa R; Savio, Luiz Eduardo Baggio; Safya, Hanaa; Nanini, Hayandra; Bernardazzi, Cláudio; Abalo, Alessandra; de Souza, Heitor S P; Kanellopoulos, Jean; Bobé, Pierre; Coutinho, Cláudia M L M; Coutinho-Silva, Robson

    2017-03-07

    P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB(low). Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut.

  9. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse

    PubMed Central

    Moran, Amy E.; Holzapfel, Keli L.; Xing, Yan; Cunningham, Nicole R.; Maltzman, Jonathan S.; Punt, Jennifer

    2011-01-01

    The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (Treg cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although Treg cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that Treg cell progenitors compete for recognition of rare stimulatory TCR self-ligands. PMID:21606508

  10. Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

    PubMed

    Kahaleh, M B; Fan, P S; Otsuka, T

    1999-05-01

    In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc.

  11. Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

    SciTech Connect

    Burrows, Scott R.; Chen, Zhenjun; Archbold, Julia K.; Tynan, Fleur E.; Beddoe, Travis; Kjer-Nielsen, Lars; Miles, John J.; Khanna, Rajiv; Moss, Denis J.; Liu, Yu Chih; Gras, Stephanie; Kostenko, Lyudmila; Brennan, Rebekah M.; Clements, Craig S.; Brooks, Andrew G.; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie

    2010-07-07

    {alpha}{beta} T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

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

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  13. KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

    PubMed

    Kamimura, Daisuke; Katsunuma, Kokichi; Arima, Yasunobu; Atsumi, Toru; Jiang, Jing-jing; Bando, Hidenori; Meng, Jie; Sabharwal, Lavannya; Stofkova, Andrea; Nishikawa, Naoki; Suzuki, Hironao; Ogura, Hideki; Ueda, Naoko; Tsuruoka, Mineko; Harada, Masaya; Kobayashi, Junya; Hasegawa, Takanori; Yoshida, Hisahiro; Koseki, Haruhiko; Miura, Ikuo; Wakana, Shigeharu; Nishida, Keigo; Kitamura, Hidemitsu; Fukada, Toshiyuki; Hirano, Toshio; Murakami, Masaaki

    2015-06-17

    KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

  14. DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo.

    PubMed

    Lehmann, Christian H K; Baranska, Anna; Heidkamp, Gordon F; Heger, Lukas; Neubert, Kirsten; Lühr, Jennifer J; Hoffmann, Alana; Reimer, Katharina C; Brückner, Christin; Beck, Simone; Seeling, Michaela; Kießling, Melissa; Soulat, Didier; Krug, Anne B; Ravetch, Jeffrey V; Leusen, Jeanette H W; Nimmerjahn, Falk; Dudziak, Diana

    2017-04-07

    Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4(+) and CD8(+) T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8(+) T cell responses was largely dependent on CD11c(+)CD8(+) DCs, whereas CD11c(+)CD8(-) DCs were critical for priming CD4(+) T cell responses.

  15. Crystal structure of a Gammadelta T-cell Receptor Specific for the Human MHC class I Homolog MICA

    SciTech Connect

    B Xu; J Pizarro; M Holmes; C McBeth; V Groh; T Spies; R Strong

    2011-12-31

    {gamma}{delta} T cells play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Human {gamma}{delta} T cells of the V{sub {delta}}1 subset predominate in intestinal epithelia and respond to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, mediating responses to tumorigenesis or viral infection. The crystal structure of an MIC-reactive V{sub {delta}}1 {gamma}{delta} T-cell receptor (TCR) showed expected overall structural homology to antibodies, {alpha}{beta}, and other {gamma}{delta} TCRs, but complementary determining region conformations and conservation of V{sub {delta}}1 use revealed an uncharacteristically flat potential binding surface. MIC, likewise, serves as a ligand for the activating immunoreceptor natural killer group 2, D (NKG2D), also expressed on {gamma}{delta} T cells. Although MIC recognition drives both the TCR-dependent stimulatory and NKG2D-dependent costimulatory signals necessary for activation, interaction analyses showed that MIC binding by the two receptors was mutually exclusive. Analysis of relative binding kinetics suggested sequential recognition, defining constraints for the temporal organization of {gamma}{delta} T-cell/target cell interfaces.

  16. Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

    PubMed

    Wolf, Insa M A; Diercks, Björn-Philipp; Gattkowski, Ellen; Czarniak, Frederik; Kempski, Jan; Werner, René; Schetelig, Daniel; Mittrücker, Hans-Willi; Schumacher, Valéa; von Osten, Manuel; Lodygin, Dimitri; Flügel, Alexander; Fliegert, Ralf; Guse, Andreas H

    2015-10-13

    The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

  17. ZAP-70, CTLA-4 and proximal T cell receptor signaling in cows infected with Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Leite, Fernando L; Eslabão, Livia B; Pesch, Bruce; Bannantine, John P; Reinhardt, Timothy A; Stabel, Judith R

    2015-09-15

    Paratuberculosis is a chronic intestinal disease of ruminant animals caused by Mycobacterium avium subsp. paratuberculosis (MAP). A hallmark of paratuberculosis is a transition from a cell-mediated Th1 type response to a humoral Th2 response with the progression of disease from a subclinical to clinical state. The objective of this study was to investigate the expression of two crucial molecules in T cell function, ZAP-70 (zeta-chain-associated protein of 70 kDa) and CTLA-4 (cytotoxic T-lymphocyte antigen-4), in cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMCs) isolated from control non-infected cows (n=5), and cows in subclinical (n=6) and clinical stages of paratuberculosis (n=6) were cultured alone (medium only), and with concanavalin A, and a whole cell sonicate of MAP for 24, 72 and 144 h to measure the dynamic changes of ZAP-70 and CTLA-4 expression on CD4, CD8, and gamma delta (γδ) T cells. Flow cytometry was also performed to measure ZAP-70 phosphorylation to examine proximal T cell receptor signaling in animals of different disease status. The surface expression of CTLA-4 was increased in animals in subclinical stage of infection while levels of ZAP-70 were decreased in CD4+ T cells of both subclinical and clinical animals, indicating a change in T cell phenotype with disease state. Interestingly, proximal T cell receptor signaling was not altered in infected animals. This study demonstrated changes in crucial signaling molecules in animals infected with MAP, thereby elucidating T cell alterations associated with disease progression.

  18. Development of CD8+ T cells expressing two distinct receptors specific for MTB and HIV-1 peptides

    PubMed Central

    Hao, Pei-Pei; Zhang, Xiao-Bing; Luo, Wei; Zhou, Chao-Ying; Wen, Qian; Yang, Zhi; Liu, Su-Dong; Jiang, Zhen-Min; Zhou, Ming-Qian; Jin, Qi; Ma, Li

    2013-01-01

    The immune response in individuals co-infected with Mycobacterium tuberculosis (MTB) and the human immunodeficiency virus (MTB/HIV) gradually deteriorates, particularly in the cellular compartment. Adoptive transfer of functional effector T cells can confer protective immunity to immunodeficient MTB/HIV co-infected recipients. However, few such effector T cells exist in vivo, and their isolation and amplification to sufficient numbers is difficult. Therefore, enhancing immune responses against both pathogens is critical for treating MTB/HIV co-infected patients. One approach is adoptive transfer of T cell receptor (TCR) gene-modified T cells for the treatment of MTB/HIV co-infections because lymphocyte numbers and their functional avidity is significantly increased by TCR gene transfer. To generate bispecific CD8+ T cells, MTB Ag85B199–207 peptide-specific TCRs (MTB/TCR) and HIV-1 Env120–128 peptide-specific TCRs (HIV/TCR) were isolated and introduced into CD8+ T cells simultaneously using a retroviral vector. To avoid mispairing among exogenous and endogenous TCRs, and to improve the function and stability of the introduced TCRs, several strategies were employed, including introducing mutations in the MTB/TCR constant (C) regions, substituting part of the HIV/TCR C regions with CD3ζ, and linking gene segments with three different 2A peptides. Results presented in this report suggest that the engineered T cells possessed peptide-specific specificity resulting in cytokine production and cytotoxic activity. This is the first report describing the generation of engineered T cells specific for two different pathogens and provides new insights into TCR gene therapy for the treatment of immunocompromised MTB/HIV co-infected patients.

  19. Preserved Activity of CD20-Specific Chimeric Antigen Receptor-Expressing T Cells in the Presence of Rituximab.

    PubMed

    Rufener, Gregory A; Press, Oliver W; Olsen, Philip; Lee, Sang Yun; Jensen, Michael C; Gopal, Ajay K; Pender, Barbara; Budde, Lihua E; Rossow, Jeffrey K; Green, Damian J; Maloney, David G; Riddell, Stanley R; Till, Brian G

    2016-06-01

    CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell-mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo CAR-binding sites on CD20(+) tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/mL. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/mL, CAR T cells retained ≥50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in >85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/mL (interquartile range, 19-72 μg/mL). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible. Cancer Immunol Res; 4(6); 509-19. ©2016 AACR

  20. Cutaneous type of adult T cell leukemia/lymphoma in a French West Indian woman. Clonal rearrangement of T-cell receptor beta and gamma genes and monoclonal integration of HTLV-I proviral DNA in the skin infiltrate.

    PubMed

    Gessain, A; Moulonguet, I; Flageul, B; Perrin, P; Capesius, C; D'Agay, M F; Gisselbrecht, C; Sigaux, F; Civatte, J

    1990-11-01

    A 45-year-old woman, a native of the French West Indies who had lived in France since 1973, developed multiple cutaneous plaques and nodules in 1987. Histopathologic studies revealed dermal infiltration with mature activated T cells (CD4+, CD25+, DR+) with nuclear convolutions and epidermatotropisim. High titers of specific human T lymphotropic virus (HTLV)-I antibodies were detected in the serum. Molecular analysis of DNA extracted from the skin tumor biopsy specimen showed a clonal integration of an HTLV-I provirus and a T-cell clonal population as demonstrated by T-cell receptor beta and gamma gene rearrangement studies. Neither HTLV-I provirus nor T-cell receptor rearrangements were detected in peripheral blood mononuclear cells DNA despite the presence of rare adult T cell leukemia cells (less than 1%) and a small excess of DR-expressing cells, and detection of HTLV-I Pol and Px sequences by in vitro gene amplification. In this case only gene analysis of the skin lesions made possible an early diagnosis of a cutaneous adult T cell leukemia. This illustrates the need for such molecular studies to differentiate, in HTLV-I seropositive patients from endemic areas, a HTLV-I-induced T cell lymphoma from HTLV-I-nonrelated cutaneous T cell lymphomas.

  1. Binding of a soluble alpha beta T-cell receptor to superantigen/major histocompatibility complex ligands.

    PubMed Central

    Kappler, J; White, J; Kozono, H; Clements, J; Marrack, P

    1994-01-01

    The genes for the alpha and beta chains of a murine T-cell receptor were truncated just prior to the portions encoding the transmembrane regions and introduced into baculovirus by recombination. Insect cells infected with the virus secreted a soluble form of the receptor that could be purified to homogeneity. This soluble receptor reacted with a set of six monoclonal antibodies originally raised to different epitopes on the natural transmembrane-region-containing receptor and bound with appropriate specificity to a cell surface complex of the human major histocompatibility complex class II molecule DR1 with the bacterial superantigen staphylococcal enterotoxin B. Images PMID:8078904

  2. High frequency of clonal IG and T-cell receptor gene rearrangements in histiocytic and dendritic cell neoplasms

    PubMed Central

    Huang, Wenting; Qiu, Tian; Zeng, Linshu; Zheng, Bo; Ying, Jianming; Feng, Xiaoli

    2016-01-01

    The 2008 World Health Organization (WHO) diagnostic criteria of histiocytic and dendritic cell neoplasms from hematopoietic and lymphoid tissues no longer required the absence of clonal B-cell/T-cell receptor gene rearrangements. It is true that the clonal B-cell/T-cell receptor gene rearrangements have been identified in rare cases of histiocytic and dendritic cell neoplasms, such as those with or following lymphoma/leukemia or in some sporadic histiocytic/dendritic cell sarcomas, but the clonal features of such group of tumor are still not clear. Here we investigated the clonal status of 33 samples including Langerhans cell histiocytosis (LCH), Langerhans cell sarcoma (LCS), follicular dendritic cell sarcoma (FDCS), interdigitating dendritic cell sarcoma (IDCS) and histiocytic sarcoma (HS). Among them, twenty-eight cases were sporadic without current or past lymphoma/leukemia. Three cases were found with a past history of T-cell lymphoma, one case was followed by extraosseous plasmacytoma, and one case was found with diffuse large B-cell lymphoma (DLBCL). Our results showed that there was a high frequency of clonal IG and T-cell receptor gene rearrangements in these cases. Notably, 4 cases of LCH and 2 cases of FDCS showed both B and T cell receptor gene rearrangements concurrently. One case of FDCS synchronous with DLBCL showed identical clonal IGH in both tumor populations and clonal TCRβ in FDCS alone. No matter if the presence of clonal receptor gene rearrangements was associated with the tumor origin or tumorigenesis, it might serve as a novel tumor marker for developing target therapy. PMID:27823979

  3. Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever.

    PubMed

    de-Oliveira-Pinto, Luzia Maria; Marinho, Cíntia Ferreira; Povoa, Tiago Fajardo; de Azeredo, Elzinandes Leal; de Souza, Luiza Assed; Barbosa, Luiza Damian Ribeiro; Motta-Castro, Ana Rita C; Alves, Ada M B; Ávila, Carlos André Lins; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Paes, Marciano Viana; Kubelka, Claire Fernandes

    2012-01-01

    Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by

  4. The T cell antigen receptor CD3:CD4 molecular complex is diminished on the surface of pulmonary lymphocytes.

    PubMed Central

    Marathias, K.; Pinto, C.; Rodberg, G.; Preffer, F.; Wong, J.; Kradin, R.

    1994-01-01

    CD4, a 55-kd cell surface glycoprotein, binds to class II major histocompatibility complex (MHC) (Ia) antigens and functions as a coreceptor for the T cell antigen receptor (Ti alpha beta)-CD3 complex. We have observed that critical elements of the T cell antigen multireceptor complex, including Ti alpha beta, CD3, CD4, but not CD8, were diminished on CD45RO+ pulmonary T lymphocytes but not CD45RO+ peripheral blood T lymphocytes (PBL). Epitopes mapping from the first (D1) to the fourth (D4) extracytoplasmic Ig-like domains of CD4 were expressed to a lesser degree on pulmonary T cells than on PBL (P = 0.002). CD4 expression on pulmonary T cells did not increase after 72 hours of ex vivo culture in complete medium but was restored toward control levels by stimulation with phytohemagglutinin, anti-CD3, or interleukin-2. CD4 mRNA isolated from lung T cells and PBL co-migrated on Northern blots and the total levels of CD4 mRNA were comparable, suggesting that diminished CD4 expression by pulmonary T cells might reflect a posttranscriptional change. To determine whether CD4bright T cells convert with mitogen stimulation to CD4dim cells, PBLs were stimulated with immobilized anti-CD3, anti-CD4, or a molecularly engineered anti-CD3:CD4 bispecific monoclonal antibody and the ratio of the CD4:CD3 mean fluorescence staining intensities was calculated at days 3 and 13. The CD4:CD3 ratio decreased primarily for cells stimulated with anti-CD3:CD4, suggesting that co-ligation of CD3 and CD4 is required for the generation of CD4dim T cells. We conclude that diminished Ti alpha beta-CD3:CD4 expression is a characteristic of T cells in lung that is not shared by peripheral blood T cells in vivo, and speculate that this change reflects T cell activation in a millieu of limited interleukin-2 availability. Images Figure 8 Figure 9 PMID:7977652

  5. Stimulation of β₂-adrenergic receptors inhibits calcineurin activity in CD4(+) T cells via PKA-AKAP interaction.

    PubMed

    Riether, Carsten; Kavelaars, Annemieke; Wirth, Timo; Pacheco-López, Gustavo; Doenlen, Raphael; Willemen, Hanneke; Heijnen, Cobi J; Schedlowski, Manfred; Engler, Harald

    2011-01-01

    The sympathetic nervous system (SNS) is able to modulate immune functions via adrenoceptor-dependent mechanisms. Activation of β₂-adrenergic receptors (AR) on CD4(+) T lymphocytes has been shown to inhibit Th1-cytokine production and cell proliferation. Here, we investigated the role of the calcium/calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway, in β₂-AR-mediated suppression of T cell function. Purified rat splenic CD4(+) T cells were stimulated with anti-CD3/anti-CD28 in presence or absence of the β₂-AR agonist terbutaline (TERB). Treatment with TERB induced a dose-dependent inhibition of cellular CaN activity, along with a reduction in IL-2 and IFN-γ production, and T cell proliferation. Co-administration of the β-AR antagonist nadolol abolished these effects. Blockade of the cAMP-dependent protein kinase A (PKA) with the inhibitor H-89 completely prevented TERB-induced CaN inhibition. However, a receptor-independent rise in the second messenger cAMP was not sufficient to suppress CaN activity. Disruption of the interaction between PKA and A-kinase anchoring protein (AKAP) by the inhibitor peptide St-Ht31 fully blocked TERB-induced CaN inhibition, demonstrating that PKA-AKAP interaction is essential for the β₂-AR-mediated CaN inhibition. Taken together, this study provides evidence for a link between the β₂-AR and TCR signaling pathways since expression of IL-2 and IFN-γ in activated T cells largely depends on dephosphorylation of the transcription factor NFAT by CaN, and identifies a novel intracellular mechanism that can lead to downregulation of T cell function after SNS activation.

  6. Sequence and diversity of rabbit T-cell receptor gamma chain genes

    SciTech Connect

    Isono, T.; Kim, C.J.; Seto, A.

    1995-03-01

    The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes. 18 refs., 4 figs., 1 tab.

  7. Isolation of monotreme T-cell receptor alpha and beta chains.

    PubMed

    Belov, Katherine; Miller, Robert D; Ilijeski, Aron; Hellman, Lars; Harrison, Gavan A

    2004-06-01

    Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the 'mammalian' immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR alpha-chain cDNAs ( TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR beta-chain cDNA ( TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.

  8. Structural Evidence for a Germline-Encoded T Cell Receptor - Major Histocompatibility Complex Interaction 'Codon'

    SciTech Connect

    Feng, D.; Bond, C.J.; Ely, L.K.; Maynard, J.; Garcia, K.C.

    2009-06-02

    All complexes of T cell receptors (TCRs) bound to peptide-major histocompatibility complex (pMHC) molecules assume a stereotyped binding 'polarity', despite wide variations in TCR-pMHC docking angles. However, existing TCR-pMHC crystal structures have failed to show broadly conserved pairwise interaction motifs. Here we determined the crystal structures of two TCRs encoded by the variable {beta}-chain 8.2 (V{sub {beta}}8.2), each bound to the MHC class II molecule I-A{sup u}, and did energetic mapping of V{sub {alpha}} and V{sub {beta}} contacts with I-A{sup u}. Together with two previously solved structures of V{sub {beta}}8.2-containing TCR-MHC complexes, we found four TCR-I-A complexes with structurally superimposable interactions between the V{sub {beta}} loops and the I-A {alpha}-helix. This examination of a narrow 'slice' of the TCR-MHC repertoire demonstrates what is probably one of many germline-derived TCR-MHC interaction 'codons'.

  9. Crossreactive αβ T cell receptors are the predominant targets of thymocyte negative selection

    PubMed Central

    McDonald, Benjamin D.; Bunker, Jeffrey J.; Erickson, Steven A.; Oh-Hora, Masatsugu; Bendelac, Albert

    2015-01-01

    SUMMARY The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual preselection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4−CD8β− intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation. PMID:26522985

  10. Age Estimation in Living Egyptians Using Signal Joint T-cell Receptor Excision Circle Rearrangement.

    PubMed

    Ibrahim, Samah F; Gaballah, Iman F; Rashed, Laila A

    2016-07-01

    Age estimation is one of the challenges in forensic sciences. There are many techniques to estimate the age. Molecular biology approach is one of these techniques. Signal joint T-cell receptor excision circles gene (sjTRECs), is one of this approach. We aimed to use sjTRECs as a suitable marker for age estimation among Egyptian population. TaqMan qPCR approach was used to quantify sjTREC levels in blood samples obtained from 153 healthy Egyptian individuals ranging from a few weeks to 70 years. Our results showed a significant negative correlation between sjTREC levels and age with p ≤ 0.05. Moreover, the individual's age can be determined through this formula Age = -30.671+ (-5.998Y) (Y is dCtTBP - sjTREC) with standard error ±7.35 years. Within the forensic context, sjTREC' levels can be used to estimate the Egyptian individual's age accurately.

  11. Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor

    NASA Astrophysics Data System (ADS)

    Kumar, Amit; Delogu, Francesco

    2017-02-01

    The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR’s cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient.

  12. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  13. T-cell receptor V alpha and C alpha alleles associated with multiple and myasthenia gravis.

    PubMed Central

    Oksenberg, J R; Sherritt, M; Begovich, A B; Erlich, H A; Bernard, C C; Cavalli-Sforza, L L; Steinman, L

    1989-01-01

    Polymorphic markers in genes encoding that alpha chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, we amplified selected sequences derived from the full-length TcR alpha cDNA probe. These PCR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of the polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, we have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V alpha and C alpha markers were identified between patients and healthy individuals. Images PMID:2915992

  14. Toll-like receptor 2 controls expansion and function of regulatory T cells

    PubMed Central

    Sutmuller, Roger P.M.; den Brok, Martijn H.M.G.M.; Kramer, Matthijs; Bennink, Erik J.; Toonen, Liza W.J.; Kullberg, Bart-Jan; Joosten, Leo A.; Akira, Shizuo; Netea, Mihai G.; Adema, Gosse J.

    2006-01-01

    Tregs play a central role in the suppression of immune reactions and prevention of autoimmune responses harmful to the host. During acute infection, however, Tregs might hinder effector T cell activity directed toward the elimination of the pathogenic challenge. Pathogen recognition receptors from the TLR family expressed by innate immune cells are crucial for the generation of effective immunity. We have recently shown the CD4+CD25+ Treg subset in TLR2–/– mice to be significantly reduced in number compared with WT littermate control mice, indicating a link between Tregs and TLR2. Here, we report that the TLR2 ligand Pam3Cys, but not LPS (TLR4) or CpG (TLR9), directly acts on purified Tregs in a MyD88-dependent fashion. Moreover, when combined with TCR stimulation, TLR2 triggering augmented Treg proliferation in vitro and in vivo and resulted in a temporal loss of the suppressive Treg phenotype in vitro by directly affecting Tregs. Importantly, WT Tregs adoptively transferred into TLR2–/– mice were neutralized by systemic administration of TLR2 ligand during the acute phase of a Candida albicans infection, resulting in a 100-fold reduced C. albicans outgrowth. This demonstrates that in vivo TLR2 also controls the function of Tregs and establishes a direct link between TLRs and the control of immune responses through Tregs. PMID:16424940

  15. Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor.

    PubMed

    Guo, Xingdong; Yan, Chengsong; Li, Hua; Huang, Wenmao; Shi, Xiaoshan; Huang, Min; Wang, Yingfang; Pan, Weiling; Cai, Mingjun; Li, Lunyi; Wu, Wei; Bai, Yibing; Zhang, Chi; Liu, Zhijun; Wang, Xinyan; Zhang, Xiaohui F; Tang, Chun; Wang, Hongda; Liu, Wanli; Ouyang, Bo; Wong, Catherine C; Cao, Yi; Xu, Chenqi

    2017-03-24

    T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3εCD). At the single-molecule level, we found that CD3εCD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS. These conformations were generated because different regions of CD3εCD had heterogeneous lipid-binding properties and therefore had heterogeneous dynamics. Live-cell imaging experiments demonstrated that different antigen stimulations could stabilize CD3εCD at different conformations. Lipid-dependent conformational dynamics thus provide structural basis for the versatile signaling property of TCR.Cell Research advance online publication 24 March 2017; doi:10.1038/cr.2017.42.

  16. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

    PubMed Central

    Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Nicovich, Philip R.; Bridgeman, John S.; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D.; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K.; Price, David A.; Acuto, Oreste; Parton, Robert G.; Gooding, J. Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-01-01

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination. PMID:27573839

  17. Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor

    PubMed Central

    Kumar, Amit; Delogu, Francesco

    2017-01-01

    The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR’s cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient. PMID:28195200

  18. Generation and reactivation of T-cell receptor A joining region pseudogenes in primates

    SciTech Connect

    Thiel, C.; Lanchbury, J.S.; Otting, N.

    1996-06-01

    Tandemly duplicated T-cell receptor (Tcr) AJ (J{alpha}) segments contribute significantly to TCRA chain junctional region diversity in mammals. Since only limited data exists on TCRA diversity in nonhuman primates, we examined the TCRAJ regions of 37 chimpanzee and 71 rhesus macaque TCRA cDNA clones derived from inverse polymerase chain reaction on peripheral blood mononuclear cell cDNA of healthy animals. Twenty-five different TCRAJ regions were characterized in the chimpanzee and 36 in the rhesus macaque. Each bears a close structural relationship to an equivalent human TCRAJ region. Conserved amino acid motifs are shared between all three species. There are indications that differences between nonhuman primates and humans exist in the generation of TCRAJ pseudogenes. The nucleotide and amino acid sequences of the various characterized TCRAJ of each species are reported and we compare our results to the available information on human genomic sequences. Although we provide evidence of dynamic processes modifying TCRAJ segments during primate evolution, their repertoire and primary structure appears to be relatively conserved. 21 refs., 2 figs.

  19. Sex-hormone receptors pattern on regulatory T-cells: clinical implications for multiple sclerosis.

    PubMed

    Aristimuño, Carol; Teijeiro, Roseta; Valor, Lara; Alonso, Bárbara; Tejera-Alhambra, Marta; de Andrés, Clara; Miñarro, Desamparados Oliver; López-Lazareno, Nieves; Faure, Florence; Sánchez-Ramón, Silvia

    2012-12-01

    Cellular mechanisms underlying sexual dimorphism in the immune response remain largely unknown. Concerning the interactions among the nervous, endocrine and immune systems, we reported that during gestation, a period during which multiple sclerosis (MS) clearly ameliorates, there is a physiological expansion of regulatory T-lymphocytes (T(Reg)). Given that alterations in T(Reg) proportions and suppressive function are involved in MS pathophysiology, we investigated the in vitro effect of sex hormones on T(Reg). Here, we show that both E2 and progesterone (P2) enhance T(Reg) function in vitro, although only E2 further induces a T(Reg) phenotype in activated responder T-cells (CD4(+)CD25(-)) (P < 0.01). E2 receptor beta (ERβ) percentages and mean fluorescence intensity (MFI) on T(Reg) were lower in MS patients than in controls (P < 0.05), in parallel with lower E2 plasma levels (P < 0.05). Importantly, percentages and MFI of ERβ were higher in T(Reg) than in T-responder cells (P < 0.0001) both in MS patients and controls. We show a unique differential pattern of higher ER and PR levels in T(Reg), which may be relevant for the in vivo responsiveness of these cells to sex hormones and hence to MS physiopathology.

  20. Genomic organization of the human T-cell antigen-receptor alpha/delta locus.

    PubMed

    Satyanarayana, K; Hata, S; Devlin, P; Roncarolo, M G; De Vries, J E; Spits, H; Strominger, J L; Krangel, M S

    1988-11-01

    Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.

  1. Characterization of horse (Equus caballus) T-cell receptor beta chain genes

    SciTech Connect

    Schrenzel, M.D.; Watson, J.L.; Ferrick, D.A.

    1994-12-31

    Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5{prime} untranslated region of one horse gene. Germline sequences included the 5{prime} region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intro. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

  2. T-cell receptor polymorphisms in Tlingit Indians with rheumatoid arthritis.

    PubMed

    Charmley, P; Nelson, J L; Hansen, J A; Branchaud, A; Barrington, R A; Templin, D; Boyer, G; Lanier, A P; Concannon, P

    1994-01-01

    Rheumatoid arthritis (RA) develops as a result of the interaction of both genetic and environmental factors. Among the genes in humans that have been suggested as candidate susceptibility genes in RA are those encoding the T cell receptor for antigen (TCR). A high prevalence and early age of onset of RA has previously been reported in Alaskan Tlingit Indians. In this study, the frequency of seven different restriction fragment length polymorphisms (RFLPs) in the TCR alpha and beta gene complexes were measured in a population of Alaskan Tlingit Indians. No statistically significant differences were noted when the frequencies of these RFLPs were compared between Tlingits with RA and healthy controls (p > 0.05). These results do not support the hypothesis of an RA-susceptibility allele in the vicinity of these TCR alpha or beta genes. Since TCR RFLPs have not been extensively studied in native American populations, TCR polymorphism frequencies in the Tlingits were also compared to the frequencies observed in a second control group of healthy Caucasians. Statistically significant differences were observed in these comparisons implying a different distribution of individuals in these populations with different TCR repertoires.

  3. Genetic variation in MHC proteins is associated with T cell receptor expression biases

    PubMed Central

    Sharon, Eilon; Sibener, Leah V.; Battle, Alexis; Fraser, Hunter B.; Garcia, K. Christopher; Pritchard, Jonathan K.

    2016-01-01

    Within each individual, a highly diverse T cell receptor (TCR) repertoire interacts with peptides presented by major histocompatibility complex (MHC) molecules. Despite extensive research, it remains controversial whether germline-encoded TCR-MHC contacts promote TCR-MHC specificity and if so, whether there exist differences in TCR V-gene compatibilities with different MHC alleles. We applied eQTL mapping to test for associations between genetic variation and TCR V-gene usage in a large human cohort. We report strong trans associations between variation in the MHC locus and TCR V-gene usage. Fine mapping of the association signals reveals specific amino acids in MHC genes that bias V-gene usage, many of which contact or are spatially proximal to the TCR or peptide. Hence, these MHC variants, several of which are linked to autoimmune diseases, can directly affect TCR-MHC interaction. These results provide the first examples of trans-QTLs mediated by protein-protein interactions, and are consistent with intrinsic TCR-MHC specificity. PMID:27479906

  4. How a T Cell Receptor-like Antibody Recognizes Major Histocompatibility Complex-bound Peptide

    SciTech Connect

    Mareeva, T.; Martinez-Hackert, E; Sykulev, Y

    2008-01-01

    We determined the crystal structures of the T cell receptor (TCR)-like antibody 25-D1.16 Fab fragment bound to a complex of SIINFEKL peptide from ovalbumin and the H-2Kb molecule. Remarkably, this antibody directly 'reads' the structure of the major histocompatibility complex (MHC)-bound peptide, employing the canonical diagonal binding mode utilized by most TCRs. This is in marked contrast with another TCR-like antibody, Hyb3, bound to melanoma peptide MAGE-A1 in association with HLA-A1 MHC class I. Hyb3 assumes a non-canonical orientation over its cognate peptide-MHC and appears to recognize a conformational epitope in which the MHC contribution is dominant. We conclude that TCR-like antibodies can recognize MHC-bound peptide via two different mechanisms: one is similar to that exploited by the preponderance of TCRs and the other requires a non-canonical antibody orientation over the peptide-MHC complex.

  5. Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.

    PubMed

    Murugesan, Sricharan; Hong, Jinsung; Yi, Jason; Li, Dong; Beach, Jordan R; Shao, Lin; Meinhardt, John; Madison, Grey; Wu, Xufeng; Betzig, Eric; Hammer, John A

    2016-11-07

    Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

  6. A novel splice variant of folate receptor 4 predominantly expressed in regulatory T cells

    PubMed Central

    2012-01-01

    Background Regulatory T cells (Tregs) are required for proper maintenance of immunological self-tolerance and immune homeostasis. Folate receptor 4 (FR4) is expressed at high levels in transforming growth factor-beta (TGF-β)-induced Tregs and natural Tregs. Moreover, antibody-mediated targeting of FR4 is sufficient to mediate Treg depletion. Results In this study, we describe a novel FR4 transcript variant, FR4D3, in which exon 3 is deleted. The mRNA of FR4D3 encodes a FR4 variant truncated by 189 bp. FR4D3 was found to be predominantly expressed in CD4+CD25+ Treg cells. Overexpression of FR4D3 in CD4+CD25+ Treg cells in vitro stimulated proliferation, which may modulate the ability of these cells to bind and incorporate folic acid. Conclusions Our results suggested that high levels of FR4D3 may be critical to support the substantial proliferative capacity of Treg cells. PMID:22694797

  7. Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling

    PubMed Central

    Jahan, Akhee S.; Lestra, Maxime; Swee, Lee Kim; Fan, Ying; Lamers, Mart M.; Tafesse, Fikadu G.; Theile, Christopher S.; Spooner, Eric; Bruzzone, Roberto; Ploegh, Hidde L.; Sanyal, Sumana

    2016-01-01

    Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12−/− Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12−/− cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades. PMID:26811477

  8. Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination.

    PubMed

    Pageon, Sophie V; Tabarin, Thibault; Yamamoto, Yui; Ma, Yuanqing; Bridgeman, John S; Cohnen, André; Benzing, Carola; Gao, Yijun; Crowther, Michael D; Tungatt, Katie; Dolton, Garry; Sewell, Andrew K; Price, David A; Acuto, Oreste; Parton, Robert G; Gooding, J Justin; Rossy, Jérémie; Rossjohn, Jamie; Gaus, Katharina

    2016-09-13

    Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

  9. Dynamical footprint of cross-reactivity in a human autoimmune T-cell receptor.

    PubMed

    Kumar, Amit; Delogu, Francesco

    2017-02-14

    The present work focuses on the dynamical aspects of cross-reactivity between myelin based protein (MBP) self-peptide and two microbial peptides (UL15, PMM) for Hy.1B11 T-cell receptor (TCR). This same TCR was isolated from a patient suffering from multiple sclerosis (MS). The study aims at highlighting the chemical interactions underlying recognition mechanisms between TCR and the peptides presented by Major Histocompatibility Complex (MHC) proteins, which form a crucial component in adaptive immune response against foreign antigens. Since the ability of a TCR to recognize different peptide antigens presented by MHC depends on its cross-reactivity, we used molecular dynamics methods to obtain atomistic detail on TCR-peptide-MHC complexes. Our results show how the dynamical basis of Hy.1B11 TCR's cross-reactivity is rooted in a similar bridging interaction pattern across the TCR-peptide-MHC interface. Our simulations confirm the importance of TCR CDR3α E98 residue interaction with MHC and a predominant role of P6 peptide residue in MHC binding affinity. Altogether, our study provides energetic and dynamical insights into factors governing peptide recognition by the cross-reactive Hy.1B11 TCR, found in MS patient.

  10. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

    PubMed

    Chen, Cheng; Li, Kesang; Jiang, Hua; Song, Fei; Gao, Huiping; Pan, Xiaorong; Shi, Bizhi; Bi, Yanyu; Wang, Huamao; Wang, Hongyang; Li, Zonghai

    2017-04-01

    Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1(+)GPC3(-) tumor cells, but they exhibited a similar cytotoxicity against GPC3(+)ASGR1(-) and GPC3(+)ASGR1(+) HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3(+)ASGR1(+) HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3(+)ASGR1(+) HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3(+)ASGR1(+) HCC.

  11. Uncoupling of T cell receptor zeta chain function during the induction of anergy by the superantigen, staphylococcal enterotoxin A.

    PubMed

    Cornwell, William D; Rogers, Thomas J

    2010-07-01

    Staphylococcus aureus enterotoxins have immunomodulatory properties. In this study, we show that Staphylococcal enterotoxin A (SEA) induces a strong proliferative response in a murine T cell clone independent of MHC class II bearing cells. SEA stimulation also induces a state of hypo-responsiveness (anergy). We characterized the components of the T cell receptor (TCR) during induction of anergy by SEA. Most interestingly, TCR zeta chain phosphorylation was absent under SEA anergizing conditions, which suggests an uncoupling of zeta chain function. We characterize here a model system for studying anergy in the absence of confounding costimulatory signals.

  12. Improving antigenic peptide vaccines for cancer immunotherapy using a dominant tumor-specific T cell receptor.

    PubMed

    Buhrman, Jonathan D; Jordan, Kimberly R; Munson, Daniel J; Moore, Brandon L; Kappler, John W; Slansky, Jill E

    2013-11-15

    Vaccines that incorporate peptide mimics of tumor antigens, or mimotope vaccines, are commonly used in cancer immunotherapy and function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, they often elicit T cells that do not cross-react with or that have low affinity for the tumor antigen. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotope vaccines for the dominant peptide antigen from a mouse colon tumor that elicits a range of tumor protection following vaccination. The TCR from this high affinity T cell clone was rarely identified in ex vivo evaluation of tumor-specific T cells elicited by mimotope vaccination. Conversely, a low affinity clone found in the tumor and following immunization was frequently identified. Using peptide libraries, we determined if this frequently identified TCR improved the discovery of efficacious mimotopes. We demonstrated that the representative TCR identified more protective mimotopes than the high affinity TCR. These results suggest that targeting a dominant fraction of tumor-specific T cells generates potent immunity and that consideration of the available T cell repertoire is necessary for targeted T cell therapy. These results have important implications when optimizing mimotope vaccines for cancer immunotherapy.

  13. CCR5 in cancer immunotherapy: More than an "attractive" receptor for T cells.

    PubMed

    González-Martín, Alicia; Mira, Emilia; Mañes, Santos

    2012-01-01

    Despite intensive study, the role of CCR5 in cancer remains elusive. We showed that CCR5 expression by both CD4+ and CD8+ T cells is necessary to boost anti-tumor responses by optimizing helper-dependent CD8+ T cell priming. Our findings could have implications for cancer treatment in patients with defective CCR5 expression.

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

    PubMed

    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. Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity

    PubMed Central

    Uto, Tomofumi; Fukaya, Tomohiro; Takagi, Hideaki; Arimura, Keiichi; Nakamura, Takeshi; Kojima, Naoya; Malissen, Bernard; Sato, Katsuaki

    2016-01-01

    Dendritic cells (DCs) comprise several subsets that are critically involved in the initiation and regulation of immunity. Clec4A4/DC immunoreceptor 2 (DCIR2) is a C-type lectin receptor (CLR) exclusively expressed on CD8α− conventional DCs (cDCs). However, how Clec4A4 controls immune responses through regulation of the function of CD8α− cDCs remains unclear. Here we show that Clec4A4 is a regulatory receptor for the activation of CD8α− cDCs that impairs inflammation and T-cell immunity. Clec4a4−/−CD8α− cDCs show enhanced cytokine production and T-cell priming following Toll-like receptor (TLR)-mediated activation. Furthermore, Clec4a4−/− mice exhibit TLR-mediated hyperinflammation. On antigenic immunization, Clec4a4−/− mice show not only augmented T-cell responses but also progressive autoimmune pathogenesis. Conversely, Clec4a4−/− mice exhibit resistance to microbial infection, accompanied by enhanced T-cell responses against microbes. Thus, our findings highlight roles of Clec4A4 in regulation of the function of CD8α− cDCs for control of the magnitude and quality of immune response. PMID:27068492

  16. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    SciTech Connect

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia . E-mail: lombardi@pharm.unipmn.it

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

  17. Haematopoietic cell lines capable of colonizing the thymus following in vivo transfer expressed T-cell receptor gamma-gene immature mRNA.

    PubMed Central

    Shimamura, M; Oku, M; Ohta, S; Yamagata, T

    1992-01-01

    To clarify the mechanism by which progenitor T (pro-T) cells recognize and enter the thymus, an attempt was made to produce haematopoietic cell lines by the fusion of BALB/c nude mouse bone marrow or foetal liver cells (gestation 14 and 15 days) with AKR thymoma BW5147, thereby immortalizing cells with potency to colonize the thymus, a characteristic of pro-T cells rarely found in adult bone marrow or foetal liver. The hybridomas thus produced were classified according to the phenotype of surface markers, T-cell receptor (TcR) gene configuration and expression. All hybridomas were negative in the surface expression of T-cell markers such as TcR alpha beta, TcR gamma delta, CD3, CD4 and CD8. They had TcR beta-, gamma- and delta-genes, each with a different status with respect to configuration and transcription. Some possessed partially rearranged TcR genes and others expressed immature TcR mRNA. The cell lines were examined for their capacity to colonize the thymus following intravenous injection into recipient mice. It was found that the cells with capacity of colonizing the thymus expressed immature TcR delta mRNA, while the cell lines lacking TcR delta-genes did not home to the thymus. These findings imply that the potency for migrating to thymus is closely associated with the particular stage of prethymic cell differentiation which could be estimated by the analysis of TcR genes, and that some cell lines with the expression of TcR delta-gene mRNA and the ability to colonize the thymus are derived from pro-T cells. Images Figure 2 Figure 3 PMID:1478683

  18. Peripheral tissue homing receptors enable T cell entry into lymph nodes and affect the anatomical distribution of memory cells

    PubMed Central

    Brinkman, C. Colin; Rouhani, Sherin J.; Srinivasan, Nithya; Engelhard, Victor H.

    2013-01-01

    Peripheral tissue homing receptors enable T cells to access inflamed non-lymphoid tissues. Here we show that two such molecules, E-selectin ligand and α4β1 integrin, enable activated and memory T cells to enter lymph nodes as well. This affects the quantitative and qualitative distribution of these cells among regional lymph node beds. CD8 memory T cells in lymph nodes that express these molecules were mostly CD62Llo, and would normally be classified as effector memory cells. However, similar to central memory cells, they expanded upon antigen re-encounter. This led to differences in the magnitude of the recall response that depended on the route of immunization. These novel cells share properties of both central and effector memory cells, and reside in lymph nodes based on previously undescribed mechanisms of entry. PMID:23926324

  19. T cell receptor/CARMA1/NF-κB signaling controls T-helper (Th) 17 differentiation

    PubMed Central

    Molinero, Luciana L.; Cubre, Alan; Mora-Solano, Carolina; Wang, Ying; Alegre, Maria-Luisa

    2012-01-01

    IL-17–producing CD4 T cells play a key role in immune responses against extracellular bacteria and autoimmunity. Nuclear factor κB (NF-κB) is required for T-cell activation and selected effector functions, but its role in Th17 differentiation is controversial. Using genetic mouse models that impede T-cell–NF-κB signaling either downstream of the T-cell receptor (TCR) or of IκB kinase β (IKKβ), we demonstrate that NF-κB signaling controls not only survival and proliferation of activated T cells, but, if cell survival and cell-cycle progression are enabled, has an additional role in promoting completion of Th17 differentiation. CARD-containing MAGUK protein 1 (CARMA1), an adapter required for TCR/NF-κB signaling, was necessary for acquisition of IL-17A, IL-17F, IL-21, IL-22, IL-23R, and CCR6 expression in T cells cultured under Th17 conditions. In proliferating cells, lack of CARMA1 selectively prevented Th17, but not Th1 or Th2 differentiation, in a cell-intrinsic manner. Consistent with these data, CARMA1-KO mice were resistant to experimental autoimmune encephalomyelitis. Surprisingly, transcription factors essential for Th17 differentiation such as RORγt, AHR, and IRF4 were normally induced in CARMA1-KO T cells activated under Th17 conditions, suggesting that the Th17 differentiation program was initiated normally. Instead, chromatin loci of Th17 effector molecules failed to acquire an open conformation in CARMA1-KO T cells. Our results demonstrate that TCR/CARMA1/NF-κB controls completion of Th17 differentiation by enabling chromatin accessibility of Th17 effector molecule loci. PMID:23091043

  20. Adoptive transfer of MART-1 T cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma

    PubMed Central

    Chodon, Thinle; Comin-Anduix, Begonya; Chmielowski, Bartosz; Koya, Richard C; Wu, Zhongqi; Auerbach, Martin; Ng, Charles; Avramis, Earl; Seja, Elizabeth; Villanueva, Arturo; McCannel, Tara A.; Ishiyama, Akira; Czernin, Johannes; Radu, Caius G.; Wang, Xiaoyan; Gjertson, David W.; Cochran, Alistair J.; Cornetta, Kenneth; Wong, Deborah J.L.; Kaplan-lefko, Paula; Hamid, Omid; Samlowski, Wolfram; Cohen, Peter A.; Daniels, Gregory A.; Mukherji, Bijay; Yang, Lili; Zack, Jerome A.; Kohn, Donald B.; Heath, James R.; Glaspy, John A.; Witte, Owen N.; Baltimore, David; Economou, James S.; Ribas, Antoni

    2014-01-01

    Purpose It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short one-week manufacture protocol to determine the feasibility, safety and antitumor efficacy of this double cell therapy. Experimnetal Design A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and re-infused with (n = 10) or without (n = 3) prior cryopreservation. Results 14 patients with metastatic melanoma were enrolled and nine out of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within two weeks of ACT indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared to cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using non-cryopreserved T cells. Conclusion Double cell therapy with ACT of TCR engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. PMID:24634374

  1. Linking the aryl hydrocarbon receptor with altered DNA methylation patterns and developmentally induced aberrant antiviral CD8+ T cell responses

    PubMed Central

    Winans, Bethany; Nagari, Anusha; Chae, Minho; Post, Christina M.; Ko, Chia-I; Puga, Alvaro; Kraus, W. Lee; Lawrence, B. Paige

    2015-01-01

    Successfully fighting infection requires a properly tuned immune system. Recent epidemiological studies link exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) during development with poorer immune responses later in life. Yet, how developmental triggering of AHR durably alters immune cell function remains unknown. Using a mouse model, we show that developmental activation of AHR leads to long-lasting reduction in the response of CD8+ T cells during influenza virus infection, cells critical for resolving primary infection. Combining genome-wide approaches, we demonstrate that developmental activation alters DNA methylation and gene expression patterns in isolated CD8+ T cells prior to and during infection. Altered transcriptional profiles in CD8+ T cells from developmentally exposed mice reflect changes in pathways involved in proliferation and immunoregulation, with an overall pattern that bears hallmarks of T cell exhaustion. Developmental exposure also changed DNA methylation across the genome, but differences were most pronounced following infection, where we observed inverse correlation between promoter methylation and gene expression. This points to altered regulation of DNA methylation as one mechanism by which AHR causes durable changes in T cell function. Discovering that distinct gene sets and pathways were differentially changed in developmentally exposed mice prior to and after infection further reveals that the process of CD8+ T cell activation is rendered fundamentally different by early life AHR signaling. These findings reveal a novel role for AHR in the developing immune system: regulating DNA methylation and gene expression as T cells respond to infection later in life. PMID:25810390

  2. The structural interactions between T cell receptors and MHC-peptide complexes place physical limits on self-nonself discrimination.

    PubMed

    Wucherpfennig, K W

    2005-01-01

    The activation and expansion of T cells in an antimicrobial immune response is based on the ability of T cell receptors (TCR) to discriminate between MHC-bound peptides derived from different microbial agents as well as self-proteins. However, the specificity of T cells is constrained by the limited number of peptide side chains that are available for TCR binding. By considering the structural requirements for peptide binding to MHC molecules and TCR recognition of MHC-peptide complexes, we demonstrated that human T cell clones could recognize a number of peptides from different organisms that were remarkably distinct in their primary sequence. These peptides were particularly diverse at those sequence positions buried in pockets of the MHC binding site, whereas a higher degree of similarity was present at a limited number of peptide residues that created the interface with the TCR. These T cell clones had been isolated from multiple sclerosis patients with human myelin basic protein, demonstrating that activation of such autoreactive T cells by microbial peptides with sufficient structural similarity may contribute to the disease process. Similar findings have now been made for a variety of human and murine T cell clones, indicating that specificity and cross-reactivity are inherent properties of TCR recognition. The observations that particular TCR are highly sensitive to changes at particular peptide positions but insensitive to many other changes in peptide sequence are not contradictory, but rather the result of structural interactions in which a relatively flat TCR surface contacts a limited number of side chains from a peptide that is deeply embedded in the MHC binding site.

  3. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

    PubMed

    Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

    2015-02-18

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

  4. LFA-1-dependent Ca2+ entry following suboptimal T cell receptor triggering proceeds without mobilization of intracellular Ca2+.

    PubMed

    Kim, Kwangmi; Wang, Lin; Hwang, Inkyu

    2009-08-14

    A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

  5. HTLV-1 bZIP Factor Enhances T-Cell Proliferation by Impeding the Suppressive Signaling of Co-inhibitory Receptors

    PubMed Central

    Shimura, Kazuya; Onishi, Chiho; Iyoda, Tomonori; Inaba, Kayo

    2017-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells. PMID:28046066

  6. V{delta}1 T cell receptor binds specifically to MHC I chain related A: Molecular and biochemical evidences

    SciTech Connect

    Zhao Jianqing; Huang Jie; Chen Hui; Cui Lianxian; He Wei . E-mail: heweiimu@public.bta.net.cn

    2006-01-06

    Human MHC class I chain-related A (MICA) is a tumor-associated antigen that can be recognized by V{delta}1 subset of tumor-infiltrating {gamma}{delta} T cells. We previously reported that immobilized recombinant MICA protein could induce the proliferation of tumor-infiltrating V{delta}1 {gamma}{delta} T cells in vitro. But there has been no direct evidence showing the engagement of {gamma}{delta} T cell receptors (TCR) of the induced cells with MICA. In the current investigation, we show that MICA induces specific cytolytic activity of the expanded {gamma}{delta} T cells. We expressed the coupled V domains from the MICA-induced T cells as a single polypeptide chain V{delta}V{gamma} TCR ({gamma}{delta} scTCR). Such scTCR can specifically bind MICA of HeLa cells. Direct interaction of {gamma}{delta} scTCRs with in vitro expressed MICA was monitored using an IAsys biosensor. We found that the V{delta}1 scTCR can specifically bind to immobilized MICA molecule and MICA{alpha}1{alpha}2 domains are responsible for the binding reaction.

  7. Mesothelin-specific Chimeric Antigen Receptor mRNA-Engineered T cells Induce Anti-Tumor Activity in Solid Malignancies

    PubMed Central

    Beatty, Gregory L.; Haas, Andrew R.; Maus, Marcela V.; Torigian, Drew A.; Soulen, Michael C.; Plesa, Gabriela; Chew, Anne; Zhao, Yangbing; Levine, Bruce L.; Albelda, Steven M.; Kalos, Michael; June, Carl H.

    2014-01-01

    Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB co-stimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was demonstrated in both patients and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel anti-self antibodies. These data demonstrate the potential of utilizing mRNA engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope-spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. PMID:24579088

  8. Kinetic discrimination in T-cell activation.

    PubMed Central

    Rabinowitz, J D; Beeson, C; Lyons, D S; Davis, M M; McConnell, H M

    1996-01-01

    We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model. PMID:8643643

  9. Immunoglobulin and T Cell Receptor Genes: IMGT® and the Birth and Rise of Immunoinformatics

    PubMed Central

    Lefranc, Marie-Paule

    2014-01-01

    IMGT®, the international ImMunoGeneTics information system®1, (CNRS and Université Montpellier 2) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989, IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and proteins of the IgSF and MhSF superfamilies. IMGT® has been built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences, and three-dimensional (3D) structures. The concepts include the IMGT® standardized keywords (concepts of identification), IMGT® standardized labels (concepts of description), IMGT® standardized nomenclature (concepts of classification), IMGT unique numbering, and IMGT Colliers de Perles (concepts of numerotation). IMGT® comprises seven databases, 15,000 pages of web resources, and 17 tools, and provides a high-quality and integrated system for the analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses. Tools and databases are used in basic, veterinary, and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. They include, for example IMGT/V-QUEST and IMGT/JunctionAnalysis for nucleotide sequence analysis and their high-throughput version IMGT/HighV-QUEST for next-generation sequencing (500,000 sequences per batch), IMGT/DomainGapAlign for amino acid sequence analysis of IG and TR variable and constant domains and of MH groove domains, IMGT/3Dstructure-DB for 3D structures, contact analysis and paratope/epitope interactions of IG/antigen and TR/peptide-MH complexes and IMGT/mAb-DB interface for therapeutic antibodies and fusion proteins for immune applications (FPIA). PMID:24600447

  10. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

  11. IMGT standardization for statistical analyses of T cell receptor junctions: the TRAV-TRAJ example.

    PubMed

    Bleakley, Kevin; Giudicelli, Véronique; Wu, Yan; Lefranc, Marie-Paule; Biau, Gérard

    2006-01-01

    The diversity of immunoglobulin (IG) and T cell receptor (TR) chains depends on several mechanisms: combinatorial diversity, which is a consequence of the number of V, D and J genes and the N-REGION diversity, which creates an extensive and clonal somatic diversity at the V-J and V-D-J junctions. For the IG, the diversity is further increased by somatic hypermutations. The number of different junctions per chain and per individual is estimated to be 10(12). We have chosen the human TRAV-TRAJ junctions as an example in order to characterize the required criteria for a standardized analysis of the IG and TR V-J and V-D-J junctions, based on the IMGT-ONTOLOGY concepts, and to serve as a first IMGT junction reference set (IMGT, http://imgt.cines.fr). We performed a thorough statistical analysis of 212 human rearranged TRAV-TRAJ sequences, which were aligned and analysed by the integrated IMGT/V-QUEST software, which includes IMGT/JunctionAnalysis, then manually expert-verified. Furthermore, we compared these 212 sequences with 37 other human TRAV-TRAJ junction sequences for which some particularities (potential sequence polymorphisms, sequencing errors, etc.) did not allow IMGT/JunctionAnalysis to provide the correct biological results, according to expert verification. Using statistical learning, we constructed an automatic warning system to predict if new, automatically analysed TRAV-TRAJ sequences should be manually re-checked. We estimated the robustness of this automatic warning system.

  12. Immunoglobulin and T Cell Receptor Genes: IMGT(®) and the Birth and Rise of Immunoinformatics.

    PubMed

    Lefranc, Marie-Paule

    2014-01-01

    IMGT(®), the international ImMunoGeneTics information system(®) (1), (CNRS and Université Montpellier 2) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989, IMGT(®) marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT(®) is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and proteins of the IgSF and MhSF superfamilies. IMGT(®) has been built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences, and three-dimensional (3D) structures. The concepts include the IMGT(®) standardized keywords (concepts of identification), IMGT(®) standardized labels (concepts of description), IMGT(®) standardized nomenclature (concepts of classification), IMGT unique numbering, and IMGT Colliers de Perles (concepts of numerotation). IMGT(®) comprises seven databases, 15,000 pages of web resources, and 17 tools, and provides a high-quality and integrated system for the analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses. Tools and databases are used in basic, veterinary, and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. They include, for example IMGT/V-QUEST and IMGT/JunctionAnalysis for nucleotide sequence analysis and their high-throughput version IMGT/HighV-QUEST for next-generation sequencing (500,000 sequences per batch), IMGT/DomainGapAlign for amino acid sequence analysis of IG and TR variable and constant domains and of MH groove domains, IMGT/3Dstructure-DB for 3D structures, contact analysis and paratope/epitope interactions of IG/antigen and TR/peptide-MH complexes and IMGT/mAb-DB interface for therapeutic antibodies and fusion proteins for immune applications (FPIA).

  13. A T-cell specific transcriptional enhancer element 3 prime of C sub. alpha. in the human T-cell receptor. alpha. locus

    SciTech Connect

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M. )

    1989-09-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3{prime} of C{sub {alpha}} (constant region {alpha} chain) in the human T-cell receptor (TCR) {alpha}-chain locus. This enhancer is active on both a TCR V{sub {alpha}} (variable region {alpha} chain) promoter and the minimal simian virus 40 promoter in TCR {alpha}/{beta} Jurkat and EL4 cells but is inactive on a V{sub {alpha}} promoter TCR {gamma}/{delta} PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and {kappa}B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR {alpha} gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR {alpha}/{beta} lineage. In addition, the TCR {alpha} enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR {alpha} locus.

  14. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation.

    PubMed

    Gross, Catharina C; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G; Wiendl, Heinz

    2016-05-24

    Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56(bright) NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4(+) T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4(+) T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor's ligand CD155 on CD4(+) T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4(+) T cells and the cytolytic activity of NK cells.

  15. Building and optimizing a virus-specific T cell receptor library for targeted immunotherapy in viral infections.

    PubMed

    Banu, Nasirah; Chia, Adeline; Ho, Zi Zong; Garcia, Alfonso Tan; Paravasivam, Komathi; Grotenbreg, Gijsbert M; Bertoletti, Antonio; Gehring, Adam J

    2014-02-25

    Restoration of antigen-specific T cell immunity has the potential to clear persistent viral infection. T cell receptor (TCR) gene therapy can reconstitute CD8 T cell immunity in chronic patients. We cloned 10 virus-specific TCRs targeting 5 different viruses, causing chronic and acute infection. All 10 TCR genetic constructs were optimized for expression using a P2A sequence, codon optimization and the addition of a non-native disulfide bond. However, maximum TCR expression was only achieved after establishing the optimal orientation of the alpha and beta chains in the expression cassette; 9/10 TCRs favored the beta-P2A-alpha orientation over alpha-P2A-beta. Optimal TCR expression was associated with a significant increase in the frequency of IFN-gamma+ T cells. In addition, activating cells for transduction in the presence of Toll-like receptor ligands further enhanced IFN-gamma production. Thus, we have built a virus-specific TCR library that has potential for therapeutic intervention in chronic viral infection or virus-related cancers.

  16. Dysregulation of Toll-Like Receptor 7 Compromises Innate and Adaptive T Cell Responses and Host Resistance to an Attenuated West Nile Virus Infection in Old Mice

    PubMed Central

    Xie, Guorui; Luo, Huanle; Pang, Lan; Peng, Bi-hung; Winkelmann, Evandro; McGruder, Brenna; Hesse, Joseph; Whiteman, Melissa; Campbell, Gerald; Milligan, Gregg N.; Cong, Yingzi; Barrett, Alan D.

    2015-01-01

    ABSTRACT The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), a mosquito-borne flavivirus, has induced severe neurological symptoms, mostly in the elderly population. No vaccines are available for human use. Recent work showed that an attenuated WNV, a nonstructural (NS) 4B-P38G mutant, induced no lethality but strong immune responses in young (6- to 10-week-old) mice. While studying protective efficacy, we found unexpectedly that old (21- to 22-month) mice were susceptible to WNV NS4B-P38G mutant infection but were protected from subsequent lethal wild-type WNV challenge. Compared to responses in young mice, the NS4B-P38G mutant triggered higher inflammatory cytokine and interleukin-10 (IL-10) production, a delayed γδ T cell expansion, and lower antibody and WNV-specific T cell responses in old mice. Toll-like receptor 7 (TLR7) is expressed on multiple types of cells. Impaired TLR7 signaling in old mice led to dendritic cell (DC) antigen-presenting function compromise and a reduced γδ T cell and regulatory T cell (Treg) expansion during NS4B-P38G mutant infection. R848, a TLR7 agonist, decreased host vulnerability in NS4B-P38G-infected old mice by enhancing γδ T cell and Treg expansion and the antigen-presenting capacity of DCs, thereby promoting T cell responses. In summary, our results suggest that dysregulation of TLR7 partially contributes to impaired innate and adaptive T cell responses and an enhanced vulnerability in old mice during WNV NS4B-P38G mutant infection. R848 increases the safety and efficacy during immunization of old mice with the WNV NS4B-P38G mutant. IMPORTANCE The elderly are known to have enhanced susceptibility to infections and an impaired capacity to respond to vaccination. West Nile virus (WNV), an emerging mosquito-borne flavivirus, has induced severe neurological symptoms more frequently in the elderly population. No vaccines are available

  17. Glycosylation of the T-cell antigen-specific receptor and its potential role in lectin-mediated cytotoxicity

    SciTech Connect

    Hubbard, S.C.; Kranz, D.M.; Longmore, G.D.; Sitkovsky, M.V.; Eisen, H.N.

    1986-03-01

    Cytotoxic T lymphocytes (CTLs) normally destroy only those cells (target cells) whose surface antigens they recognize. However, in the presence of lectins such as Con A, CTLs destroy virtually any cell, regardless of its antigens. The oligosaccharides of the T-cell antigen-specific receptor, a dimeric surface glycoprotein composed of disulfide-linked ..cap alpha.. and ..beta.. subunits, are of interest because of their potential involvement in this lectin-dependent cytotoxic activity. The authors report here that three or four asparagine-linked oligosaccharides could be enzymatically removed from each of the receptor subunits expressed by a cloned line of murine CTLs (clone 2C), consistent with the presence of glycosylation sites deduced from cDNA sequences of the ..cap alpha.. and ..beta.. genes expressed in this clone. All the N-linked glycans on the ..cap alpha.. subunit were of the complex type, but the ..beta.. subunit carried two or three endoglycosidase H-sensitive oligosaccharides. High-mannose glycans can bind tightly to Con A and, indeed, this lectin was found to bind specifically to solubilized 2C T-cell receptor. The Con A-dependent cytotoxic activity of clone 2C, but not of other CTL clones, was inhibited by a monoclonal antibody (1B2) that is specific for the T-cell receptor of clone 2C. Antibody 1B2 also inhibited clone 2C cytotoxicity mediated by phytohemagglutinin, lentil-lectin, and wheat-germ agglutinin. These results suggest that, although lectin-dependent lysis of target cells by CTLs is antigen nonspecific, the cytolytic activity can be triggered by binding of the lectin to the T-cell antigen-specific receptor.

  18. T Cell Receptors and the Evolution of Recognition Mechanisms in Immunity.

    ERIC Educational Resources Information Center

    Inchley, C. J.

    1986-01-01

    Discusses recent advances in the study of mammalian immunology. Explains the roles of two families of lymphocytes, the B cells and T cells. Also examines evolutionary mechanisms related to the immune system. (ML)

  19. T cell receptor (TCR) V gene usage in patients with systemic necrotizing vasculitis.

    PubMed Central

    Giscombe, R; Grunewald, J; Nityanand, S; Lefvert, A K

    1995-01-01

    Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL-2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V-specific MoAbs to investigate the number of cells expressing certain V alpha and V beta gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR V alpha or V beta gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long-term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease-mediating T cells. PMID:7648706

  20. Prostaglandin E2 suppresses allergic sensitization and lung inflammation by targeting the E prostanoid 2 receptor on T cells

    PubMed Central

    Zaslona, Zbigniew; Okunishi, Katsuhide; Bourdonnay, Emilie; Domingo-Gonzalez, Racquel; Moore, Bethany B.; Lukacs, Nicholas W.; Aronoff, David M.; Peters-Golden, Marc

    2013-01-01

    Background Endogenous prostanoids have been suggested to modulate sensitization during experimental allergic asthma, but the specific role of prostaglandin E2 (PGE2) or of specific E prostanoid (EP) receptors is not known. Objective Here we tested the role of EP2 signaling in allergic asthma. Methods Wild type (WT) and EP2−/− mice were subjected to ovalbumin sensitization and acute airway challenge. The PGE2 analog misoprostol was administered during sensitization in both genotypes. In vitro culture of splenocytes and of flow-sorted dendritic cells and T cells defined the mechanism by which EP2 exerted its protective effect. Adoptive transfer of WT and EP2−/− CD4 T cells was used to validate the importance of EP2 expression on T cells. Results As compared to WT mice, EP2−/− mice had exaggerated airway inflammation in this model. Splenocytes and lung lymph node cells from sensitized EP2−/− mice produced more IL-13 than did WT cells, suggesting increased sensitization. In WT but not EP2−/− mice, subcutaneous administration of a stable PGE2 analog during sensitization inhibited allergic inflammation. PGE2 decreased cytokine production and inhibited STAT6 phosphorylation by CD3/CD28-stimulated CD4pos T cells. Co-culture of flow cytometry-sorted splenic CD4pos T cells and CD11cpos dendritic cells from WT or EP2−/− mice suggested that the increased IL-13 production in EP2−/− mice was due to the lack of EP2 specifically on T cells. Adoptive transfer of CD4pos EP2−/− T cells caused greater cytokine production in the lungs of WT mice than did transfer of WT CD4pos T cells. Conclusion We conclude that the PGE2-EP2 axis is an important endogenous brake on allergic airway inflammation, primarily targets T cells, and its agonism represents a potential novel therapeutic approach to asthma. PMID:24075232

  1. Strong and sustained effector function of memory- versus naïve-derived T cells upon T-cell receptor RNA transfer: implications for cellular therapy.

    PubMed

    Thomas, Simone; Klobuch, Sebastian; Besold, Katrin; Plachter, Bodo; Dörrie, Jan; Schaft, Niels; Theobald, Matthias; Herr, Wolfgang

    2012-12-01

    Current protocols used to select CMV-specific T cells for adoptive immunotherapy focus on virus-specifi