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Sample records for partial t-cell receptor

  1. Designer T cells by T cell receptor replacement.

    PubMed

    Sommermeyer, Daniel; Neudorfer, Julia; Weinhold, Monika; Leisegang, Matthias; Engels, Boris; Noessner, Elfriede; Heemskerk, Mirjam H M; Charo, Jehad; Schendel, Dolores J; Blankenstein, Thomas; Bernhard, Helga; Uckert, Wolfgang

    2006-11-01

    T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong--in terms of cell surface expression--and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRalpha/beta combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering. PMID:17051621

  2. Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy

    PubMed Central

    Smith, Judith A.; Tso, J. Yun; Clark, Marcus R.; Cole, Michael S.; Bluestone, Jeffrey A.

    1997-01-01

    Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including ζ chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of ζ (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cγ-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 × anti-CD4 F(ab)′2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3. PMID:9126922

  3. T cell receptor usage in rheumatic disease.

    PubMed

    Richardson, B C

    1992-01-01

    Protection against microbial attack or invasion is a fundamental function of the immune system. Crucial to this function is the ability to distinguish "self" from the invading organism, and tolerate "self" while removing "non-self". The ability to distinguish self from non-self is not inherent in the immune system, but rather is acquired and continuously maintained. Unfortunately, the mechanisms maintaining self-tolerance are not perfect, and at times break down. In these instances an autoimmune disease results. T cells initiate normal immune responses, and it is now clear that T cells can also initiate pathologic immune responses. In animal models, T cells produce diseases resembling rheumatoid arthritis (RA) (1-3), systemic lupus erythematosus (4-6) and progressive systemic sclerosis (7,8). It is likely that T cells participate in human autoimmune diseases as well. The molecular basis of T cell antigen recognition has been clarified over the past decade. These advances now allow direct examination of the T cell receptor (TCR) molecules participating in autoimmune responses, and raise the exciting possibility that the cells inducing autoimmune responses may finally be identified. Selective agents might then be developed which would interfere with or inhibit the cells. Understanding these developments requires detailed knowledge of how T cells recognize antigen, and of the receptors involved in autoimmune diseases. This article reviews the current literature on T cell receptor structure, and summarizes what is currently known about the usage of specific T cell receptors in autoimmune rheumatic disease. PMID:1582073

  4. Inhibitory Receptors Beyond T Cell Exhaustion

    PubMed Central

    Fuertes Marraco, Silvia A.; Neubert, Natalie J.; Verdeil, Grégory; Speiser, Daniel E.

    2015-01-01

    Inhibitory receptors (iRs) are frequently associated with “T cell exhaustion”. However, the expression of iRs is also dependent on T cell differentiation and activation. Therapeutic blockade of various iRs, also referred to as “checkpoint blockade”, is showing ­unprecedented results in the treatment of cancer patients. Consequently, the clinical potential in this field is broad, calling for increased research efforts and rapid refinements in the understanding of iR function. In this review, we provide an overview on the significance of iR expression for the interpretation of T cell functionality. We summarize how iRs have been strongly associated with “T cell exhaustion” and illustrate the parallel evidence on the importance of T cell differentiation and activation for the expression of iRs. The differentiation subsets of CD8 T cells (naïve, effector, and memory cells) show broad and inherent differences in iR expression, while activation leads to strong upregulation of iRs. Therefore, changes in iR expression during an immune response are often concomitant with T cell differentiation and activation. Sustained expression of iRs in chronic infection and in the tumor microenvironment likely reflects a specialized T cell differentiation. In these situations of prolonged antigen exposure and chronic inflammation, T cells are “downtuned” in order to limit tissue damage. Furthermore, we review the novel “checkpoint blockade” treatments and the potential of iRs as biomarkers. Finally, we provide recommendations for the immune monitoring of patients to interpret iR expression data combined with parameters of activation and differentiation of T cells. PMID:26167163

  5. CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

    PubMed Central

    Lindquist, Jonathan A.; Arhel, Nathalie; Felder, Edward; Karl, Sabine; Haas, Tobias L.; Fulda, Simone; Walczak, Henning; Kirchhoff, Frank; Debatin, Klaus-Michael

    2009-01-01

    CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion. PMID:19487421

  6. Force Generation upon T Cell Receptor Engagement

    PubMed Central

    Husson, Julien; Chemin, Karine; Bohineust, Armelle; Hivroz, Claire; Henry, Nelly

    2011-01-01

    T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically

  7. Regulation of T cell apoptosis via T cell receptors and steroid receptors.

    PubMed

    Iwata, M; Ohoka, Y; Kuwata, T; Asada, A

    1996-11-01

    Less than 5% of immature CD4/CD8 double-positive (DP) thymocytes are positively selected to survive and differentiate into single-positive CD4 and CD8 T cells, while self-reactive DP thymocytes undergo apoptosis (negative selection). Both positive and negative selection events are active processes that involve signaling through the T cell receptors (TCRs) and through some accessory molecules. The two events differ quantitatively in the strength of the interaction between TCR and peptide/major histocompatibility complex molecules. We established an in vitro model of positive selection that can be analyzed quantitatively. Positive selection is likely to inhibit glucocorticoid-induced apoptosis in DP thymocytes. Proper crosslinking of TCR together with CD4, CD8, or LFA-1 inhibits the death, and its inhibitory activity is mimicked by proper combinations of ionomycin, a calcium ionophore, and phorbol myristate acetate (PMA), a protein kinase C (PKC) activator. The drug concentrations are within narrow ranges, and are lower than those which are required for the proliferation of mature T cells. Transient stimulation with the combinations of ionomycin and PMA induces differentiation and commitment of isolated DP thymocytes to the CD4 or CD8 T cell lineage in suspension cultures. The level of PKC activity appears to determine the lineage to commit. Functional mature T cells are induced from the committed cells upon secondary stimulation. Activation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, also appears to be essential for positive selection as well as for the inhibition of glucocorticoid-induced apoptosis. Negative selection and the regulation of mature T cell apoptosis through TCR and steroid receptors are also discussed. PMID:8948021

  8. 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

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

    PubMed

    Koh, Sarene; Shimasaki, Noriko; Bertoletti, Antonio

    2016-01-01

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

  10. 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

  11. T-cell receptors in ectothermic vertebrates.

    PubMed

    Charlemagne, J; Fellah, J S; De Guerra, A; Kerfourn, F; Partula, S

    1998-12-01

    The structure and expression of genes encoding molecules homologous to mammalian T-cell receptors (TCR) have been recently studied in ectothermic vertebrate species representative of chondrychthians, teleosts, and amphibians. The overall TCR chain structure is well conserved in phylogeny: TCR beta- and TCR alpha-like chains were detected in all the species analyzed; TCR gamma- and TCR delta-like chains were also present in a chondrychthian species. The diversity potential of the variable (V) and joining (J) segments is rather large and, as in mammals, conserved diversity (D) segments are associated to the TCR beta and TCR delta chains. An important level of junctional diversity occurred at the V-(D)-J junctions, with the potential addition of N- and P-nucleotides. Thus, the conservation of the structure and of the potential of diversity of TCR molecules have been under a permanent selective pressure during vertebrate evolution. The structure of MHC class I and class II molecules was also well conserved in jawed vertebrates. TCR and MHC molecules are strongly functionally linked and play a determinant role in the initiation and the regulation of the specific immune responses; thus, it is not surprising that their structures have been reciprocally frozen during evolution. PMID:9914905

  12. T cell receptor gene deletion circles identify recent thymic emigrants in the peripheral T cell pool

    PubMed Central

    Kong, Fan-kun; Chen, Chen-lo H.; Six, Adrien; Hockett, Richard D.; Cooper, Max D.

    1999-01-01

    Progenitor cells undergo T cell receptor (TCR) gene rearrangements during their intrathymic differentiation to become T cells. Rearrangements of the variable (V), diversity (D), and joining (J) segments of the TCR genes result in deletion of the intervening chromosomal DNA and the formation of circular episomes as a byproduct. Detection of these extrachromosomal excision circles in T cells located in the peripheral lymphoid tissues has been viewed as evidence for the existence of extrathymic T cell generation. Because all of the T cells in chickens apparently are generated in the thymus, we have employed this avian model to determine the fate of the V(D)J deletion circles. In normal animals we identified TCR Vγ-Jγ and Vβ-Dβ deletion circles in the blood, spleen, and intestines, as well as in the thymus. Thymectomy resulted in the gradual loss of these DNA deletion circles in all of the peripheral lymphoid tissues. A quantitative PCR analysis of Vγ1-Jγ1 and Vβ1-Dβ deletion circles in splenic γδ and Vβ1+ αβ T cells indicated that their numbers progressively decline after thymectomy with a half-life of approximately 2 weeks. Although TCR deletion circles therefore cannot be regarded as reliable indicators of in situ V(D)J rearrangement, measuring their levels in peripheral T cell samples can provide a valuable index of newly generated T cells entering the T cell pool. PMID:9990059

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

    PubMed

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

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

  14. Chronic Tumor Necrosis Factor Alters T Cell Responses by Attenuating T Cell Receptor Signaling

    PubMed Central

    Cope, Andrew P.; Liblau, Roland S.; Yang, Xiao-Dong; Congia, Mauro; Laudanna, Carlo; Schreiber, Robert D.; Probert, Lesley; Kollias, George; McDevitt, Hugh O.

    1997-01-01

    Repeated injections of adult mice with recombinant murine TNF prolong the survival of NZB/W F1 mice, and suppress type I insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To determine whether repeated TNF injections suppress T cell function in adult mice, we studied the responses of influenza hemagglutinin-specific T cells derived from T cell receptor (HNT-TCR) transgenic mice. Treatment of adult mice with murine TNF for 3 wk suppressed a broad range of T cell responses, including proliferation and cytokine production. Furthermore, T cell responses of HNT-TCR transgenic mice also expressing the human TNF-globin transgene were markedly reduced compared to HNT-TCR single transgenic littermates, indicating that sustained p55 TNF-R signaling is sufficient to suppress T cell function in vivo. Using a model of chronic TNF exposure in vitro, we demonstrate that (a) chronic TNF effects are dose and time dependent, (b) TNF suppresses the responses of both Th1 and Th2 T helper subsets, (c) the suppressive effects of endogenous TNF produced in T cell cultures could be reversed with neutralizing monoclonal antibodies to TNF, and (d) prolonged TNF exposure attenuates T cell receptor signaling. The finding that anti-TNF treatment in vivo enhances T cell proliferative responses and cytokine production provides evidence for a novel regulatory effect of TNF on T cells in healthy laboratory mice. These effects are more pronounced in chronic inflammatory disease. In addition, our data provide a mechanism through which prolonged TNF exposure suppresses disease in animal models of autoimmunity. PMID:9151895

  15. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    PubMed

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy. PMID:10811469

  16. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. PMID:27163336

  17. 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.

  18. 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

  19. 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. PMID:26819347

  20. 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.

  1. 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. PMID:26377367

  2. Chimeric Antigen Receptor T Cell Therapy in Hematology

    PubMed Central

    Ataca, Pınar; Arslan, Önder

    2015-01-01

    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. PMID:26377367

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

    PubMed

    Hoseini, S S; Hapke, M; Herbst, J; Wedekind, D; Baumann, R; Heinz, N; Schiedlmeier, B; Vignali, D A A; van den Brink, M R M; Schambach, A; Blazar, B R; Sauer, M G

    2015-07-01

    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. As 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

  4. 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. PMID:17637721

  5. Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells

    PubMed Central

    Smoligovets, Alexander A.; Smith, Adam W.; Wu, Hung-Jen; Petit, Rebecca S.; Groves, Jay T.

    2012-01-01

    T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell–nanopatterned supported lipid bilayer system to quantitatively probe the actin–TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations. PMID:22389407

  6. Role of T Cell Receptor Affinity in the Efficacy and Specificity of Adoptive T Cell Therapies

    PubMed Central

    Stone, Jennifer D.; Kranz, David M.

    2013-01-01

    Over the last several years, there has been considerable progress in the treatment of cancer using gene modified adoptive T cell therapies. Two approaches have been used, one involving the introduction of a conventional αβ T cell receptor (TCR) against a pepMHC cancer antigen, and the second involving introduction of a chimeric antigen receptor (CAR) consisting of a single-chain antibody as an Fv fragment linked to transmembrane and signaling domains. In this review, we focus on one aspect of TCR-mediated adoptive T cell therapies, the impact of the affinity of the αβ TCR for the pepMHC cancer antigen on both efficacy and specificity. We discuss the advantages of higher-affinity TCRs in mediating potent activity of CD4 T cells. This is balanced with the potential disadvantage of higher-affinity TCRs in mediating greater self-reactivity against a wider range of structurally similar antigenic peptides, especially in synergy with the CD8 co-receptor. Both TCR affinity and target selection will influence potential safety issues. We suggest pre-clinical strategies that might be used to examine each TCR for possible on-target and off-target side effects due to self-reactivities, and to adjust TCR affinities accordingly. PMID:23970885

  7. Chemokine receptor expression by inflammatory T cells in EAE

    PubMed Central

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4+ T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4+ T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4+ T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8+ T cells. CD8+ T cells expressed CXCR3, which was also expressed by CD4+ T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6+ and CXCR3+ CD4+ T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8+ T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4+ T cells expressed CCR6 within infiltrates. CD4-negative CCR6+ cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4+ and CD8+ T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE. PMID:25071447

  8. 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. PMID:17855129

  9. Discrimination of T-cell subsets and T-cell receptor repertoire distribution.

    PubMed

    Bretschneider, Isabell; Clemente, Michael J; Meisel, Christian; Guerreiro, Manuel; Streitz, Mathias; Hopfenmüller, Werner; Maciejewski, Jaroslav P; Wlodarski, Marcin W; Volk, Hans-Dieter

    2014-01-01

    Flow cytometry-based analysis of T-cell receptor (TCR) repertoires is an essential tool for the detection of clonal T-cell expansions in physiologic and pathologic conditions. Individual T-cell subsets can be investigated based on their surface properties. The aims of our study were to provide reference values for various disease settings and delineate the contribution of individual TCR repertoires to the human T-cell differentiation pathway. We analyzed blood of 66 healthy subjects aged 0 (cord blood) to 72 years. Lymphocyte subpopulations and TCR repertoires were simultaneously explored using antibodies specific to CD3, CD4, CD8, CD45RA, CCR7, CD27, CD57 and a set of 25 antibodies detecting human TCR-Vβ chains. Statistical analysis included Wilcoxon, paired t and ANOVA tests. Initially, TCR expansion values were calculated based on the analysis of TCR-Vβ distribution on CD4+ and CD8+ T cells. We then established gating strategies and an algorithm for data analysis allowing for discrimination of T-cell subsets and TCR distribution. Dominant TCR expansions were present within effector as opposed to central/effector memory or naive cells, e.g., median TCR-Vβ expansion rate was highest on CD45RA+/CCR7- effector CD4+/8+ cells (eight and 11-fold, respectively). Remarkably, TCR expansions were missing (0) or very low (0.5) on CD4+ and CD8+ central memory population, respectively. No significant gender-related variability of TCR repertoires was identified, and significant impact of chronic cytomegalovirus infection was shown. Our results serve as reference for future studies elucidating clonal TCR dominance of T-cell subsets. PMID:24272857

  10. Clinicopathologic Spectrum of Gastrointestinal T-cell Lymphoma: Reappraisal Based on T-cell Receptor Immunophenotypes.

    PubMed

    Tanaka, Tsutomu; Yamamoto, Hideko; Elsayed, Ahmed Ali; Satou, Akira; Asano, Naoko; Kohno, Kei; Kinoshita, Tomohiro; Niwa, Yasumasa; Goto, Hidemi; Nakamura, Shigeo; Kato, Seiichi

    2016-06-01

    The differential diagnosis of primary gastrointestinal EBV T-cell lymphoma (GITCL) includes enteropathy-associated T-cell lymphoma (EATL), peripheral T-cell lymphoma, not otherwise specified, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma. Type II EATL is considered to be a tumor of intraepithelial lymphocytes. However, the evaluation of intraepithelial lymphocytosis by biopsy specimens is challenging, which poses a diagnostic problem between the EATL and peripheral T-cell lymphoma, not otherwise specified. This situation requested us to establish a pragmatic diagnostic approach for the classification of GITCL. We identified 42 cases of GITCL and analyzed clinicopathologic features, especially addressing their T-cell receptor (TCR) phenotype. Nine (21%) of 42 GITCL cases were positive for TCRγ protein expression. Among these TCRγ cases, TCRβ expression or not was detected in 5 and 4, respectively, but resulted in no further clinicopathologic differences. TCRβ positivity without TCRγ expression (βγ) was seen in 9 GITCL patients (21%). Twenty-four patients (57%) were negative for TCRβ and γ expression (βγ). Compared with TCRβγ or βγ type, TCRγ cases were characterized by exclusive involvement of intestinal sites (100% vs. 11%, P<0.001; 100% vs. 58%, P=0.032, respectively), but not of stomach (0% vs. 78%, P=0.002; 0% vs. 38%, P=0.039, respectively). Notably, TCRγ positivity was an independent unfavorable prognostic factor among our GITCL patients (P<0.001). Considering our results, TCRγ GITCL, that is, intestinal γδ T-cell lymphoma, appears to constitute a distinct disease entity. PMID:26975035

  11. T cell receptor interactions with class I heavy-chain influence T cell selection

    PubMed Central

    Kuhns, Scott T.; Tallquist, Michelle D.; Johnson, Aaron J.; Mendez-Fernandez, Yanice; Pease, Larry R.

    2000-01-01

    The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the Kb molecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, Kbm3, a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire. PMID:10639152

  12. Targeting human melanoma neoantigens by T cell receptor gene therapy.

    PubMed

    Leisegang, Matthias; Kammertoens, Thomas; Uckert, Wolfgang; Blankenstein, Thomas

    2016-03-01

    In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy. PMID:26808500

  13. Chimeric antigen receptor (CAR) and T cell receptor (TCR) Modified T cells Enter Main Street and Wall Street

    PubMed Central

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

    2015-01-01

    The field of adoptive cell transfer (ACT) is currently comprised of 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 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. Here, we discuss some of the challenges and opportunities that face the field of ACT. PMID:26188068

  14. Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis.

    PubMed Central

    Allegretta, M; Albertini, R J; Howell, M D; Smith, L R; Martin, R; McFarland, H F; Sriram, S; Brostoff, S; Steinman, L

    1994-01-01

    The selection of T cell clones with mutations in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene has been used to isolate T cells reactive to myelin basic protein (MBP) in patients with multiple sclerosis (MS). These T cell clones are activated in vivo, and are not found in healthy individuals. The third complementarity determining regions (CDR3) of the T cell receptor (TCR) alpha and beta chains are the putative contact sites for peptide fragments of MBP bound in the groove of the HLA molecule. The TCR V gene usage and CDR3s of these MBP-reactive hprt-T cell clones are homologous to TCRs from other T cells relevant to MS, including T cells causing experimental allergic encephalomyelitis (EAE) and T cells found in brain lesions and in the cerebrospinal fluid (CSF) of MS patients. In vivo activated MBP-reactive T cells in MS patients may be critical in the pathogenesis of MS. PMID:8040252

  15. Partial defects of T cell development associated with poor T cell function

    PubMed Central

    Notarangelo, Luigi D.

    2013-01-01

    For many years, Severe Combined Immune Deficiency (SCID) diseases, characterized by virtual lack of circulating T cells and severe predisposition to infections since early in life, have been considered the prototypic forms of genetic defects of T cell development. More recently, advances in genome sequencing have allowed identification of a growing number of gene defects that cause severe, but incomplete, defects in T cell development and/or function. Along with recurrent and severe infections, and especially cutaneous viral infections, the clinical phenotype of these conditions is characterized by prominent immune dysregulation. PMID:23465662

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

    PubMed

    Morris, Emma C; Stauss, Hans J

    2016-06-30

    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

  17. Optimal T-cell receptor affinity for inducing autoimmunity.

    PubMed

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

    2014-12-01

    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

  18. Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8+ T Cells and Efficiently Reduces Chronic Retroviral Loads

    PubMed Central

    Dietze, Kirsten K.; Zelinskyy, Gennadiy; Liu, Jia; Kretzmer, Freya; Schimmer, Simone; Dittmer, Ulf

    2013-01-01

    Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8+ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8+ T cells and expansion of regulatory T cells (Tregs) that suppress CD8+ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8+ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8+ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8+ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases. PMID:24339778

  19. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    PubMed Central

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  20. Functional Development of the T Cell Receptor for Antigen

    PubMed Central

    Ebert, Peter J.R.; Li, Qi-Jing; Huppa, Johannes B.; Davis, Mark M.

    2016-01-01

    For over three decades now, the T cell receptor (TCR) for antigen has not ceased to challenge the imaginations of cellular and molecular immunologists alike. T cell antigen recognition transcends every aspect of adaptive immunity: it shapes the T cell repertoire in the thymus and directs T cell-mediated effector functions in the periphery, where it is also central to the induction of peripheral tolerance. Yet, despite its central position, there remain many questions unresolved: how can one TCR be specific for one particular peptide-major histocompatibility complex (pMHC) ligand while also binding other pMHC ligands with an immunologically relevant affinity? And how can a T cell’s extreme specificity (alterations of single methyl groups in their ligand can abrogate a response) and sensitivity (single agonist ligands on a cell surface are sufficient to trigger a measurable response) emerge from TCR–ligand interactions that are so low in affinity? Solving these questions is intimately tied to a fundamental understanding of molecular recognition dynamics within the many different contexts of various T cell–antigen presenting cell (APC) contacts: from the thymic APCs that shape the TCR repertoire and guide functional differentiation of developing T cells to the peripheral APCs that support homeostasis and provoke antigen responses in naïve, effector, memory, and regulatory T cells. Here, we discuss our recent findings relating to T cell antigen recognition and how this leads to the thymic development of foreign-antigen-responsive αβT cells. PMID:20800817

  1. Chimeric antigen receptor-modified T cells strike back.

    PubMed

    Frigault, Matthew J; Maus, Marcela V

    2016-07-01

    Chimeric antigen receptors (CARs) are engineered molecules designed to endow a polyclonal T-cell population with the ability to recognize tumor-associated surface antigens. In their simplest form, CARs comprise a targeting moiety in the form of a single-chain variable fragment from an antibody connected to various intracellular signaling domains allowing for T-cell activation. This powerful approach combines the specificity of an antibody with the cytotoxic ability of a T cell. There has been much excitement since early phase trials of CAR-T cells targeting CD19 expressed on B-cell malignancies demonstrated remarkable efficacy in inducing long-term, stable remissions in otherwise relapsed/refractory disease. Despite these successes, we have just begun to understand the intricacies of CAR biology with efforts underway to utilize this platform in the treatment of other, previously refractory malignancies. Challenges currently include identification of viable cancer targets, management strategies for potentially severe and irreversible toxicities and overcoming the immunosuppressive nature of the tumor microenvironment. This review will focus on basic CAR structure and function, previous success and new approaches aimed at the broader application of CAR-T-cell therapy. PMID:27021308

  2. 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.

  3. A logical model provides insights into T cell receptor signaling.

    PubMed

    Saez-Rodriguez, Julio; Simeoni, Luca; Lindquist, Jonathan A; Hemenway, Rebecca; Bommhardt, Ursula; Arndt, Boerge; Haus, Utz-Uwe; Weismantel, Robert; Gilles, Ernst D; Klamt, Steffen; Schraven, Burkhart

    2007-08-01

    Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications. PMID:17722974

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

    PubMed

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

    2016-03-01

    Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs. PMID:26526988

  5. 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

  6. Recombinant T Cell Receptor Ligand (RTL) Treats Experimental Stroke

    PubMed Central

    Subramanian, Sandhya; Zhang, Bing; Kosaka, Yasuharu; Burrows, Gregory G.; Grafe, Marjorie R.; Vandenbark, Arthur A.; Hurn, Patricia D.; Offner, Halina

    2009-01-01

    Background and Purpose Experimental stroke induces a biphasic effect on the immune response that involves early activation of peripheral leukocytes followed by severe immunodepression and atrophy of spleen and thymus. In tandem, the developing infarct is exacerbated by influx of numerous inflammatory cell types, including T and B lymphocytes. These features of stroke prompted our use of Recombinant T Cell Receptor Ligands (RTL), partial MHC class II molecules covalently bound to myelin peptides. We tested the hypothesis that RTL would improve ischemic outcome in brain without exacerbating defects in peripheral immune system function. Methods Four daily doses of RTL were administered subcutaneously to C57BL/6 mice after middle cerebral artery occlusion (MCAO), and lesion size and cellular composition were assessed in brain, and cell numbers were assessed in spleen and thymus. Results Treatment with RTL551 (I-Ab molecule linked to MOG-35−55 peptide) reduced cortical and total stroke lesion size by ∼50%, inhibited the accumulation of inflammatory cells, particularly macrophages/activated microglial cells and dendritic cells, and mitigated splenic atrophy. Treatment with RTL1000 (HLA-DR2 moiety linked to human MOG-35−55 peptide) similarly reduced the stroke lesion size in HLA-DR2 transgenic mice. In contrast, control RTL with a non-neuroantigen peptide or a mismatched MHC class II moiety had no effect on stroke lesion size. Conclusions These data are the first to demonstrate successful treatment of experimental stroke using a neuroantigen specific immunomodulatory agent administered after ischemia, suggesting therapeutic potential in human stroke. PMID:19443805

  7. 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. PMID:26056165

  8. Direct measurement of T cell receptor affinity and sequence from naïve antiviral T cells.

    PubMed

    Zhang, Shu-Qi; Parker, Patricia; Ma, Ke-Yue; He, Chenfeng; Shi, Qian; Cui, Zhonghao; Williams, Chad M; Wendel, Ben S; Meriwether, Amanda I; Salazar, Mary Alice; Jiang, Ning

    2016-06-01

    T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCRs). 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. 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

  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. The role of CD4-Lck in T-cell receptor antagonism: evidence for negative signaling.

    PubMed Central

    Racioppi, L; Matarese, G; D'Oro, U; De Pascale, M; Masci, A M; Fontana, S; Zappacosta, S

    1996-01-01

    Small changes in the complex between a peptide and a molecule of the major histocompatibility complex generate ligands able to partially activate (partial agonist) or even inhibit (antagonist) T-cell functions. T-cell receptor engagement of antagonist complex results in a partial zeta chain phosphorylation without activation of the associated ZAP-70 kinase. Herein we show that, despite a strong inhibition of both inositol phospholipid hydrolysis and extracellular increasing antagonist concentrations increased the activity of the CD4-Lck kinase. Addition of anti-CD4 antibody to culture medium prevented inhibitory effects induced by antagonist ligand. We propose that CD4-Lck activation triggered by antagonist complexes may act in a dominant negative mode, thus overriding stimulatory signals coming from agonist ligand. These findings identify a new T-cell signaling profile that may explain the ability of some T-cell receptor variant ligands to inhibit specific biological activities or trigger alternative activation programs. Images Fig. 3 Fig. 4 PMID:8816805

  11. 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.

  12. The Vitamin D Receptor and T Cell Function

    PubMed Central

    Kongsbak, Martin; Levring, Trine B.; Geisler, Carsten; von Essen, Marina Rode

    2013-01-01

    The vitamin D receptor (VDR) is a nuclear, ligand-dependent transcription factor that in complex with hormonally active vitamin D, 1,25(OH)2D3, regulates the expression of more than 900 genes involved in a wide array of physiological functions. The impact of 1,25(OH)2D3-VDR signaling on immune function has been the focus of many recent studies as a link between 1,25(OH)2D3 and susceptibility to various infections and to development of a variety of inflammatory diseases has been suggested. It is also becoming increasingly clear that microbes slow down immune reactivity by dysregulating the VDR ultimately to increase their chance of survival. Immune modulatory therapies that enhance VDR expression and activity are therefore considered in the clinic today to a greater extent. As T cells are of great importance for both protective immunity and development of inflammatory diseases a variety of studies have been engaged investigating the impact of VDR expression in T cells and found that VDR expression and activity plays an important role in both T cell development, differentiation and effector function. In this review we will analyze current knowledge of VDR regulation and function in T cells and discuss its importance for immune activity. PMID:23785369

  13. 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

  14. Transient Receptor Potential (TRP) channels in T cells.

    PubMed

    Bertin, Samuel; Raz, Eyal

    2016-05-01

    The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases. PMID:26468011

  15. Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background WC1 co-receptors belong to the scavenger receptor cysteine-rich superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1+ '' T cells. Our previous study identified partial sequences for 13 different WC1 genes by annota...

  16. 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

  17. 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.

  18. VDJtools: Unifying Post-analysis of T Cell Receptor Repertoires

    PubMed Central

    Bolotin, Dmitriy A.; Britanova, Olga V.; Putintseva, Ekaterina V.; Pogorelyy, Mikhail V.; Nazarov, Vadim I.; Zvyagin, Ivan V.; Kirgizova, Vitalina I.; Kirgizov, Kirill I.; Skorobogatova, Elena V.; Chudakov, Dmitriy M.

    2015-01-01

    Despite the growing number of immune repertoire sequencing studies, the field still lacks software for analysis and comprehension of this high-dimensional data. Here we report VDJtools, a complementary software suite that solves a wide range of T cell receptor (TCR) repertoires post-analysis tasks, provides a detailed tabular output and publication-ready graphics, and is built on top of a flexible API. Using TCR datasets for a large cohort of unrelated healthy donors, twins, and multiple sclerosis patients we demonstrate that VDJtools greatly facilitates the analysis and leads to sound biological conclusions. VDJtools software and documentation are available at https://github.com/mikessh/vdjtools. PMID:26606115

  19. 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.

  20. 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

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

    PubMed

    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 "CCT6A(m) 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 CCT6A(m) 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

  2. 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

  3. 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

  4. 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

  5. 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. PMID:25864915

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

    PubMed Central

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

    2015-01-01

    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. PMID:25864915

  7. 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

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

    PubMed Central

    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; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT 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-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi 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-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  9. A molecular basis underpinning the T cell receptor heterogeneity of mucosal-associated invariant T cells

    PubMed Central

    Eckle, Sidonia B.G.; Birkinshaw, Richard W.; Kostenko, Lyudmila; Corbett, Alexandra J.; McWilliam, Hamish E.G.; Reantragoon, Rangsima; Chen, Zhenjun; Gherardin, Nicholas A.; Beddoe, Travis; Liu, Ligong; Patel, Onisha; Meehan, Bronwyn; Fairlie, David P.; Villadangos, Jose A.; Godfrey, Dale I.

    2014-01-01

    Mucosal-associated invariant T (MAIT) cells express an invariant T cell receptor (TCR) α-chain (TRAV1-2 joined to TRAJ33, TRAJ20, or TRAJ12 in humans), which pairs with an array of TCR β-chains. MAIT TCRs can bind folate- and riboflavin-based metabolites restricted by the major histocompatibility complex (MHC)-related class I−like molecule, MR1. However, the impact of MAIT TCR and MR1-ligand heterogeneity on MAIT cell biology is unclear. We show how a previously uncharacterized MR1 ligand, acetyl-6-formylpterin (Ac-6-FP), markedly stabilized MR1, potently up-regulated MR1 cell surface expression, and inhibited MAIT cell activation. These enhanced properties of Ac-6-FP were attributable to structural alterations in MR1 that subsequently affected MAIT TCR recognition via conformational changes within the complementarity-determining region (CDR) 3β loop. Analysis of seven TRBV6-1+ MAIT TCRs demonstrated how CDR3β hypervariability impacted on MAIT TCR recognition by altering TCR flexibility and contacts with MR1 and the Ag itself. Ternary structures of TRBV6-1, TRBV6-4, and TRBV20+ MAIT TCRs in complex with MR1 bound to a potent riboflavin-based antigen (Ag) showed how variations in TRBV gene usage exclusively impacted on MR1 contacts within a consensus MAIT TCR-MR1 footprint. Moreover, differential TRAJ gene usage was readily accommodated within a conserved MAIT TCR-MR1-Ag docking mode. Collectively, MAIT TCR heterogeneity can fine-tune MR1 recognition in an Ag-dependent manner, thereby modulating MAIT cell recognition. PMID:25049336

  10. 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

  11. The T cell receptor beta genes of Xenopus.

    PubMed

    Chretien, I; Marcuz, A; Fellah, J; Charlemagne, J; Du Pasquier, L

    1997-03-01

    cDNA of the T cell receptor beta (TCRB) have been isolated from the anuran amphibian Xenopus and they show strong structural homology to TCRB sequences of other vertebrates. Ten BV families, two D segments, ten J segments, and a single C region have been defined so far. Each V family consists of one to two members per haploid genome. A unique feature of the Xenopus TCRB constant region is the lack of N-linked carbohydrate glycosylation sites. The recombination signal sequences suggest that the mechanism of rearrangements are identical to those of mammals. The locus is inherited in a diploid manner despite the pseudotetraploidy of the Xenopus laevis and X. gilli used in this study. PMID:9079820

  12. Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells

    PubMed Central

    Zerrahn, Jens; Volkmann, Ariane; Coles, Mark C.; Held, Werner; Lemonnier, Francois A.; Raulet, David H.

    1999-01-01

    The identity of cells that mediate positive selection of CD8+ T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated β2-microglobulin mutant mice or mice with mutations in both the Kb and Db genes were repopulated with fetal liver cells from class I+ TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8+ T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8+ T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8+ T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)–TCR+ CD8+ T cells was observed in class I+ into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed. PMID:10500200

  13. T-cell receptor accessory and co-receptor molecules in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell receptor (TCR) associated invariant chains CD3gamma/delta,epsilon, and zeta as well as TCR co-receptors CD8alpha and CD8beta were isolated from the channel catfish, Ictalurus punctatus, at both the gene and cDNA levels. All of catfish CD3 sequences encode for proteins that resemble their resp...

  14. 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. PMID:26860370

  15. Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation

    PubMed Central

    Ribeiro, Sérgio T.; Ribot, Julie C.; Silva-Santos, Bruno

    2015-01-01

    The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy. PMID:25674089

  16. T Cell Receptor (TCR) Antagonism without a Negative Signal: Evidence from T Cell Hybridomas Expressing Two Independent TCRs

    PubMed Central

    Stotz, Sabine H.; Bolliger, Luca; Carbone, Francis R.; Palmer, Ed

    1999-01-01

    Antagonist peptides inhibit T cell responses by an unknown mechanism. By coexpressing two independent T cell receptors (TCRs) on a single T cell hybridoma, we addressed the question of whether antagonist ligands induce a dominant-negative signal that inhibits the function of a second, independent TCR. The two receptors, Vα2Vβ5 and Vα2Vβ10, restricted by H-2Kb and specific for the octameric peptides SIINFEKL and SSIEFARL, respectively, were coexpressed on the same cell. Agonist stimulation demonstrated that the two receptors behaved independently with regard to antigen-induced TCR downregulation and intracellular biochemical signaling. The exposure of one TCR (Vα2Vβ5) to antagonist peptides could not inhibit a second independent TCR (Vα2Vβ10) from responding to its antigen. Thus, our data clearly demonstrate that these antagonist ligands do not generate a dominant-negative signal which affects the responsiveness of the entire cell. In addition, a kinetic analysis showed that even 12 h after engagement with their cognate antigen and 10 h after reaching a steady-state of TCR internalization, T cells were fully inhibited by the addition of antagonist peptides. The window of susceptibility to antagonist ligands correlated exactly with the time required for the responding T cells to commit to interleukin 2 production. The data support a model where antagonist ligands can competitively inhibit antigenic peptides from productively engaging the TCR. This competitive inhibition is effective during the entire commitment period, where sustained TCR engagement is essential for full T cell activation. PMID:9892608

  17. Selective Induction of Apoptosis in Mature T Lymphocytes by Variant T Cell Receptor Ligands

    PubMed Central

    Combadière, Behazine; e Sousa, Caetano Reis; Germain, Ronald N.; Lenardo, Michael J.

    1998-01-01

    Activation, anergy, and apoptosis are all possible outcomes of T cell receptor (TCR) engagement. The first leads to proliferation and effector function, whereas the others can lead to partial or complete immunological tolerance. Structural variants of immunizing peptide–major histocompatibility complex molecule ligands that induce selective lymphokine secretion or anergy in mature T cells in association with altered intracellular signaling events have been described. Here we describe altered ligands for mature mouse CD4+ T helper 1 cells that lead to T cell apoptosis by the selective expression of Fas ligand (FasL) and tumor necrosis factor (TNF) without concomitant IL-2, IL-3, or interferon γ production. All ligands that stimulated cell death were found to induce FasL and TNF mRNA expression and TCR aggregation (“capping”) at the cell surface, but did not elicit a common pattern of tyrosine phosphorylation of the TCR-associated signal transduction chains. Thus, TCR ligands that uniquely trigger T cell apoptosis without inducing cytokines that are normally associated with activation can be identified. PMID:9449715

  18. Sequence variation in the human T-cell receptor loci.

    PubMed

    Mackelprang, Rachel; Carlson, Christopher S; Subrahmanyan, Lakshman; Livingston, Robert J; Eberle, Michael A; Nickerson, Deborah A

    2002-12-01

    Identifying common sequence variations known as single nucleotide polymorphisms (SNPs) in human populations is one of the current objectives of the human genome project. Nearly 3 million SNPs have been identified. Analysis of the relative allele frequency of these markers in human populations and the genetic associations between these markers, known as linkage disequilibrium, is now underway to generate a high-density genetic map. Because of the central role T cells play in immune reactivity, the T-cell receptor (TCR) loci have long been considered important candidates for common disease susceptibility within the immune system (e.g., asthma, atopy and autoimmunity). Over the past two decades, hundreds of SNPs in the TCR loci have been identified. Most studies have focused on defining SNPs in the variable gene segments which are involved in antigenic recognition. On average, the coding sequence of each TCR variable gene segment contains two SNPs, with many more found in the 5', 3' and intronic sequences of these segments. Therefore, a potentially large repertoire of functional variants exists in these loci. Association between SNPs (linkage disequilibrium) extends approximately 30 kb in the TCR loci, although a few larger regions of disequilibrium have been identified. Therefore, the SNPs found in one variable gene segment may or may not be associated with SNPs in other surrounding variable gene segments. This suggests that meaningful association studies in the TCR loci will require the analysis and typing of large marker sets to fully evaluate the role of TCR loci in common disease susceptibility in human populations. PMID:12493004

  19. Targeting of cancer neoantigens with donor-derived T cell receptor repertoires.

    PubMed

    Strønen, Erlend; Toebes, Mireille; Kelderman, Sander; van Buuren, Marit M; Yang, Weiwen; van Rooij, Nienke; Donia, Marco; Böschen, Maxi-Lu; Lund-Johansen, Fridtjof; Olweus, Johanna; Schumacher, Ton N

    2016-06-10

    Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)-A*02:01-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such "outsourced" immune responses in cancer immunotherapy. PMID:27198675

  20. Sympathetic neural signaling via the β2-adrenergic receptor suppresses T-cell receptor-mediated human and mouse CD8(+) T-cell effector function.

    PubMed

    Estrada, Leonardo D; Ağaç, Didem; Farrar, J David

    2016-08-01

    Postganglionic sympathetic neurons innervate secondary lymphoid organs and secrete norepinephrine (NE) as the primary neurotransmitter. NE binds and signals through five distinct members of the adrenergic receptor family. In this study, we show elevated expression of the β2-adrenergic receptor (ADRB2) on primary human CD8(+) effector memory T cells. Treatment of both human and murine CD8(+) T cells with NE decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. The effects of NE were specifically reversed by β2-specific antagonists. Adrb2(-/-) CD8(+) T cells were completely resistant to the effects of NE. Further, the ADRB2-specific pharmacological ligand, albuterol, significantly suppressed effector functions in both human and mouse CD8(+) T cells. While both TCR activation and stimulation with IL-12 + IL-18 were able to induce inflammatory cytokine secretion, NE failed to suppress IFN-γ secretion in response to IL-12 + IL18. Finally, the long-acting ADRB2-specific agonist, salmeterol, markedly reduced the cytokine secretion capacity of CD8(+) T cells in response to infection with vesicular stomatitis virus. This study reveals a novel intrinsic role for ADRB2 signaling in CD8(+) T-cell function and underscores the novel role this pathway plays in adaptive T-cell responses to infection. PMID:27222010

  1. 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

  2. Complex T-Cell Receptor Repertoire Dynamics Underlie the CD8+ T-Cell Response to HIV-1

    PubMed Central

    Costa, Ana I.; Koning, Dan; Ladell, Kristin; McLaren, James E.; Grady, Bart P. X.; Schellens, Ingrid M. M.; van Ham, Petra; Nijhuis, Monique; Borghans, José A. M.; Keşmir, Can; Price, David A.

    2014-01-01

    ABSTRACT Although CD8+ T cells are important for the control of HIV-1 in vivo, the precise correlates of immune efficacy remain unclear. In this study, we conducted a comprehensive analysis of viral sequence variation and T-cell receptor (TCR) repertoire composition across multiple epitope specificities in a group of antiretroviral treatment-naive individuals chronically infected with HIV-1. A negative correlation was detected between changes in antigen-specific TCR repertoire diversity and CD8+ T-cell response magnitude, reflecting clonotypic expansions and contractions related to alterations in cognate viral epitope sequences. These patterns were independent of the individual, as evidenced by discordant clonotype-specific transitions directed against different epitopes in single subjects. Moreover, long-term asymptomatic HIV-1 infection was characterized by evolution of the TCR repertoire in parallel with viral replication. Collectively, these data suggest a continuous bidirectional process of adaptation between HIV-1 and virus-specific CD8+ T-cell clonotypes orchestrated at the TCR-antigen interface. IMPORTANCE We describe a relation between viral epitope mutation, antigen-specific T-cell expansion, and the repertoire of responding clonotypes in chronic HIV-1 infection. This work provides insights into the process of coadaptation between the human immune system and a rapidly evolving lentivirus. PMID:25320304

  3. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

    PubMed Central

    Dik, Willem A.; Pike-Overzet, Karin; Weerkamp, Floor; de Ridder, Dick; de Haas, Edwin F.E.; Baert, Miranda R.M.; van der Spek, Peter; Koster, Esther E.L.; Reinders, Marcel J.T.; van Dongen, Jacques J.M.; Langerak, Anton W.; Staal, Frank J.T.

    2005-01-01

    To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before. PMID:15928199

  4. Dissociation of peripheral T cell responses from thymocyte negative selection by weak agonists supports a spare receptor model of T cell activation

    PubMed Central

    McNeil, Lisa K.; Evavold, Brian D.

    2002-01-01

    We have focused on stability of the peptide-MHC complex as a determining factor of ligand potency for thymocytes and peripheral CD4+ T cell responses. MHC variant peptides that have low affinities and fast dissociation rates are different in that they stimulate proliferation and cytolysis of mature T cells (classifying the variant peptides as weak agonists) but do not induce thymocyte negative selection. The MHC variant weak agonists require significant receptor reserve, because decreasing the level of T cell receptor on mature T cells blocks the proliferative response. These results demonstrate that peripheral T cells are more sensitive to MHC variant ligands by virtue of increased T cell receptor expression; in addition, the data support a T cell model of the spare receptor theory. PMID:11904393

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

    PubMed Central

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

    2013-01-01

    Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157–165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8+ T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, KD = ∼1 − 5 μM. Beyond the affinity threshold at KD < 1 μM we observed an attenuation in cellular function, in line with the “half-life” model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method

  6. T cell receptor diversity in the human thymus.

    PubMed

    Vanhanen, Reetta; Heikkilä, Nelli; Aggarwal, Kunal; Hamm, David; Tarkkila, Heikki; Pätilä, Tommi; Jokiranta, T Sakari; Saramäki, Jari; Arstila, T Petteri

    2016-08-01

    A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRα and TCRβ gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3×10(6) unique TCRβ and 0.5×10(6) TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3×10(6) unique TCRβ sequences and 3.7×10(6) TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70×10(6) unique TCRβ sequences and 60 to 100×10(6) TCRα sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times. PMID:27442982

  7. 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.

  8. Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

    PubMed Central

    Howell, M D; Diveley, J P; Lundeen, K A; Esty, A; Winters, S T; Carlo, D J; Brostoff, S W

    1991-01-01

    Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA. PMID:1660155

  9. 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

  10. Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

    PubMed

    Sreeramkumar, Vinatha; Hons, Miroslav; Punzón, Carmen; Stein, Jens V; Sancho, David; Fresno, Manuel; Cuesta, Natalia

    2016-01-01

    Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings. PMID:26051593

  11. Diversity-oriented approaches for interrogating T-cell receptor repertoire, ligand recognition, and function

    PubMed Central

    Birnbaum, Michael E.; Dong, Shen; Garcia, K. Christopher

    2012-01-01

    Summary Molecular diversity lies at the heart of adaptive immunity. T-cell receptors and peptide-major histocompatibility complex molecules utilize and rely upon an enormous degree of diversity at the levels of genetics, chemistry, and structure to engage one another and carry out their functions. This high level of diversity complicates the systematic study of important aspects of T-cell biology, but recent technical advances have allowed for the ability to study diversity in a comprehensive manner. In this review, we assess insights gained into T-cell receptor function and biology from our increasingly precise ability to assess the T-cell repertoire as a whole or to perturb individual receptors with engineered reagents. We conclude with a perspective on a new class of high-affinity, non-stimulatory peptide ligands we have recently discovered using diversity-oriented techniques that challenges notions for how we think about T-cell receptor signaling. PMID:23046124

  12. Rejection of cardiac allografts by T cells expressing a restricted repertoire of T-cell receptor V beta genes.

    PubMed Central

    Shirwan, H; Barwari, L; Cramer, D V

    1997-01-01

    We have recently shown that T cells infiltrating cardiac allografts early in graft rejection use a limited T-cell receptor (TCR) V beta repertoire. In this study we tested whether this limited repertoire of V beta genes is important for graft rejection. A cell line, AL2-L3, was established from LEW lymphocytes infiltrating ACI heart allografts 2 days after transplantation. This cell line is composed of CD4+ T cells that primarily recognize the class II RTI.B major histocompatibility complex (MHC) molecule expressed by the donor graft. This cell line precipitated acute rejection of donor hearts with a median survival time (MST) of 10.5 days following adoptive transfer to sublethally irradiated LEW recipients. This rate of graft rejection was significantly (P < 0.0007) accelerated when compared with a MST of 60 days for allografts in irradiated control recipients. The AL2-L3-mediated acceleration of graft rejection was donor specific as WF third-party heart allografts were rejected with a delayed tempo (MST = 28.5 days). The V beta repertoire of this cell line was primarily restricted to the expression of V beta 4, 15 and 19 genes. The nucleotide sequence analysis of the beta-chain cDNAs from this cell line demonstrated that the restricted use of the V gene repertoire was not shared with the N, D and J regions. A wide variety of CDR3 loops and J beta genes were used in association with selected V beta genes. These data provide evidence for the role a restricted repertoire of V beta genes plays in cardiac allograft rejection in this model. The restricted usage of the V beta repertoire in an early T-cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T-cell populations for elimination at the time of organ transplantation. Images Figure 2 PMID:9176111

  13. 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

  14. 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

  15. 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

  16. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  17. Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology

    PubMed Central

    Suzuki, Maya; Curran, Kevin J.; Cheung, Nai-Kong V.

    2016-01-01

    Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging. PMID:25832831

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

    PubMed

    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; Maus, Marcela V

    2016-03-01

    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

  19. Identification of a T-cell-specific transcriptional enhancer located 3' of C gamma 1 in the murine T-cell receptor gamma locus.

    PubMed Central

    Spencer, D M; Hsiang, Y H; Goldman, J P; Raulet, D H

    1991-01-01

    A transcriptional enhancer element has been localized 3 kilobases 3' of the murine T-cell receptor C gamma 1 locus using a chloramphenicol acetyltransferase reporter gene construct. As a monomer the enhancer functions only in PEER gamma delta cells and Jurkat alpha beta cells of the T-cell lines tested. However, a tetramer of the enhancer functions in virtually all T-cell lines tested, including alpha beta T-cell lines, but not in other cell types. These results suggest that elements other than the enhancer are responsible for the failure of rearranged C gamma 1 genes to be expressed in alpha beta T cells. The enhancer has been localized to a 200-base-pair Rsa I restriction fragment, which contains sequence motifs similar to those found in the other T-cell receptor enhancers but not in the immunoglobulin enhancers. Images PMID:1992471

  20. Linking T-cell receptor sequence to functional phenotype at the single-cell level

    PubMed Central

    Han, Arnold; Glanville, Jacob; Hansmann, Leo; Davis, Mark M

    2015-01-01

    Although each T lymphocyte expresses a T-cell receptor (TCR) that recognizes cognate antigen and controls T-cell activation, different T cells bearing the same TCR can be functionally distinct. Each TCR is a heterodimer, and both α- and β-chains contribute to determining TCR antigen specificity. Here we present a methodology enabling integration of information about TCR specificity with information about T cell function. This method involves sequencing of TCRα and TCRβ genes, and amplifying functional genes characteristic of different T cell subsets, in single T cells. Because this approach retains information about individual TCRα-TCRβ pairs, TCRs of interest can be expressed and used in functional studies, for antigen discovery, or in therapeutic applications. We apply this approach to study the clonal ancestry and differentiation of T lymphocytes infiltrating a human colorectal carcinoma. PMID:24952902

  1. Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

    PubMed

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

    2016-04-01

    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 redirecting 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

  2. Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer.

    PubMed

    Fujiwara, H; Ochi, T; Ochi, F; Miyazaki, Y; Asai, H; Narita, M; Okamoto, S; Mineno, J; Kuzushima, K; Shiku, H; Yasukawa, M

    2015-12-01

    To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/β genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia. PMID:26104661

  3. IL-33 Receptor-Expressing Regulatory T Cells Are Highly Activated, Th2 Biased and Suppress CD4 T Cell Proliferation through IL-10 and TGFβ Release

    PubMed Central

    Datsi, Angeliki; Hegazy, Ahmed N.; Varga, Domonkos V.; Holecska, Vivien; Saito, Hirohisa; Nakae, Susumu; Löhning, Max

    2016-01-01

    Immunomodulatory Foxp3+ regulatory T cells (Tregs) form a heterogeneous population consisting of subsets with different activation states, migratory properties and suppressive functions. Recently, expression of the IL-33 receptor ST2 was shown on Tregs in inflammatory settings. Here we report that ST2 expression identifies highly activated Tregs in mice even under homeostatic conditions. ST2+ Tregs preferentially accumulate at non-lymphoid sites, likely mediated by their high expression of several chemokine receptors facilitating tissue homing. ST2+ Tregs exhibit a Th2-biased character, expressing GATA-3 and producing the Th2 cytokines IL-5 and IL-13 –especially in response to IL-33. Yet, IL-33 is dispensable for the generation and maintenance of these cells in vivo. Furthermore, ST2+ Tregs are superior to ST2− Tregs in suppressing CD4+ T cell proliferation in vitro independent of IL-33. This higher suppressive capacity is partially mediated by enhanced production and activation of the anti-inflammatory cytokines IL-10 and TGFβ. Thus, ST2 expression identifies a highly activated, strongly suppressive Treg subset preferentially located in non-lymphoid tissues. Here ST2+ Tregs may be well positioned to immediately react to IL-33 alarm signals. Their specific properties may render ST2+ Tregs useful targets for immunomodulatory therapies. PMID:27548066

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

    PubMed

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  5. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  6. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy.

    PubMed

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. PMID:26993168

  7. Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy.

    PubMed

    Maus, Marcela V; June, Carl H

    2016-04-15

    Chimeric antigen receptors (CAR) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early-phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of approximately 90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into the mechanisms regulating the persistence of CAR T cells. In addition, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy.Clin Cancer Res; 22(8); 1875-84. ©2016 AACR SEE ALL ARTICLES IN THIS CCR FOCUS SECTION, "OPPORTUNITIES AND CHALLENGES IN CANCER IMMUNOTHERAPY". PMID:27084741

  8. T cell receptor gamma and delta rearrangements in hematologic malignancies. Relationship to lymphoid differentiation.

    PubMed Central

    Griesinger, F; Greenberg, J M; Kersey, J H

    1989-01-01

    We have studied recombinatorial events of the T cell receptor delta and gamma chain genes in hematopoietic malignancies and related these to normal stages of lymphoid differentiation. T cell receptor delta gene recombinatorial events were found in 91% of acute T cell lymphoblastic leukemia, 68% of non-T, non-B lymphoid precursor acute lymphoblastic leukemia (ALL) and 80% of mixed lineage acute leukemias. Mature B-lineage leukemias and acute nonlymphocytic leukemias retained the T-cell receptor delta gene in the germline configuration. The incidence of T cell receptor gamma and delta was particularly high in CD10+CD19+ non-T, non-B lymphoid precursor ALL. In lymphoid precursor ALL, T cell receptor delta was frequently rearranged while T cell receptor gamma was in the germline configuration. This suggests that TCR delta rearrangements may precede TCR gamma rearrangements in lymphoid ontogeny. In T-ALL, only concordant T cell receptor delta and gamma rearrangements were observed. Several distinct rearrangements were defined using a panel of restriction enzymes. Most of the rearrangements observed in T-ALL represented joining events of J delta 1 to upstream regions. In contrast, the majority of rearrangements in lymphoid precursor ALL most likely represented D-D or V-D rearrangements, which have been found to be early recombinatorial events of the TCR delta locus. We next analyzed TCR delta rearrangements in five CD3+TCR gamma/delta+ ALL and cell lines. One T-ALL, which demonstrated a different staining pattern with monoclonal antibodies against the products of the TCR gamma/delta genes than the PEER cell line, rearranges J delta 1 to a currently unidentified variable region. Images PMID:2547833

  9. Defective T cell Receptor-mediated Signal Transduction in Memory CD4 T Lymphocytes Exposed to Superantigen or anti-T cell Receptor Antibodies

    PubMed Central

    Watson, Andrew R.O.; Lee, William T.

    2007-01-01

    Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR1) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance. PMID:17083922

  10. Human CD4+ T-Cells: A Role for Low-Affinity Fc Receptors

    PubMed Central

    Chauhan, Anil K.

    2016-01-01

    Both lymphoid and myeloid cells express Fc receptors (FcRs). Low-affinity FcRs engage circulating immune complexes, which results in the cellular activation and pro-inflammatory cytokine production. FcRs participate in the internalization, transport, and/or recycling of antibodies and antigens. Cytosolic FcRs also route these proteins to proteasomes and antigen-presentation pathways. Non-activated CD4+ T-cells do not express FcRs. Once activated, naive CD4+ T-cells express FcγRIIIa, which, upon IC ligation, provide a costimulatory signal for the differentiation of these cells into effector cell population. FcγRIIIa present on CD4+ T-cell membrane could internalize nucleic acid-containing ICs and elicit a cross-talk with toll-like receptors. FcγRIIIa common γ-chain forms a heterodimer with the ζ-chain of T-cell receptor complex, suggesting a synergistic role for these receptors. This review first summarizes our current understanding of FcRs on CD4+ T-cells. Thereafter, I will attempt to correlate the findings from the recent literature on FcRs and propose a role for these receptors in modulating adaptive immune responses via TLR signaling, nucleic acid sensing, and epigenetic changes in CD4+ T-cells. PMID:27313579

  11. 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

  12. Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

    PubMed

    Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

    2013-01-01

    Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy. PMID:23296935

  13. 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

  14. IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo

    PubMed Central

    Koneru, Mythili; Purdon, Terence J.; Spriggs, David; Koneru, Susmith; Brentjens, Renier J.

    2015-01-01

    A novel approach for the treatment of ovarian cancer includes immunotherapy with genetically engineered T cells targeted to ovarian cancer cell antigens. Using retroviral transduction, T cells can be created that express an artificial T cell receptor (TCR) termed a chimeric antigen receptor (CAR). We have generated a CAR, 4H11-28z, specific to MUC-16ecto antigen, which is the over-expressed on a majority of ovarian tumor cells and is the retained portion of MUC-16 after cleavage of CA-125. We previously demonstrated that T cells modified to express the 4H11-28z CAR eradicate orthotopic human ovarian cancer xenografts in SCID-Beige mice. However, despite the ability of CAR T cells to localize to tumors, their activation in the clinical setting can be inhibited by the tumor microenvironment, as is commonly seen for endogenous antitumor immune response. To potentially overcome this limitation, we have recently developed a construct that co-expresses both MUC16ecto CAR and IL-12 (4H11-28z/IL-12). In vitro, 4H11-28z/IL-12 CAR T cells show enhanced proliferation and robust IFNγ secretion compared to 4H11-28z CAR T cells. In SCID-Beige mice with human ovarian cancer xenografts, IL-12 secreting CAR T cells exhibit enhanced antitumor efficacy as determined by increased survival, prolonged persistence of T cells, and higher systemic IFNγ. Furthermore, in anticipation of translating these results into a phase I clinical trial which will be the first to study IL-12 secreting CAR T cells in ovarian cancer, an elimination gene has been included to allow for deletion of CAR T cells in the context of unforeseen or off-tumor on-target toxicity. PMID:25949921

  15. Induction of Inhibitory Receptors on T Cells During Plasmodium vivax Malaria Impairs Cytokine Production.

    PubMed

    Costa, Pedro A C; Leoratti, Fabiana M S; Figueiredo, Maria M; Tada, Mauro S; Pereira, Dhelio B; Junqueira, Caroline; Soares, Irene S; Barber, Daniel L; Gazzinelli, Ricardo T; Antonelli, Lis R V

    2015-12-15

    The function and regulation of the immune response triggered during malaria is complex and poorly understood, and there is a particular paucity of studies conducted in humans infected with Plasmodium vivax. While it has been proposed that T-cell-effector responses are crucial for protection against blood-stage malaria in mice, the mechanisms behind this in humans remain poorly understood. Experimental models of malaria have shown that the regulatory molecules, cytotoxic T-lymphocyte attenuator-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and programmed death-1 (PD-1) are involved in the functional impairment of T cells during infection. Our goal was to define the role of these molecules during P. vivax malaria. We demonstrate that infection triggers the expression of regulatory molecules on T cells. The pattern of expression differs in CD4(+) and CD8(+) T cells. Higher frequencies of CD4(+) express more than 1 regulatory molecule compared to CD8(+) T cells. Moreover, lower proportions of CD4(+) T cells coexpress regulatory molecules, but are still able to proliferate. Importantly, simultaneously blockade of the CLTA-4, PD-1, and T-cell immunoglobulin and mucin-3 signaling restores the cytokine production by antigen-specific cells. These data support the hypothesis that upregulation of inhibitory receptors on T cells during P. vivax malaria impairs parasite-specific T-cell effector function. PMID:26019284

  16. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR

    PubMed Central

    Torikai, Hiroki; Reik, Andreas; Liu, Pei-Qi; Zhou, Yuanyue; Zhang, Ling; Maiti, Sourindra; Huls, Helen; Miller, Jeffrey C.; Kebriaei, Partow; Rabinovitch, Brian; Lee, Dean A.; Champlin, Richard E.; Bonini, Chiara; Naldini, Luigi; Rebar, Edward J.; Gregory, Philip D.; Holmes, Michael C.

    2012-01-01

    Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR+ T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies. PMID:22535661

  17. T Cell Receptor Sequencing Reveals the Clonal Diversity and Overlap of Colonic Effector and FOXP3+ T Cells in Ulcerative Colitis

    PubMed Central

    Lord, James; Chen, Janice; Thirlby, Richard C.; Sherwood, Anna M.; Carlson, Christopher S.

    2015-01-01

    Background & Aims FOXP3+ regulatory T cell (Tregs) prevent inflammation, but are paradoxically increased in ulcerative colitis (UC). Local T cell activation has been hypothesized to account for increased FOXP3 expression in colon lamina propria (LP) T cells. Methods To see if human FOXP3+ LP T cells are an activated fraction of otherwise FOXP3− effector T cells (Teff) and explore their clonal diversity in health and disease, we deep sequenced clonally unique T cell receptor (TCR) hypervariable regions of FOXP3+ and FOXP3− CD4+ T cell subpopulations from inflamed versus non-inflamed colon LP or mesenteric lymph nodes (MLN) of patients with or without UC. Results The clonal diversity of each LP T cell population was no different between patients with versus without UC. Repertoire overlap was only seen between a minority of FOXP3+ and FOXP3− cells, including recently activated CD38+ cells and Th17-like CD161+ Teff, but this repertoire overlap was no different between patients with versus without UC, and was no larger than the overlap between Helios− and Helios+ FOXP3+ cells. Conclusions Thus, at steady state, only a minority of FOXP3+, and particularly Helios+, T cells share a TCR sequence with FOXP3− effector populations in the colon LP, even in UC, revealing distinct clonal origins for LP Tregs and effector T cells in humans. PMID:25437819

  18. 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

  19. 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.

  20. HIV-specific Cytotoxic T Cells from Long-Term Survivors Select a Unique T Cell Receptor

    PubMed Central

    Dong, Tao; Stewart-Jones, Guillaume; Chen, Nan; Easterbrook, Philippa; Xu, Xiaoning; Papagno, Laura; Appay, Victor; Weekes, Michael; Conlon, Chris; Spina, Celsa; Little, Susan; Screaton, Gavin; van der Merwe, Anton; Richman, Douglas D.; McMichael, Andrew J.; Jones, E. Yvonne; Rowland-Jones, Sarah L.

    2004-01-01

    HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vβ13.2 T cell receptors (TCRs) with very similar and unusually long β-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8–restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vβ13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8–specific CTL using other TCRs. Selection of Vβ13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome. PMID:15596521

  1. Regulation of T cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm

    PubMed Central

    Beemiller, Peter; Krummel, Matthew F.

    2013-01-01

    Summary The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering, signalosome assembl, y and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its ‘scaffolding’ function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  2. Regulation of T-cell receptor signaling by the actin cytoskeleton and poroelastic cytoplasm.

    PubMed

    Beemiller, Peter; Krummel, Matthew F

    2013-11-01

    The actin cytoskeleton plays essential roles in modulating T-cell activation. Most models of T-cell receptor (TCR) triggering signalosome assembly and immune synapse formation invoke actin-dependent mechanisms. As T cells are constitutively motile cells, TCR triggering and signaling occur against a cytoskeletal backdrop that is constantly remodeling. While the interplay between actin dynamics and TCR signaling have been the focus of research for many years, much of the work in T cells has considered actin largely for its 'scaffolding' function. We examine the roles of the actin cytoskeleton in TCR signaling and immune synapse formation with an emphasis on how poroelasticity, an ensemble feature of actin dynamics with the cytosol, relates to how T cells respond to stimulation. PMID:24117819

  3. 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

  4. Litomosoides sigmodontis induces TGF-β receptor responsive, IL-10-producing T cells that suppress bystander T-cell proliferation in mice.

    PubMed

    Hartmann, Wiebke; Schramm, Christoph; Breloer, Minka

    2015-09-01

    Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth-specific but also nonhelminth-specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T-cell suppression. When OT-II T cells specific for the third-party antigen ovalbumin are transferred into helminth-infected mice, these cells respond to antigen-specific stimulation with reduced proliferation compared to activation within non-infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two-staged process. Parasite products first engage the TGF-β receptor on host-derived T cells that are central to suppression. In a second step, host-derived T cells produce IL-10 and subsequently suppress the adoptively transferred OT-II T cells. Terminal suppression was IL-10-dependant but independent of intrinsic TGF-β receptor- or PD-1-mediated signaling in the suppressed OT-II T cells. Blockade of the same key suppression mediators, i.e. TGF-β- and IL-10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis-infected mice. PMID:26138667

  5. 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

  6. 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

    2016-07-01

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

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

    PubMed

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

    2015-10-16

    There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control. PMID:26405231

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

    PubMed Central

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

    2016-01-01

    There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen specificity. In these split receptors, antigen binding and intracellular signaling components only assemble in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate both cell autonomous recognition and user control. PMID:26405231

  9. Gene-modified T-cell therapy using chimeric antigen receptors for pediatric hematologic malignancies.

    PubMed

    Nakazawa, Yozo

    2016-06-01

    Chimeric antigen receptor (CAR) is the generic name for synthetic T cell receptors redirected to tumor-associated antigens. Most CARs consist of an ectodomain (scFv or ligand), a hinge region, a transmembrane domain, and signaling endodomains derived from one or two co-stimulatory molecules (CD28, 4-1BB, etc) and from a CD3-ζ chain. CD19-targeted CAR T cell therapy has achieved major success in the treatment of B cell malignancies. CD19 CAR-T cells elicited complete remission in 70-90% of adult and pediatric patients with relapsed/refractory acute lymphoblastic leukemia (ALL). CD19 CAR T cell therapy from allogeneic donors including third party donors is a potential option for B-cell malignancies. CAR T cell therapies for myeloma, acute myeloid leukemia, and T-cell leukemia are still under development. Our group is currently preparing a phase I study of CD19 CAR T cell therapy in pediatric and young adult patients with ALL using a non-viral gene transfer method, the piggyBac-transposon system. PMID:27384848

  10. Cytokine release syndrome in cancer immunotherapy with chimeric antigen receptor engineered T cells.

    PubMed

    Xu, Xiao-Jun; Tang, Yong-Min

    2014-02-28

    Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells is a promising therapy for cancers. However, the safety of this approach is concerned. Cytokine release syndrome (CRS) is a common but lethal complication of CAR-T cell therapy. The development of CRS correlates with CAR structures, tumor type and burden, and patients' genetic polymorphisms. CRS related adverse events may be reduced by designing safer CARs and CAR-T cells and following strict dose-escalation scheme. Timely and effective cytokine-directed treatment with corticosteroid and various cytokine antagonists is important to avoid CRS associated death. PMID:24141191

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

    PubMed

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

    2016-05-01

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

  12. 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

  13. 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. PMID:27392648

  14. Tissue microarray-based screening for chromosomal breakpoints affecting the T-cell receptor gene loci in mature T-cell lymphomas.

    PubMed

    Leich, E; Haralambieva, E; Zettl, A; Chott, A; Rüdiger, T; Höller, S; Müller-Hermelink, H-K; Ott, G; Rosenwald, A

    2007-09-01

    The pathogenesis of mature T-cell non-Hodgkin lymphomas (T-NHLs) is poorly understood. Analogous to B-cell lymphomas, in which the immunoglobulin (IgH) receptor loci are frequently targeted by chromosomal translocations, the T-cell receptor (TCR) gene loci are affected by translocations in a subset of precursor T-cell malignancies. In a large-scale analysis of 245 paraffin-embedded mature T-NHLs, arranged in a tissue microarray format and using improved FISH assays for the detection of breakpoints in the TCRalpha/delta, TCRbeta, and TCRgamma loci, we provide evidence that mature T-NHLs other than T-cell prolymphocytic leukaemia (T-PLL) also occasionally show a chromosomal rearrangement that involves the TCRalpha/delta locus. In particular, one peripheral T-cell lymphoma (not otherwise specified, NOS) with the morphological variant of Lennert lymphoma displayed a chromosomal translocation t(14;19) involving the TCRalpha/delta and the BCL3 loci. A second case, an angio-immunoblastic T-cell lymphoma (AILT), carried an inv(14)(q11q32) affecting the TCRalpha/delta and IgH loci. FISH signal constellations as well as concomitant comparative genomic hybridization (CGH) data were also suggestive of the occurrence of an isochromosome 7, previously described to be pathognomonic for hepatosplenic T-cell lymphomas, in rare cases of enteropathy-type T-cell lymphoma. PMID:17582237

  15. T cells expressing the V beta 1 T-cell receptor are required for IgA production in the chicken.

    PubMed Central

    Cihak, J; Hoffmann-Fezer, G; Ziegler-Heibrock, H W; Stein, H; Kaspers, B; Chen, C H; Cooper, M D; Lösch, U

    1991-01-01

    While alpha beta T cells in mammals may express one of many variable (V) gene families in the beta locus, chickens have only two V beta gene families. The avian V beta 2+ T cells are recognized by the T-cell receptor 3 (TCR3) monoclonal antibody and V beta 1+ T cells are recognized by the TCR2 antibody, which we used to selectively suppress development of V beta 1+ T cells in order to examine their functional role. Suppression was accomplished by multiple injections of anti-TCR2 antibodies beginning in embryonic life and perpetuated by thymectomy 8 days after hatching. Young birds thus depleted of V beta 1+ T cells had greater than normal numbers of V beta 2+ T cells and appeared as healthy as thymectomized and untreated controls. While production of IgM and IgG antibodies was unimpaired, IgA antibody production was severely compromised in the V beta 1-depleted birds. The levels of secretory IgA in bile and lung lavage fluid were reduced 1000- to 10,000-fold and secretory IgA antibodies were not produced in response to mucosal immunization. B-cell production of IgA antibodies thus appears to require T cells expressing the V beta 1 genes, whereas T cells that express the V beta 2 genes lack this capacity. Images PMID:1835793

  16. IL12-mediated sensitizing of T-cell receptor-dependent and -independent tumor cell killing.

    PubMed

    Braun, Matthias; Ress, Marie L; Yoo, Young-Eun; Scholz, Claus J; Eyrich, Matthias; Schlegel, Paul G; Wölfl, Matthias

    2016-07-01

    Interleukin 12 (IL12) is a key inflammatory cytokine critically influencing Th1/Tc1-T-cell responses at the time of initial antigen encounter. Therefore, it may be exploited for cancer immunotherapy. Here, we investigated how IL12, and other inflammatory cytokines, shape effector functions of human T-cells. Using a defined culture system, we followed the gradual differentiation and function of antigen-specific CD8(+) T cells from their initial activation as naïve T cells through their expansion phase as early memory cells to full differentiation as clonally expanded effector T cells. The addition of IL12 8 days after the initial priming event initiated two mechanistically separate events: First, IL12 sensitized the T-cell receptor (TCR) for antigen-specific activation, leading to an approximately 10-fold increase in peptide sensitivity and, in consequence, enhanced tumor cell killing. Secondly, IL12 enabled TCR/HLA-independent activation and cytotoxicity: this "non-specific" effect was mediated by the NK cell receptor DNAM1 (CD226) and dependent on ligand expression of the target cells. This IL12 regulated, DNAM1-mediated killing is dependent on src-kinases as well as on PTPRC (CD45) activity. Thus, besides enhancing TCR-mediated activation, we here identified for the first time a second IL12 mediated mechanism leading to activation of a receptor-dependent killing pathway via DNAM1. PMID:27622043

  17. 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

  18. Acetylcholine receptor-specific suppressive T-cell factor from a retrovirally transformed T-cell line.

    PubMed Central

    Sinigaglia, F; Gotti, C; Castagnoli, R; Clementi, F

    1984-01-01

    In both experimental and human myasthenia gravis an impairment in the immune regulation leads to an increased synthesis of antibodies against the nicotinic acetylcholine receptor (AcChoR). The present work reports the establishment of an AcChoR-specific suppressive T-cell line obtained by viral transformation of AcChoR-enriched murine T lymphocytes. Enriched T cells from Torpedo AcChoR-primed mice, prestimulated in vitro with antigen, were infected with radiation leukemia viruses and injected intravenously in congeneic recipient mice. Six months later lymphomas were observed in 20% of the injected mice and two of them, of donor origin, were established as permanent continuous cell lines in vitro. One of these lines, named LA41, expresses Thy-1.2, Lyt-2, and I-Jb surface markers. Culture supernatants of LA41 cells suppress the antigen-specific in vitro proliferation of Torpedo AcChoR-primed lymphocytes. This suppression is antigen-specific since the response induced by fetal calf AcChoR and by other antigens is not affected by addition of LA41 culture supernatant in the proliferative assay. LA41 culture supernatant injected in vivo at the time of antigen-priming suppresses also significantly the production of anti-AcChoR antibodies but not the synthesis of antibodies against other antigens--i.e., fetal calf AcChoR or alpha-bungarotoxin. These data show that LA41 cells constitutively produce Torpedo AcChoR-specific suppressor factor. PMID:6095305

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

    PubMed

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

    2016-04-01

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

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

    PubMed

    Levine, B L

    2015-03-01

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

  1. Perturbed T cell IL7 receptor-signaling in chronic Chagas disease

    PubMed Central

    Albareda, M. Cecilia; Perez-Mazliah, Damián; Natale, M. Ailén; Castro-Eiro, Melisa; Alvarez, María G.; Viotti, Rodolfo; Bertocchi, Graciela; Lococo, Bruno; Tarleton, Rick L; Laucella, Susana A.

    2015-01-01

    We have previously demonstrated that immune responses in subjects with chronic Trypanosoma cruzi infection display features common to other persistent infections with signs of T cell exhaustion. Alterations in cytokine receptor signal transduction have emerged as one of the cell-intrinsic mechanisms of T cell exhaustion. Herein, we performed an analysis of the expression of IL-7R components (CD127 and CD132) on CD4+ and CD8+ T cells, and evaluated IL-7-dependent signaling events in patients at different clinical stages of chronic chagasic heart disease. Subjects with no signs of cardiac disease showed a decrease in CD127+CD132+ cells and a reciprocal gain of CD127-CD132+ in CD8+ and CD4+ T cells compared to either patients exhibiting heart enlargement or uninfected controls. T. cruzi infection, in vitro, was able to stimulate the downregulation of CD127 and the upregulation of CD132 on T cells. IL-7-induced phosphorylation of STAT5 as well as Bcl-2 and CD25 expression were lower in T. cruzi-infected subjects compared with uninfected controls. The serum levels of IL-7 was also increased in chronic chagasic patients. The present study highlights perturbed IL-7/IL-7R T cell signaling through STAT5 as a potential mechanism of T cell exhaustion in chronic T. cruzi infection. PMID:25769928

  2. Reactive oxygen species differentially affect T cell receptor-signaling pathways.

    PubMed

    Cemerski, Saso; Cantagrel, Alain; Van Meerwijk, Joost P M; Romagnoli, Paola

    2002-05-31

    Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others. PMID:11916964

  3. Critical role for BIM in T cell receptor restimulation-induced death

    PubMed Central

    Snow, Andrew L; Oliveira, João B; Zheng, Lixin; Dale, Janet K; Fleisher, Thomas A; Lenardo, Michael J

    2008-01-01

    Background Upon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals. Results Upon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption. Conclusion Our data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis. Reviewers This article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr. PMID:18715501

  4. Functional cloning of a gp100-reactive T-cell receptor from vitiligo patient skin.

    PubMed

    Klarquist, Jared; Eby, Jonathan M; Henning, Steven W; Li, Mingli; Wainwright, Derek A; Westerhof, Wiete; Luiten, Rosalie M; Nishimura, Michael I; Le Poole, I Caroline

    2016-05-01

    We isolated gp100-reactive T cells from perilesional skin of a patient with progressive vitiligo with superior reactivity toward melanoma cells compared with tumor-infiltrating lymphocytes 1520, a melanoma-derived T-cell line reactive with the same cognate peptide. After dimer enrichment and limited dilution cloning, amplified cells were subjected to reverse transcription and 5' RACE to identify the variable TCRα and TCRβ subunit sequences. The full-length sequence was cloned into a retroviral vector separating both subunits by a P2A slippage sequence and introduced into Jurkat cells and primary T cells. Cytokine secreted by transduced cells in response to cognate peptide and gp100-expressing targets signifies that we have successfully cloned a gp100-reactive T-cell receptor from actively depigmenting skin. PMID:26824221

  5. Ly-6A is required for T cell receptor expression and protein tyrosine kinase fyn activity.

    PubMed Central

    Lee, S K; Su, B; Maher, S E; Bothwell, A L

    1994-01-01

    To characterize the function of the Ly-6A antigen in T cell activation, antisense Ly-6 RNA was expressed in a stably transfected antigen-specific T cell clone. Reduced Ly-6A expression results in inhibition of responses to antigen, anti-TCR (anti-T cell receptor) crosslinking and concanavalin A plus recombinant interleukin 1 and causes impairment of in vitro fyn tyrosine kinase activity. More substantial reduction of Ly-6A results in reduction of TCR expression. Analysis of mRNA species indicates that the reduction is specific for the TCR beta chain. These data demonstrate that Ly-6A may regulate TCR expression and may be involved in early events of T cell activation via regulation of fyn tyrosine kinase activity. Images PMID:8187770

  6. 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

  7. Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor.

    PubMed

    Geisler, C; Pallesen, G; Platz, P; Odum, N; Dickmeiss, E; Ryder, L P; Svejgaard, A; Plesner, T; Larsen, J K; Koch, C

    1989-07-01

    Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected. A thymic biopsy specimen showed severe abnormalities of both the thymic lymphoid and epithelial component with abortive medullary differentiation and almost an entire lack of Hassall's corpuscles. This patient represents a case of primary immune deficiency syndrome not previously described. Thymic deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue. PMID:2527256

  8. Survivin blockade sensitizes rhabdomyosarcoma cells for lysis by fetal acetylcholine receptor-redirected T cells.

    PubMed

    Simon-Keller, Katja; Paschen, Annette; Hombach, Andreas A; Ströbel, Philipp; Coindre, Jean-Michel; Eichmüller, Stefan B; Vincent, Angela; Gattenlöhner, Stefan; Hoppe, Florian; Leuschner, Ivo; Stegmaier, Sabine; Koscielniak, Ewa; Leverkus, Martin; Altieri, Dario C; Abken, Hinrich; Marx, Alexander

    2013-06-01

    Cellular immunotherapy may provide a strategy to overcome the poor prognosis of metastatic and recurrent rhabdomyosarcoma (RMS) under the current regimen of polychemotherapy. Because little is known about resistance mechanisms of RMS to cytotoxic T cells, we investigated RMS cell lines and biopsy specimens for expression and function of immune costimulatory receptors and anti-apoptotic molecules by RT-PCR, Western blot analysis, IHC, and cytotoxicity assays using siRNA or transfection-modified RMS cell lines, together with engineered RMS-directed cytotoxic T cells specific for the fetal acetylcholine receptor. We found that costimulatory CD80 and CD86 were consistently absent from all RMSs tested, whereas inducible T-cell co-stimulator ligand (ICOS-L; alias B7H2) was expressed by a subset of RMSs and was inducible by tumor necrosis factor α in two of five RMS cell lines. Anti-apoptotic survivin, along with other inhibitor of apoptosis (IAP) family members (cIAP1, cIAP2, and X-linked inhibitor of apoptosis protein), was overexpressed by RMS cell lines and biopsy specimens. Down-regulation of survivin by siRNA or pharmacologically in RMS cells increased their susceptibility toward a T-cell attack, whereas induction of ICOS-L did not. Treatment of RMS-bearing Rag(-/-) mice with fetal acetylcholine receptor-specific chimeric T cells delayed xenograft growth; however, this happened without definitive tumor eradication. Combined blockade of survivin and application of chimeric T cells in vivo suppressed tumor proliferation during survivin inhibition. In conclusion, survivin blockade provides a strategy to sensitize RMS cells for T-cell-based therapy. PMID:23562272

  9. 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. PMID:24122851

  10. Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

    PubMed

    Lynn, Rachel C; Poussin, Mathilde; Kalota, Anna; Feng, Yang; Low, Philip S; Dimitrov, Dimiter S; Powell, Daniel J

    2015-05-28

    T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo. m909 CAR T cells exhibited selective activation and lytic function against engineered C30-FRβ as well as endogenous FRβ(+) AML cell lines in vitro. In mouse models of human AML, m909 CAR T cells mediated the regression of engrafted FRβ(+) THP1 AML in vivo. In addition, we demonstrated that treatment of AML with all-trans retinoic acid (ATRA) enhanced FRβ expression, resulting in improved immune recognition by m909 CAR T cells. Because many cell surface markers are shared between AML blasts and healthy hematopoietic stem and progenitor cells (HSCs), we evaluated FRβ expression and recognition of HSCs by CAR T cells. m909 CAR T cells were not toxic against healthy human CD34(+) HSCs in vitro. Our results indicate that FRβ is a promising target for CAR T-cell therapy of AML, which may be augmented by combination with ATRA. PMID:25887778

  11. Expression of Chemokine Receptors on Peripheral Blood T Cells in Children with Chronic Kidney Disease

    PubMed Central

    Szczepańska, Maria; Sędek, Łukasz; Makulska, Irena; Szprynger, Krystyna; Mazur, Bogdan; Zwolińska, Danuta; Karpe, Jacek; Ziora, Katarzyna; Szczepański, Tomasz

    2015-01-01

    Chemokine receptors play a role in leukocyte recruitment, activation, and maintaining effector functions and regulate adaptive immune response and angiogenesis. The study aimed at flow cytometric analysis of T cell subsets with selected surface chemokine receptors (CCR4, CCR5, CCR7, CXCR3, and CXCR4) or receptor combination in peripheral blood of children with chronic kidney disease (CKD) on hemodialysis (HD). The percentage of T lymphocytes with CD8 and combined CD28,CCR7 expression was higher in HD children. The percentage of T lymphocytes expressing CCR7, CD28,CCR7, and CXCR4,CD8 was increased in children on conservative treatment. Total number (tn) of CXCR4+ cells was reduced in children on hemodialysis. The tn of T CXCR3+ cells was lower in children on conservative treatment. During HD the percentage of T CD4+ cells was higher and of T CXCR3+ lymphocytes was lower after HD session as compared to 15 min of session duration. During HD tn of T cells with expression of CCR4, CCR5, CCR7, CXCR3, and CXCR4 was constant. The alteration of chemokine receptors expression in children with CKD occurs early in the development. Diminished expression of CXCR3, CXCR4 on T cells in patients with CKD on HD might result in impaired inflammatory response. Increased CCR7+ T cell percentage could be responsible for the alteration of migration of cells into secondary lymphatic organs. PMID:25866451

  12. TALEN-mediated genetic inactivation of the glucocorticoid receptor in cytomegalovirus-specific T cells.

    PubMed

    Menger, Laurie; Gouble, Agnes; Marzolini, Maria A V; Pachnio, Annette; Bergerhoff, Katharina; Henry, Jake Y; Smith, Julianne; Pule, Martin; Moss, Paul; Riddell, Stanley R; Quezada, Sergio A; Peggs, Karl S

    2015-12-24

    Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator-like effector nuclease (TALEN) messenger RNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer-selected CMV-specific CD8(+) T cells (HLA-A02/NLV peptide), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic GVHD model in sublethally irradiated NOD-scid IL2rγ(null) mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids. PMID:26508783

  13. Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor

    PubMed Central

    Jena, Bipulendu; Dotti, Gianpietro

    2010-01-01

    Infusions of antigen-specific T cells have yielded therapeutic responses in patients with pathogens and tumors. To broaden the clinical application of adoptive immunotherapy against malignancies, investigators have developed robust systems for the genetic modification and characterization of T cells expressing introduced chimeric antigen receptors (CARs) to redirect specificity. Human trials are under way in patients with aggressive malignancies to test the hypothesis that manipulating the recipient and reprogramming T cells before adoptive transfer may improve their therapeutic effect. These examples of personalized medicine infuse T cells designed to meet patients' needs by redirecting their specificity to target molecular determinants on the underlying malignancy. The generation of clinical grade CAR+ T cells is an example of bench-to-bedside translational science that has been accomplished using investigator-initiated trials operating largely without industry support. The next-generation trials will deliver designer T cells with improved homing, CAR-mediated signaling, and replicative potential, as investigators move from the bedside to the bench and back again. PMID:20439624

  14. 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 Central

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

    2016-01-01

    ABSTRACT 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). PMID:27195186

  15. Charged MVB protein 5 is involved in T-cell receptor signaling.

    PubMed

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-01

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling. PMID:26821576

  16. Charged MVB protein 5 is involved in T-cell receptor signaling

    PubMed Central

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-01

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)–mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5KD) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5KD Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5KD Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling. PMID:26821576

  17. 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. PMID:26827842

  18. Leptin receptor signaling in T cells is required for Th17 differentiation

    PubMed Central

    Reis, Bernardo S; Lee, Kihyun; Fanok, Melania H; Mascaraque, Cristina; Amoury, Manal; Cohn, Lillian; Rogoz, Aneta; Dallner, Olof S; Moraes-Vieira, Pedro M; Domingos, Ana I; Mucida, Daniel

    2015-01-01

    The hormone leptin plays a key role in energy homeostasis, and the absence of either leptin or its receptor (lepR) leads to severe obesity and metabolic disorders. To avoid indirect effects and to address the cell-intrinsic role of leptin signaling in the immune system, we conditionally targeted lepR in T cells. In contrast to pleiotropic immune disorders reported in obese mice with leptin or lepR deficiency, we found that lepR deficiency in CD4+ T cells resulted in a selective defect in both autoimmune and protective Th17 responses. Reduced capacity for differentiation towards a Th17 phenotype by lepr-deficient T cells was attributed to reduced activation of the signal transducer and activator of transcription 3 (STAT3) and its downstream targets. This study establishes cell-intrinsic roles for leptin receptor signaling in the immune system and suggests that leptin signaling during T cell differentiation plays a crucial role in T cell peripheral effector function. PMID:25917102

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

    PubMed

    Castro, Caitlin D; Luoma, Adrienne M; Adams, Erin J

    2015-09-01

    The structure and amino acid diversity of the T-cell receptor (TCR), similar in nature to that of Fab portions of antibodies, would suggest that 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

  20. 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

  1. Sequence and diversity of the rat delta T-cell receptor.

    PubMed

    Watson, D; Ando, T; Knight, J F

    2000-07-01

    The cDNA sequence of the delta T-cell receptor (TCRD) in the adult Lewis rat thymus was determined using the technique of rapid amplification of cDNA ends. Sixteen variable region genes (TCRDV), two diversity regions (TCRDD), two joining regions (TCRDJ), and a single constant region gene (TCRDC) were identified. The sixteen unique TCRDV genes identified represented eight different subfamilies in the rat and were highly conserved (>80% nucleotide identity) to corresponding mouse sequences. Extensive junctional diversity was observed in the rat, with both TCRDD regions (TCRDD1 and TCRDD2) utilized in the majority of cDNA clones identified. The two TCRDJ genes were highly conserved and corresponded to TCRDJ1 and TCRDJ2 in the mouse; the majority of clones utilized TCRDJ1. The TCRDC region in the rat was 91.1% identical to the mouse TCRDC gene and was highly conserved to other species. Although extensive sequence information about mouse gamma-delta T-cell receptor genes is available, current knowledge of rat gamma-delta T-cells is limited. The sequence analysis presented in this study adds to our understanding of gamma-delta T-cells in general, and it may be utilized to study the role of gamma-delta T-cells in immune-mediated disease and transplantation models previously established in the rat. PMID:10941843

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

    PubMed

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

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  3. A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

    PubMed Central

    Shutter, J; Cain, J A; Ledbetter, S; Rogers, M D; Hockett, R D

    1995-01-01

    T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell. PMID:8524269

  4. Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.

    PubMed

    Dudek, Nadine L; Thomas, Helen E; Mariana, Lina; Sutherland, Robyn M; Allison, Janette; Estella, Eugene; Angstetra, Eveline; Trapani, Joseph A; Santamaria, Pere; Lew, Andrew M; Kay, Thomas W H

    2006-09-01

    Cytotoxic T-cells are the major mediators of beta-cell destruction in type 1 diabetes, but the molecular mechanisms are not definitively established. We have examined the contribution of perforin and Fas ligand to beta-cell destruction using islet-specific CD8(+) T-cells from T-cell receptor transgenic NOD8.3 mice. NOD8.3 T-cells killed Fas-deficient islets in vitro and in vivo. Perforin-deficient NOD8.3 T-cells were able to destroy wild-type but not Fas-deficient islets in vitro. These results imply that NOD8.3 T-cells use both pathways and that Fas is required for beta-cell killing only when perforin is missing. Consistent with this theory, transgenic NOD8.3 mice with beta-cells that do not respond to Fas ligation were not protected from diabetes. We next investigated the mechanism of protection provided by overexpression of suppressor of cytokine signaling-1 (SOCS-1) in beta-cells of NOD8.3 mice. SOCS-1 islets remained intact when grafted into NOD8.3 mice and were less efficiently killed in vitro. However, addition of exogenous peptide rendered SOCS-1 islets susceptible to 8.3 T-cell-mediated lysis. Therefore, NOD8.3 T-cells use both perforin and Fas pathways to kill beta-cells and the surprising blockade of NOD8.3 T-cell-mediated beta-cell death by SOCS-1 overexpression may be due in part to reduced target cell recognition. PMID:16936188

  5. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  6. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

    PubMed

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  7. Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

    PubMed Central

    Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

    2014-01-01

    Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960

  8. Adoptive Therapy with Chimeric Antigen Receptor Modified T Cells of Defined Subset Composition

    PubMed Central

    Riddell, Stanley R.; Sommermeyer, Daniel; Berger, Carolina; Liu, Lingfeng (Steven); Balakrishnan, Ashwini; Salter, Alex; Hudecek, Michael; Maloney, David G.; Turtle, Cameron J.

    2014-01-01

    The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in in targeting CD19 on B cell malignancies. The clinical trials of CD19 CAR therapy have thus far not attempted to select defined subsets prior to transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to utilizing adoptive therapy with genetically modified T cells of defined subset and phenotypic composition. PMID:24667960

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

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

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

    PubMed

    Davodeau, F; Peyrat, M A; Gaschet, J; Hallet, M M; Triebel, F; Vié, H; Kabelitz, D; Bonneville, M

    1994-11-01

    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

  11. Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq.

    PubMed

    Redmond, David; Poran, Asaf; Elemento, Olivier

    2016-01-01

    Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments. Source code is available at https://github.com/ElementoLab/scTCRseq . PMID:27460926

  12. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli.

    PubMed Central

    Weaver, C T; Pingel, J T; Nelson, J O; Thomas, M L

    1991-01-01

    CD45 is a high-molecular-weight transmembrane protein tyrosine phosphatase expressed only by nucleated cells of hematopoietic origin. To examine function, mouse CD8+ cytolytic T-cell clones were derived that had a specific defect in the expression of CD45. Northern (RNA) blot analysis indicates that the CD45 deficiency is due to either a transcriptional defect or mRNA instability. The CD45-deficient cells were greatly diminished in their ability to respond to antigen. All functional parameters of T-cell receptor signalling analyzed (cytolysis of targets, proliferation, and cytokine production) were markedly diminished. A CD45+ revertant was isolated, and the ability to respond to antigen was restored. These results support a central and immediate role for this transmembrane protein tyrosine phosphatase in T-cell receptor signalling. Images PMID:1652055

  13. High-throughput pairing of T cell receptor α and β sequences.

    PubMed

    Howie, Bryan; Sherwood, Anna M; Berkebile, Ashley D; Berka, Jan; Emerson, Ryan O; Williamson, David W; Kirsch, Ilan; Vignali, Marissa; Rieder, Mark J; Carlson, Christopher S; Robins, Harlan S

    2015-08-19

    The T cell receptor (TCR) protein is a heterodimer composed of an α chain and a β chain. TCR genes undergo somatic DNA rearrangements to generate the diversity of T cell binding specificities needed for effective immunity. Recently, high-throughput immunosequencing methods have been developed to profile the TCR α (TCRA) and TCR β (TCRB) repertoires. However, these methods cannot determine which TCRA and TCRB chains combine to form a specific TCR, which is essential for many functional and therapeutic applications. We describe and validate a method called pairSEQ, which can leverage the diversity of TCR sequences to accurately pair hundreds of thousands of TCRA and TCRB sequences in a single experiment. Our TCR pairing method uses standard laboratory consumables and equipment without the need for single-cell technologies. We show that pairSEQ can be applied to T cells from both blood and solid tissues, such as tumors. PMID:26290413

  14. Chimeric antigen receptor-engineered T cells for cancer immunotherapy: progress and challenges.

    PubMed

    Han, Ethan Q; Li, Xiu-ling; Wang, Chun-rong; Li, Tian-fang; Han, Shuang-yin

    2013-01-01

    Recent years have witnessed much progress in both basic research and clinical trials regarding cancer immunotherapy with chimeric antigen receptor (CAR)-engineered T cells. The unique structure of CAR endows T cell tumor specific cytotoxicity and resistance to immunosuppressive microenvironment in cancers, which helps patients to better tackle the issue of immunological tolerance. Adoptive immunotherapy (AIT) using this supernatural T cell have gained momentum after decades of intense debates because of the promising results obtained from preclinical models and clinical trials. However, it is very important for us to evaluate thoroughly the challenges/obstacles before widespread clinical application, which clearly warrants more studies to improve our understanding of the mechanism underlying AIT. In this review, we focus on the critical issues related to the clinical outcomes of CAR-based adoptive immunotherapy and discuss the rationales to refine this new cancer therapeutic modality. PMID:23829929

  15. 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...

  16. 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. PMID:27378344

  17. 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

  18. T-cell receptor Vbeta gene expression in experimental lupus nephritis.

    PubMed Central

    Sutmuller, M; Baelde, H J; Ouellette, S; De Heer, E; Bruijn, J A

    1998-01-01

    A limited T-cell receptor (TCR) Vbeta repertoire employed by autoreactive T cells may be related to the development and course of autoimmune diseases. Vbeta repertoire skewing has been observed not only in man, but also in animal models of several human autoimmune diseases, such as MRL-lpr mice, which spontaneously develop a systemic lupus erythematosus (SLE)-like disease. Murine chronic graft-versus-host disease (GVHD) is an inducible model for SLE, involving direct interaction between donor T cells and recipient B cells. It is not known whether Vbeta-specific T-cell subsets are pathogenically involved in this model. Retroviral superantigens such as Mls-1 are known to have a profound impact on the TCR Vbeta repertoire in mice. Restriction of the peripheral TCR repertoire may result from intrathymic expression of Mls-1, which causes deletion of T cells expressing Vbeta6, -7, -8.1, or -9. Mls-1 incompatibility between donor and recipient can be used to determine the involvement of these TCR Vbeta families in GVHD. In the present study we induced GVHD in several strain combinations to investigate TCR Vbeta gene expression during GVHD, and the effect of Mls-1 incompatibility on the TCR Vbeta repertoire. TCR Vbeta gene expression was determined using an RNase protection assay. Our results indicate that T cells expressing the Vbeta2 or Vbeta16 chain play an important pathogenetic role, while T cells bearing the Vbeta1 or Vbeta6 chain may be related to self-limitation of the lupus-like disease in this model. Images Figure 1 Figure 4 PMID:9767452

  19. Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8(+) T cell receptor alpha repertoire following allogeneic transplantation.

    PubMed

    Link, C S; Eugster, A; Heidenreich, F; Rücker-Braun, E; Schmiedgen, M; Oelschlägel, U; Kühn, D; Dietz, S; Fuchs, Y; Dahl, A; Domingues, A M J; Klesse, C; Schmitz, M; Ehninger, G; Bornhäuser, M; Schetelig, J; Bonifacio, E

    2016-06-01

    Allogeneic stem cell transplantation is potentially curative, but associated with post-transplantation complications, including cytomegalovirus (CMV) infections. An effective immune response requires T cells recognizing CMV epitopes via their T cell receptors (TCRs). Little is known about the TCR repertoire, in particular the TCR-α repertoire and its clinical relevance in patients following stem cell transplantation. Using next-generation sequencing we examined the TCR-α repertoire of CD8(+) T cells and CMV-specific CD8(+) T cells in four patients. Additionally, we performed single-cell TCR-αβ sequencing of CMV-specific CD8(+) T cells. The TCR-α composition of human leucocyte antigen (HLA)-A*0201 CMVpp65- and CMVIE -specific T cells was oligoclonal and defined by few dominant clonotypes. Frequencies of single clonotypes reached up to 11% of all CD8(+) T cells and half of the total CD8(+) T cell repertoire was dominated by few CMV-reactive clonotypes. Some TCR-α clonotypes were shared between patients. Gene expression of the circulating CMV-specific CD8(+) T cells was consistent with chronically activated effector memory T cells. The CD8(+) T cell response to CMV reactivation resulted in an expansion of a few TCR-α clonotypes to dominate the CD8(+) repertoires. These results warrant further larger studies to define the ability of oligoclonally expanded T cell clones to achieve an effective anti-viral T cell response in this setting. PMID:26800118

  20. Analysis of T cells bearing different isotypic forms of the gamma/delta T cell receptor in patients with systemic autoimmune diseases.

    PubMed

    Gerli, R; Agea, E; Bertotto, A; Tognellini, R; Flenghi, L; Spinozzi, F; Velardi, A; Grignani, F

    1991-10-01

    The expression of gamma/delta T cell receptor (TCR) on peripheral blood CD3+ cells circulating in 74 patients with different systemic autoimmune diseases was evaluated. There was a significant increase in the gamma/delta T cell number only in patients with primary Sjögren's syndrome (SS) and in untreated patients with systemic lupus erythematosus (SLE). Unlike healthy subjects, a subgroup of patients with SLE and SS displayed a marked increase in gamma/delta T cells. Immunosuppressive treatment of patients with active SLE led to a normalization of the gamma/delta T cell number. Analysis of surface phenotype showed that when patient gamma/delta T cells were expanded in the peripheral blood, they were not activated but bore "memory" markers. In addition, they preferentially expressed the disulfide linked form of the TCR, except in progressive systemic sclerosis where the nondisulfide form was displayed. Serial determinations in single patients demonstrated that the gamma/delta T cell increase is a persistent immunological feature in these patient subgroups. PMID:1837314

  1. 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

  2. Specific increase in potency via structure-based design of a T cell receptor

    PubMed Central

    Malecek, Karolina; Grigoryan, Arsen; Zhong, Shi; Gu, Wei Jun; Johnson, Laura A.; Rosenberg, Steven A.; Cardozo, Timothy; Krogsgaard, Michelle

    2014-01-01

    Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are non-reactive “self” proteins, which presents an immunotherapy design challenge. Recent studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells in vivo. Although encouraging, the limited clinical responses underscore the need for enrichment of T cells with desirable anti-tumor capabilities prior to patient transfer. In this study, we used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. We analyzed the effects of selected TCR point mutations on T cell activation potency and analyzed cross-reactivity with related antigens. Our results showed that the mutated TCRs had improved T cell activation potency, while retaining a high degree of specificity. Such affinity-optimized TCRs have demonstrated to be very specific for Mart-1 (27L), the epitope for which they were structurally designed. And even though of limited clinical relevance, these studies open the possibility for future structural-based studies that could potentially be used in adoptive immunotherapy to treat melanoma while avoiding adverse autoimmunity-derived effects. PMID:25070852

  3. Multiple Receptor-Ligand Interactions Direct Tissue-Resident γδ T Cell Activation

    PubMed Central

    Witherden, Deborah. A.; Ramirez, Kevin; Havran, Wendy L.

    2014-01-01

    γδ T cells represent a major T cell population in epithelial tissues, such as skin, intestine, and lung, where they function in maintenance of the epithelium and provide a crucial first line defense against environmental and pathogenic insults. Despite their importance, the molecular mechanisms directing their activation and function have remained elusive. Epithelial-resident γδ T cells function through constant communication with neighboring cells, either via direct cell-to-cell contact or cell-to-matrix interactions. These intimate relationships allow γδ T cells to facilitate the maintenance of epithelial homeostasis, tissue repair following injury, inflammation, and protection from malignancy. Recent studies have identified a number of molecules involved in these complex interactions, under both homeostatic conditions, as well as following perturbation of these barrier tissues. These interactions are crucial to the timely production of cytokines, chemokines, growth factors, and extracellular matrix proteins for restoration of homeostasis. In this review, we discuss recent advances in understanding the mechanisms directing epithelial-T cell crosstalk and the distinct roles played by individual receptor-ligand pairs of cell surface molecules in this process. PMID:25505467

  4. 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.Clin Cancer Res; 22(7); 1559-64. ©2016 AACR. PMID:27037253

  5. Effects of dietary n-3 fatty acids on T cell activation and T cell receptor-mediated signaling in a murine model.

    PubMed

    McMurray, D N; Jolly, C A; Chapkin, R S

    2000-09-01

    A short-term feeding paradigm in mice, with diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was used to study the modulation of T cell activation via the T cell receptor (TcR) and the downstream pathways of intracellular signaling. Diets enriched in EPA and DHA suppressed antigen-specific delayed hypersensitivity reactions and mitogen-induced proliferation of T cells. Cocultures of accessory cells and T cells from mice given different diets revealed that purified fatty acid ethyl esters acted directly on the T cell, rather than through the accessory cell. The loss of proliferative capacity was accompanied by reductions in interleukin (IL)-2 secretion and IL-2 receptor alpha chain mRNA transcription, suggesting that dietary EPA and DHA act, in part, by interrupting the autocrine IL-2 activation pathway. Dietary EPA and DHA blunted the production of intracellular second messengers, including diacylglycerol and ceramide, following mitogen stimulation in vitro. Dietary effects appear to vary with the agonist employed (i.e., anti-CD3 [TcR], anti-CD28, exogenous IL-2, or phorbol myristate acetate and ionomycin). PMID:10944491

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

    PubMed

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

    1992-12-01

    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

  7. Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies.

    PubMed

    Biagi, Ettore; Marin, Virna; Giordano Attianese, Greta Maria Paola; Dander, Erica; D'Amico, Giovanna; Biondi, Andrea

    2007-03-01

    Chimeric T-cell receptors (ChTCR), are a fascinating technological step in the field of immunotherapy for orienting the activity of immune cells towards specific molecular targets expressed on the cell surface of various tumors, including hematologic malignancies. The main characteristics of ChTCR are their ability to redirect T-cell specificity and their killing/effector activity toward a selected target in a non MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. ChTCR are, in fact, artificial T-cell receptors constituted by an antigen-recognizing antibody molecule linked to a T-cell triggering domain. Various hematologic malignancies represent optimal targets for the exploitation of ChTCR, because of the bright expression of specific antigens on the surface of tumor cells. Thus, CD19 and CD20 have been targeted for B-cell lymphoid tumors (acute lymphoblastic leukemia-ALL, lymphomas and chronic lymphocytic leukemia-CLL), CD33 for myeloid leukemia, and CD30 for lymphomas. Even though technical and safety progresses are still needed to improve the profile of gene transfer and protein expression of ChTCR, phase 1 trials will be carried out in the near future to demonstrate the feasibility of their clinical translation and, it is be hoped, give preliminary indications about their anti-tumor efficacy. PMID:17339188

  8. Expression of the alpha/beta and gamma/delta T-cell receptors in 57 cases of peripheral T-cell lymphomas. Identification of a subset of gamma/delta T-cell lymphomas.

    PubMed Central

    Gaulard, P.; Bourquelot, P.; Kanavaros, P.; Haioun, C.; Le Couedic, J. P.; Divine, M.; Goossens, M.; Zafrani, E. S.; Farcet, J. P.; Reyes, F.

    1990-01-01

    Fifty-seven cases of peripheral T-cell lymphoma were studied for cell expression of the T-cell receptor (TCR) chains, using monoclonal antibodies specific for the beta chain (beta F1) of the alpha/beta TCR, and for the delta chain (anti-TCR delta-1) of the gamma/delta TCR. Three different patterns were demonstrated: in 39 cases (69%), the phenotype (CD3+beta F1+TCR delta-1-) was that of most normal T cells. A second pattern was found on six cases (10%), which were of CD3+beta F1-TCR delta-1+ phenotype, and in which DNA analysis showed a clonal rearrangement of the delta locus in the five cases studied. It is suggested that these cases are the neoplastic counterpart of the small subpopulation of normal T cells that express gamma delta receptor. It is of considerable interest that these gamma delta lymphomas had unusual clinicopathologic presentations, as one case corresponded to a lethal midline granuloma and the five others to hepatosplenic lymphomas with a sinusal/sinusoidal infiltration in spleen, marrow, and liver. The fact that the distribution of the neoplastic gamma delta cells in the splenic red pulp resembles that of normal gamma delta cells reinforces the concept of a preferential homing of gamma delta T cells to this tissue. A third pattern (CD3 +/- beta F1-TCR delta-1-) was seen in 12 cases (21%), in which, by contrast to normal post-thymic T cells, no evidence of either alpha beta or gamma delta T cell receptor was found. Images Figure 1 Figure 2 Figure 2 Figure 3 PMID:1698028

  9. Quantification of a Selective Expansion of T Cell Receptor Vβ by Superantigen Using Real-Time PCR.

    PubMed

    Park, Joo Youn; Seo, Keun Seok

    2016-01-01

    Selective expansion of T cells bearing specific T cell receptor Vβ segments is a hallmark of superantigens. Analyzing Vβ specificity of superantigens is important for characterizing newly discovered superantigens and understanding differential T cell responses to each toxin. Here, we describe a real-time PCR method using SYBR green I and primers specific to Cβ and Vβ genes for an absolute quantification. The established method was applied to quantify a selective expansion of T cell receptor Vβ expansion by superantigens and generated accurate, reproducible, and comparable results. PMID:26676047

  10. T cell receptor excision circle assessment of thymopoiesis in aging mice.

    PubMed

    Sempowski, Gregory D; Gooding, Maria E; Liao, H X; Le, Phong T; Haynes, Barton F

    2002-03-01

    Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in naïve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of naïve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 naïve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7. PMID:11922942

  11. Immunomodulation by Gut Microbiota: Role of Toll-Like Receptor Expressed by T Cells

    PubMed Central

    Valentini, Mariagrazia; Piermattei, Alessia; Di Sante, Gabriele; Delogu, Giovanni; Ria, Francesco

    2014-01-01

    A close relationship exists between gut microbiota and immune responses. An imbalance of this relationship can determine local and systemic immune diseases. In fact the immune system plays an essential role in maintaining the homeostasis with the microbiota that normally resides in the gut, while, at the same time, the gut microbiota influences the immune system, modulating number and function of effector and regulatory T cells. To achieve this aim, mutual regulation between immune system and microbiota is achieved through several mechanisms, including the engagement of toll-like receptors (TLRs), pathogen-specific receptors expressed on numerous cell types. TLRs are able to recognize ligands from commensal or pathogen microbiota to maintain the tolerance or trigger the immune response. In this review, we summarize the latest evidences about the role of TLRs expressed in adaptive T cells, to understand how the immune system promotes intestinal homeostasis, fights invasion by pathogens, and is modulated by the intestinal microbiota. PMID:25147831

  12. Phase separation of signaling molecules promotes T cell receptor signal transduction.

    PubMed

    Su, Xiaolei; Ditlev, Jonathon A; Hui, Enfu; Xing, Wenmin; Banjade, Sudeep; Okrut, Julia; King, David S; Taunton, Jack; Rosen, Michael K; Vale, Ronald D

    2016-04-29

    Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micrometer- or submicrometer-sized clusters. However, the functional consequences of such clustering have been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phosphorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling. PMID:27056844

  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. Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

    PubMed Central

    Krimpenfort, P; de Jong, R; Uematsu, Y; Dembic, Z; Ryser, S; von Boehmer, H; Steinmetz, M; Berns, A

    1988-01-01

    To characterize cis-acting elements controlling the expression of T cell receptor beta-chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta-chain with different extensions of 5' and 3' flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements. Images PMID:3396541

  15. Using bioinformatics tools for the sequence analysis of immunoglobulins and T cell receptors.

    PubMed

    Lefranc, Marie-Paule

    2006-03-01

    The huge potential repertoire of 10(12) immunoglobulins and 10(12) T cell receptors per individual results from complex mechanisms of combinatorial diversity between the variable (V), diversity (D), and junction (J) genes, nucleotide deletions and insertions (N-diversity) at the junctions and, for the immunoglobulins, somatic hypermutations. The accurate analysis of rearranged immunoglobulin and T cell receptor sequences, and the annotation of the junctions, therefore represent a huge challenge. The IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts, were the prerequisites for the implementation of the IMGT/V-QUEST and IMGT/JunctionAnalysis tools. IMGT/V-QUEST analyzes germline V and rearranged V-J or V-D-J nucleotide sequences. IMGT/JunctionAnalysis is the first tool that automatically analyzes the complex junctions in detail. These interactive tools are easy to use and freely available on the Web (http://imgt.cines.fr), either separately or integrated. PMID:18432961

  16. HIV-induced T-cell activation/exhaustion in rectal mucosa is controlled only partially by antiretroviral treatment.

    PubMed

    Rueda, Cesar Mauricio; Velilla, Paula Andrea; Chougnet, Claire A; Montoya, Carlos Julio; Rugeles, Maria Teresa

    2012-01-01

    Peripheral blood T-cells from untreated HIV-1-infected patients exhibit reduced immune responses, usually associated with a hyperactivated/exhausted phenotype compared to HAART treated patients. However, it is not clear whether HAART ameliorates this altered phenotype of T-cells in the gastrointestinal-associated lymphoid tissue (GALT), the main site for viral replication. Here, we compared T-cells from peripheral blood and GALT of two groups of chronically HIV-1-infected patients: untreated patients with active viral replication, and patients on suppressive HAART. We characterized the T-cell phenotype by measuring PD-1, CTLA-4, HLA-DR, CD25, Foxp3 and granzyme A expression by flow cytometry; mRNA expression of T-bet, GATA-3, ROR-γt and Foxp3, and was also evaluated in peripheral blood mononuclear cells and rectal lymphoid cells. In HIV-1+ patients, the frequency of PD-1(+) and CTLA-4(+) T-cells (both CD4+ and CD8+ T cells) was higher in the GALT than in the blood. The expression of PD-1 by T-cells from GALT was higher in HIV-1-infected subjects with active viral replication compared to controls. Moreover, the expression per cell of PD-1 and CTLA-4 in CD4(+) T-cells from blood and GALT was positively correlated with viral load. HAART treatment decreased the expression of CTLA-4 in CD8(+) T cells from blood and GALT to levels similar as those observed in controls. Frequency of Granzyme A(+) CD8(+) T-cells in both tissues was low in the untreated group, compared to controls and HAART-treated patients. Finally, a switch towards Treg polarization was found in untreated patients, in both tissues. Together, these findings suggest that chronic HIV-1 infection results in an activated/exhausted T-cell phenotype, despite T-cell polarization towards a regulatory profile; these alterations are more pronounced in the GALT compared to peripheral blood, and are only partiality modulated by HAART. PMID:22276176

  17. On the logic of restrictive recognition of peptide by the T-cell antigen receptor

    PubMed Central

    2011-01-01

    This essay provides an analysis of the inadequacy of the current view of restrictive recognition of peptide by the T-cell antigen receptor. A competing model is developed, and the experimental evidence for the prevailing model is reinterpreted in the new framework. The goal is to contrast the two models with respect to their consistency, coverage of the data, explanatory power, and predictability. PMID:20931295

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

    PubMed Central

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

    2015-01-01

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

  19. Interplay between T Cell Receptor Binding Kinetics and the Level of Cognate Peptide Presented by Major Histocompatibility Complexes Governs CD8+ T Cell Responsiveness*

    PubMed Central

    Irving, Melita; Zoete, Vincent; Hebeisen, Michael; Schmid, Daphné; Baumgartner, Petra; Guillaume, Philippe; Romero, Pedro; Speiser, Daniel; Luescher, Immanuel; Rufer, Nathalie; Michielin, Olivier

    2012-01-01

    Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1157–165-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8+ T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8+ T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca2+ flux for CD8+ T cells expressing TCR within a dissociation constant (KD) range of ∼1–5 μm. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with KD < ∼1 μm, irrespective of CD8 co-engagement and of half-life (t1/2 = ln 2/koff) values. With increased peptide concentration, however, the activity levels of CD8+ T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8+ T cells, as well as for peptide vaccination strategies. PMID:22549784

  20. 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

  1. The common γ-chain cytokine receptor: tricks-and-treats for T cells.

    PubMed

    Waickman, Adam T; Park, Joo-Young; Park, Jung-Hyun

    2016-01-01

    Originally identified as the third subunit of the high-affinity IL-2 receptor complex, the common γ-chain (γc) also acts as a non-redundant receptor subunit for a series of other cytokines, collectively known as γc family cytokines. γc plays essential roles in T cell development and differentiation, so that understanding the molecular basis of its signaling and regulation is a critical issue in T cell immunology. Unlike most other cytokine receptors, γc is thought to be constitutively expressed and limited in its function to the assembly of high-affinity cytokine receptors. Surprisingly, recent studies reported a series of findings that unseat γc as a simple housekeeping gene, and unveiled γc as a new regulatory molecule in T cell activation and differentiation. Cytokine-independent binding of γc to other cytokine receptor subunits suggested a pre-association model of γc with proprietary cytokine receptors. Also, identification of a γc splice isoform revealed expression of soluble γc proteins (sγc). sγc directly interacted with surface IL-2Rβ to suppress IL-2 signaling and to promote pro-inflammatory Th17 cell differentiation. As a result, endogenously produced sγc exacerbated autoimmune inflammatory disease, while the removal of endogenous sγc significantly ameliorated disease outcome. These data provide new insights into the role of both membrane and soluble γc in cytokine signaling, and open new venues to interfere and modulate γc signaling during immune activation. These unexpected discoveries further underscore the perspective that γc biology remains largely uncharted territory that invites further exploration. PMID:26468051

  2. Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation

    PubMed Central

    Wu, Qiong; Pesenacker, Anne M; Stansfield, Alka; King, Douglas; Barge, Dawn; Foster, Helen E; Abinun, Mario; Wedderburn, Lucy R

    2014-01-01

    Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T-cell-depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T-cell receptor (TCR) diversity by analysis of the β variable region (TCRVb) complementarity determining region-3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11·5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8+ TCRVb repertoire was highly oligoclonal early in immune reconstitution and re-emergence of pre-transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re-emergence of pre-ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T-cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis. PMID:24405357

  3. Immunological characteristics and T-cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T-cell-depleted autologous stem cell transplantation.

    PubMed

    Wu, Qiong; Pesenacker, Anne M; Stansfield, Alka; King, Douglas; Barge, Dawn; Foster, Helen E; Abinun, Mario; Wedderburn, Lucy R

    2014-06-01

    Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T-cell-depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T-cell receptor (TCR) diversity by analysis of the β variable region (TCRVb) complementarity determining region-3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11.5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8(+) TCRVb repertoire was highly oligoclonal early in immune reconstitution and re-emergence of pre-transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re-emergence of pre-ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T-cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis. PMID:24405357

  4. Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly.

    PubMed

    Zalli, A; Bosch, J A; Goodyear, O; Riddell, N; McGettrick, H M; Moss, P; Wallace, G R

    2015-03-01

    It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells. PMID:25526818

  5. 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. PMID:26968708

  6. CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia

    PubMed Central

    Maude, Shannon L.; Teachey, David T.; Porter, David L.

    2015-01-01

    Relapsed and refractory acute lymphoblastic leukemia (ALL) remains difficult to treat, with minimal improvement in outcomes seen in more than 2 decades despite advances in upfront therapy and improved survival for de novo ALL. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a powerful targeted immunotherapy, showing striking responses in highly refractory populations. Complete remission (CR) rates as high as 90% have been reported in children and adults with relapsed and refractory ALL treated with CAR-modified T cells targeting the B-cell–specific antigen CD19. Distinct CAR designs across several studies have produced similar promising CR rates, an encouraging finding. Even more encouraging are durable remissions observed in some patients without additional therapy. Duration of remission and CAR-modified T-cell persistence require further study and more mature follow-up, but emerging data suggest these factors may distinguish CAR designs. Supraphysiologic T-cell proliferation, a hallmark of this therapy, contributes to both efficacy and the most notable toxicity, cytokine release syndrome (CRS), posing a unique challenge for toxicity management. This review will discuss the current landscape of CD19 CAR clinical trials, CRS pathophysiology and management, and remaining challenges. PMID:25999455

  7. Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

    PubMed

    Cao, Yu; Rodgers, David T; Du, Juanjuan; Ahmad, Insha; Hampton, Eric N; Ma, Jennifer S Y; Mazagova, Magdalena; Choi, Sei-Hyun; Yun, Hwa Young; Xiao, Han; Yang, Pengyu; Luo, Xiaozhou; Lim, Reyna K V; Pugh, Holly M; Wang, Feng; Kazane, Stephanie A; Wright, Timothy M; Kim, Chan Hyuk; Schultz, Peter G; Young, Travis S

    2016-06-20

    Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response. PMID:27145250

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

    PubMed Central

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

    2016-01-01

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

  9. Hyperresponse to T-cell receptor signaling and apoptosis of Id1 transgenic thymocytes.

    PubMed

    Qi, Zengbiao; Sun, Xiao-Hong

    2004-09-01

    The basic helix-loop-helix transcription factors, E2A and HEB, play important roles in T-cell development at multiple checkpoints. Expression of their inhibitor, Id1, abolishes the function of both transcription factors in a dose-dependent manner. The Id1 transgenic thymus is characterized by an accumulation of CD4- CD8- CD44+ CD25- thymocytes, a dramatic reduction of CD4+ CD8+ thymocytes, and an abundance of apoptotic cells. Here we show that these apoptotic cells carry functional T-cell receptors (TCRs), suggesting that apoptosis occurs during T-cell maturation. In contrast, viable Id1 transgenic CD4 single positive T cells exhibit costimulation-independent proliferation upon treatment with anti-CD3 antibody, probably due to a hyperresponse to TCR signaling. Furthermore, Id1 expression causes apoptosis of CD4 and CD8 double- or single-positive thymocytes in HY- or AND-TCR transgenic mice under conditions that normally support positive selection. Collectively, these results suggest that E2A and HEB proteins are crucial for controlling the threshold for TCR signaling, and Id1 expression lowers the threshold, resulting in apoptosis of developing thymocytes. PMID:15314144

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

    PubMed

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

    2016-06-01

    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

  11. αβ T-cell receptor bias in disease and therapy (Review).

    PubMed

    Wang, Chun-Yan; Yu, Pei-Fa; He, Xiao-Bing; Fang, Yong-Xiang; Cheng, Wen-Yu; Jing, Zhi-Zhong

    2016-06-01

    The diversity and specificity of T cell receptors (TCR), the characteristics of T-cell surface marker, are central to the adaptive immunity. TCR variability is required for successful immunization coverage because this structural foundation is indispensable for the valid identification of short antigen peptides (derived from degraded antigens) that are presented by major histocompatibility molecules on the surfaces of antigen-presenting cells. Despite the vast T-cell repertoire, biased αβ TCR has become a common theme in immunology. To date, numerous examples of TCR bias have been observed in various diseases. Immunotherapy strategies that are based on αβ T cell responses are also emerged as a prominent component of clinical treatment. In the present review, we briefly summarize the current knowledge regarding basic structural information and the molecular mechanisms underlying TCR diversity. Moreover, we outline the role of TCR repertoire bias in some diseases, and its application for therapeutic interventions, as these play significant roles in disease progression, even with patients with a good prognosis. PMID:27098221

  12. 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

  13. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

    PubMed

    Beringer, Dennis X; Kleijwegt, Fleur S; Wiede, Florian; van der Slik, Arno R; Loh, Khai Lee; Petersen, Jan; Dudek, Nadine L; Duinkerken, Gaby; Laban, Sandra; Joosten, Antoinette; Vivian, Julian P; Chen, Zhenjun; Uldrich, Adam P; Godfrey, Dale I; McCluskey, James; Price, David A; Radford, Kristen J; Purcell, Anthony W; Nikolic, Tatjana; Reid, Hugh H; Tiganis, Tony; Roep, Bart O; Rossjohn, Jamie

    2015-11-01

    Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition. PMID:26437244

  14. Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

    PubMed

    Hinz, T; Flindt, S; Marx, A; Janssen, O; Kabelitz, D

    2001-05-01

    The T cell death associated gene 51 (TDAG51) was shown to be required for T cell receptor (TCR)-dependent induction of Fas/Apo1/CD95 expression in a murine T cell hybridoma. Despite the absence of a nuclear localization sequence and a nucleic acid binding domain, it was suggested to be localized in the nucleus and to function as a transcription factor regulating Fas-expression. However, we demonstrate that the human (h)TDAG51 protein is localized in the cytoplasm and the nucleoli, suggesting a role in ribosome biogenesis and/or translation regulation. Indeed, it strongly inhibited translation of a luciferase mRNA in a reticulocyte translational extract. Furthermore, cotransfection of hTDAG51 and the luciferase gene into 293T cells resulted in a strong inhibition of luciferase mRNA translation. Our findings were further strengthened by isolating in a yeast two-hybrid screen three proteins which are involved in the regulation of translation. We speculate that hTDAG51 couples TCR signaling to inhibition of protein biosynthesis in activated T lymphocytes. PMID:11369516

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

    PubMed

    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

    2016-06-21

    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

  16. Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

    PubMed

    Bunders, Madeleine J; van Hamme, John L; Jansen, Machiel H; Boer, Kees; Kootstra, Neeltje A; Kuijpers, Taco W

    2014-01-01

    Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4(+) T cells from HIV-1-exposed children and healthy controls. At baseline CD4(+) T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4(+) T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4(+) T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7(+) CD4(+) T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4(+) T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant. PMID:25341640

  17. 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

  18. Extranodal multiple involvement of enteropathy-type T-cell lymphoma without expression of CC chemokine receptor 7.

    PubMed

    Shiratsuchi, Motoaki; Suehiro, Youko; Yoshikawa, Yasuji; Ohshima, Koichi; Shiokawa, Satoshi; Nishimura, Junji

    2004-01-01

    Enteropathy-type T-cell lymphoma (ETCL) is a rare extranodal lymphoma that tends to disseminate into the intestines and other extranodal organs. We present a case of ETCL with involvement of the lungs and kidneys and report CC chemokine receptor 7 (CCR7) expression of lymphoma cells. A 73-year-old man was admitted to the hospital with a complaint of abdominal pain. Multiple ulcers and perforations were observed in the small intestine, and partial resection of the ileum was performed. Histological examination of the resected specimen revealed diffuse proliferation of atypical large lymphoid cells. The diagnosis was ETCL with dissemination into the lungs and kidney. Lymphoma cells of the small intestine and in pleural effusion were CD3+, CD4+, CD7+, CD8-, CD25-, CD56-, CD103 +/-, and TIA-1+. Rearrangement of the T-cell receptor beta gene was detected, and human T-lymphotropic virus was not integrated. Combination chemotherapy did not result in a sustained response. The results for CCR7 expression of lymphoma cells in the lung and pleural effusion were negative. Therefore we concluded that lymphoma cells did not migrate into the lymph nodes but instead spread into the extranodal organs. PMID:14979477

  19. Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

    PubMed Central

    Krantic, S; Enjalbert, A; Rabourdin-Combe, C

    1997-01-01

    The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM

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

    PubMed

    Hiyane, M I; Boscardin, S B; Rodrigues, M M

    2006-03-01

    Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vbeta genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable beta chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice. PMID:16501814

  1. Synthetic autoantigens of immunoglobulins and T-cell receptors: their recognition in aging, infection, and autoimmunity.

    PubMed

    Marchalonis, J J; Schluter, S F; Wang, E; Dehghanpisheh, K; Lake, D; Yocum, D E; Edmundson, A B; Winfield, J B

    1994-11-01

    Immunoglobulins and their close relatives, the antigen-specific T-cell receptors, are recognition proteins that express structures which readily serve as self-immunogens. Healthy humans can produce antibodies against variable region-defined recognition structures termed idiotypes, as well as against constant region structures, and the levels of these can increase markedly in autoimmune disease; e.g., rheumatoid factors are autoantibodies directed against a conformational determinant of the gamma heavy chain. More recent analyses employing synthetic peptide technologies and construction of recombinant T-cell receptors document that autoantibodies directed against both variable and constant region markers of the alpha/beta T-cell receptor occur in healthy individuals. Alterations in levels of antibody, usage of IgM or IgG isotypes, and specificity for particular peptide-defined regions vary with natural physiological processes (aging, pregnancy), with artificial allografting, with retroviral infection, and with the inception and progression of autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus). Two of the major autoimmunogeneic regions of the Tcr alpha/beta are "constitutive" markers inasmuch as all individuals tested produce antibodies against these regions. The most frequently observed autoantibodies are against Tcr V beta CDR1 and Fr3 markers. It is hypothesized that these are normally involved in immunoregulation. Autoantibodies usually are not detected against CDR2 region determinants, or the "private idiotypes" defined by the CDR3 region, or the highly conserved FR4 segment specified by the joining gene segment. However, autoantibodies against the CDR2 of the Tcr alpha chain occur in some SLE patients, and healthy pregnant women produce antibodies against the common peptide determinant expressed by the joining gene and the beginning of the C alpha or C beta domain. Although the precise role of the naturally occurring autoantibodies in

  2. 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

  3. 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

  4. Immunochemical Proof that a Novel Rearranging Gene Encodes the T Cell Receptor δ Subunit

    NASA Astrophysics Data System (ADS)

    Band, Hamid; Hochstenbach, Frans; McLean, Joanne; Hata, Shingo; Krangel, Michael S.; Brenner, Michael B.

    1987-10-01

    The T cell receptor (TCR) δ protein is expressed as part of a heterodimer with TCR γ , in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR δ was produced that binds specifically to the surface of several TCR γ δ cell lines and immunoprecipitates the TCR γ δ as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR δ subunit alone after chain separation. A candidate human TCR δ complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR γ δ cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR δ . This complementary DNA clone thus corresponds to the gene that encodes the TCR δ subunit.

  5. [Rhabdomyosarcoma lysis by T cells expressing a human autoantibody based chimeric receptor targeting the fetal acetylcholine receptors].

    PubMed

    Gattenlöhner, S

    2006-01-01

    Rhabdomyosarcomas (RMSs) are the most frequent malignant soft tissue tumors of childhood. Since even aggressive multimodality treatments including autologous stem cell rescue have failed to improve the < 20 % overall survival rate of children with metastatic RMS, novel treatment approaches are urgently needed. Looking for potential targets for immunotherapies, we identified the gamma subunit of the fetal acetylcholine receptor (fAChR) as a specific and overexpressed membrane antigen in RMS. Additionally we established a duplex RT-PCR with simultaneous amplification of alpha and gamma subunit message of the fAChR and the quantification of both transcripts resulting in alpha/gammaAChR ratio > 1 was 100% sensitive in alveolar and embryonal rhabdomyosarcoma. Since the fAChR was the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle and therefore implies, that the fAChR may be a target for immunotherapeutic strategies, we synthesized a scFv antibody fragment directed against the fAChR and enigineered both a Pseudomonas exotoxin A based immunotoxin as well as a chimeric T cell receptor composed of the antigen-binding domain of the scFv fragment joined to the signaling domain of the T cell receptor zeta chain. The interaction of fAChzeta-transduced T cells with several RMS cell lines but not with fAChR-negative controls induced strong T cell activation, characterized by secretion of high amounts of interferon-gamma. Moreover after co-incubations with RMS cell lines fAChRzeta-transduced T cells as well fAChR specific immunotoxin induced specific receptor-concentration dependent tumor cell lysis. Therefore, fAChRzeta-transduced T cells and the fAChR specific immunotoxin respectively are promising new tools for the immunotherapy of rhabdomyosarcomas and may provide an effective complementary approach to eradicate residual or metastatic RMS cells in patients, since 1. RMS-direceted chemotherapies

  6. Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

    PubMed

    O'Boyle, Graeme; Fox, Christopher R J; Walden, Hannah R; Willet, Joseph D P; Mavin, Emily R; Hine, Dominic W; Palmer, Jeremy M; Barker, Catriona E; Lamb, Christopher A; Ali, Simi; Kirby, John A

    2012-03-20

    The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis. PMID:22392992

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

    PubMed

    Rappl, Gunter; Riet, Tobias; Awerkiew, Sabine; Schmidt, Annette; Hombach, Andreas A; Pfister, Herbert; Abken, Hinrich

    2012-01-01

    Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter. PMID:22292024

  8. Organ transplantation: modulation of T-cell activation pathways initiated by cell surface receptors to suppress graft rejection.

    PubMed

    Weatherly, Kathleen; Braun, Michel Y

    2011-01-01

    T-cell activation depends upon two types of signals: a T-cell-receptor-mediated antigen-specific signal and several non-antigen-specific ones provided by the engagement of costimulatory and/or inhibitory T-cell surface molecules. In clinical transplantation, T-cell costimulatory/inhibitory molecules are involved in determining cytokine production, vascular endothelial cell damage, and induction of transplant rejection. Several of the latest new immunotherapeutic strategies being currently developed to control graft rejection aim at inhibiting alloreactive T-cell function by regulating activating and costimulatory/inhibitory signals to T cells. This article describes the recent development and potential application of these therapies in experimental and pre-clinical transplantation. PMID:20941624

  9. T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

    PubMed

    VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela G M; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

    2015-10-01

    Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

  10. 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

  11. Engrafted maternal T cells in a severe combined immunodeficiency patient express T-cell receptor variable beta segments characterized by a restricted V-D-J junctional diversity.

    PubMed

    Sottini, A; Quiròs-Roldan, E; Notarangelo, L D; Malagoli, A; Primi, D; Imberti, L

    1995-04-15

    To better understand the peculiar functional behavior of engrafted maternal T cells in a severe combined immunodeficiency (SCID) patient, we characterized, at the molecular level, the T-cell repertoire of a SCID child with a high number of engrafted, mature, activated lymphocytes. We found that, although these transplacentally acquired T cells express a random set of T-cell receptor variable beta (TCRBV) segments, the TCRBV transcripts are characterized by an extremely restricted V-D-J junctional diversity. Only a few cDNA clones were dominant among the TCRBV4+, TCRBV6+, and TCRBV20+ populations in engrafted cells, whereas the same TCRBV chains expressed by the mother's lymphocytes had the expected junctional hetero-geneity. Highly diverse and polyclonal junctions were also expressed by maternal cells activated in mixed lymphocyte reaction by Epstein-Barr virus (EBV)-transformed B lymphocytes from the patient, indicating that the strong clonal selection that characterizes the engrafted cells repertoire is probably not due to allorecognition. Furthermore, we report that the repertoire of the transplacentally acquired lymphocytes is dynamic over time and is characterized by waves of expression and contraction of selected clones, expressing different TCRBV segments. These results help to explain some of the abnormal functional behaviors of engrafted maternal cells and raise new questions regarding the mechanisms responsible for the restricted clonal diversity. PMID:7718881

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

    PubMed

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

    2015-11-01

    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

  13. SLAM family receptors and the SLAM-associated protein (SAP) modulate T cell functions

    PubMed Central

    Keszei, Marton; Romero, Xavier; Tsokos, George C.

    2010-01-01

    One or more of the signaling lymphocytic activation molecule (SLAM) family (SLAMF) of cell surface receptors, which consists of nine transmembrane proteins, i.e., SLAMF1-9, are expressed on most hematopoietic cells. While most SLAMF receptors serve as self-ligands, SLAMF2 and SLAMF4 use each other as counter structures. Six of the receptors carry one or more copies of a unique intracellular tyrosine-based switch motif, which has high affinity for the single SH2-domain signaling molecules SLAM-associated protein and EAT-2. Whereas SLAMF receptors are costimulatory molecules on the surface of CD4+, CD8+, and natural killer (NK) T cells, they also involved in early phases of lineage commitment during hematopoiesis. SLAMF receptors regulate T lymphocyte development and function and modulate lytic activity, cytokine production, and major histocompatibility complex-independent cell inhibition of NK cells. Furthermore, they modulate B cell activation and memory generation, neutrophil, dendritic cell, macrophage and eosinophil function, and platelet aggregation. In this review, we will discuss the role of SLAM receptors and their adapters in Tcell function, and we will examine the role of these receptors and their adapters in X-linked lymphoproliferative disease and their contribution to disease susceptibility in systemic lupus erythematosus. PMID:20146065

  14. 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. PMID:24231368

  15. Characterization of human platelet binding of recombinant T cell receptor ligand

    PubMed Central

    2010-01-01

    Background Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. Methods We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. Results Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. Conclusions Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation. PMID:21059245

  16. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity.

    PubMed

    Ayres, Cory M; Scott, Daniel R; Corcelli, Steven A; Baker, Brian M

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  17. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity

    PubMed Central

    Ayres, Cory M.; Scott, Daniel R.; Corcelli, Steven A.; Baker, Brian M.

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

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

    PubMed Central

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

    2016-01-01

    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. PMID:26999600

  19. T-cell receptor gene homologs are present in the most primitive jawed vertebrates.

    PubMed Central

    Rast, J P; Litman, G W

    1994-01-01

    The phylogenetic origins of T-cell immunity and T-cell antigen receptor (TCR) genes have not been established. A PCR approach using short, minimally degenerate oligodeoxynucleotide primers complementing conserved variable region segments amplifies TCR-like products from the genomic DNA of Heterodontus francisci (horned shark), a representative phylogenetically primitive cartilaginous fish. One of these products has been used as a probe to screen a Heterodontus spleen cDNA library and a clone was identified that is most related at the nucleotide sequence and predicted peptide levels to higher vertebrate TCR beta-chain genes. Genomic analyses of the TCR homologs indicate that recombining variable and joining region segments as well as constant region exons are encoded by extensive gene families, organized in the multicluster form, characteristic of both the immunoglobulin heavy- and light-chain gene loci in the cartilaginous fishes. Greater numbers of homologous products were identified when a probe complementing the putative constant region of the TCR homolog was used to screen the same cDNA library. A high degree of intergenic variation is associated with the putative variable region segments of these isolates. Direct evidence is presented for TCR-like genes, which presumably are associated with T-cell function, at the earliest stages in the phylogenetic emergence of jawed vertebrates. Images PMID:7937749

  20. 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

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

    PubMed

    Benati, Daniela; Galperin, Moran; Lambotte, Olivier; Gras, Stéphanie; Lim, Annick; Mukhopadhyay, Madhura; Nouël, Alexandre; 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

    2016-06-01

    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

  2. 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. PMID:26253731

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

    PubMed

    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

  4. 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

  5. NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist.

    PubMed

    Dammermann, Werner; Zhang, Bo; Nebel, Merle; Cordiglieri, Chiara; Odoardi, Francesca; Kirchberger, Tanja; Kawakami, Naoto; Dowden, James; Schmid, Frederike; Dornmair, Klaus; Hohenegger, Martin; Flügel, Alexander; Guse, Andreas H; Potter, Barry V L

    2009-06-30

    The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca(2+) signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca(2+) entry. BZ194 specifically and effectively blocked NAADP-stimulated [(3)H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca(2+) mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca(2+) mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4(+) effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases. PMID:19541638

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

    PubMed Central

    Fujiwara, Hiroshi

    2014-01-01

    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. PMID:25517545

  7. T-Cell Receptor-Engineered Cells for the Treatment of Hematologic Malignancies.

    PubMed

    Hossain, Nasheed M; Chapuis, Aude G; Walter, Roland B

    2016-08-01

    Recent attention in adoptive immunotherapy for hematologic malignancies has focused on lymphocytes expressing chimeric antigen receptors. An alternative technique to redirect the immune system toward cancer cells involves the use of T-cells carrying an engineered tumor-recognizing T-cell receptor (TCR). This approach allows targeting of surface or intracellular/nuclear proteins as long as they are processed and presented on the cell surface by human leukocyte antigen molecules. Several trials in advanced solid tumors, particularly melanoma and synovial sarcoma, support the validity of this strategy, although tumor responses have often been short-lived. Emerging data from patients with multiple myeloma and myeloid neoplasms suggest that the benefit of TCR-modified cells may extend to blood cancers. Methodological refinements may be necessary to increase the in vivo persistence and functionality of these cells. Particularly with affinity-enhanced TCRs, however, more effective therapies may increase the potential for serious toxicity due to the unexpected on- or off-target reactivity. PMID:27095318

  8. Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.

    PubMed Central

    Grégoire, C; Lin, S Y; Mazza, G; Rebai, N; Luescher, I F; Malissen, B

    1996-01-01

    We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels. Images Fig. 3 Fig. 4 PMID:8692966

  9. Sequence variation and linkage disequilibrium in the human T-cell receptor beta (TCRB) locus.

    PubMed

    Subrahmanyan, L; Eberle, M A; Clark, A G; Kruglyak, L; Nickerson, D A

    2001-08-01

    The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association. PMID:11438886

  10. Sequence Variation and Linkage Disequilibrium in the Human T-Cell Receptor β (TCRB) Locus

    PubMed Central

    Subrahmanyan, Lakshman; Eberle, Michael A.; Clark, Andrew G.; Kruglyak, Leonid; Nickerson, Deborah A.

    2001-01-01

    The T-cell receptor (TCR) plays a central role in the immune system, and >90% of human T cells present a receptor that consists of the α TCR subunit (TCRA) and the β subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations—African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r2>0.1) over distances of ∼30 kb in TCRB, and, in general, the distribution of r2 as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in “blocks” of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association. PMID:11438886