Sample records for t-cell receptor threshold

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Manz, Boryana N.; Jackson, Bryan L.; Petit, Rebecca S.

    2011-05-31

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

  2. T Cell Activation Thresholds are Affected by Gravitational

    NASA Technical Reports Server (NTRS)

    Adams, Charley; Gonzalez, M.; Nelman-Gonzalez, M.

    1999-01-01

    T cells stimulated in space flight by various mitogenic signals show a dramatic reduction in proliferation and expression of early activation markers. Similar results are also obtained in a ground based model of microgravity, clinorotation, which provides a vector-averaged reduction of the apparent gravity on cells without significant shear force. Here we demonstrate that T cell inhibition is due to an increase in the required threshold for activation. Dose response curves indicate that cells activated during clinorotation require higher stimulation to achieve the same level of activation, as measured by CD69 expression. Interleukin 2 receptor expression, and DNA synthesis. The amount of stimulation necessary for 50% activation is 5 fold in the clinostat relative to static. Correlation of TCR internalization with activation also exhibit a dramatic right shift in clinorotation, demonstrating unequivocally that signal transduction mechanism independent of TCR triggering account for the increased activation threshold. Previous results from space flight experiments are consistent with the dose response curves obtained for clinorotation. Activation thresholds are important aspects of T cell memory, autoimmunity and tolerance Clinorotation is a useful, noninvasive tool for the study of cellular and biochemical event regulating T cell activation threshold and the effects of gravitation forces on these systems.

  3. Mathematical Model of Naive T Cell Division and Survival IL-7 Thresholds.

    PubMed

    Reynolds, Joseph; Coles, Mark; Lythe, Grant; Molina-París, Carmen

    2013-01-01

    We develop a mathematical model of the peripheral naive T cell population to study the change in human naive T cell numbers from birth to adulthood, incorporating thymic output and the availability of interleukin-7 (IL-7). The model is formulated as three ordinary differential equations: two describe T cell numbers, in a resting state and progressing through the cell cycle. The third is introduced to describe changes in IL-7 availability. Thymic output is a decreasing function of time, representative of the thymic atrophy observed in aging humans. Each T cell is assumed to possess two interleukin-7 receptor (IL-7R) signaling thresholds: a survival threshold and a second, higher, proliferation threshold. If the IL-7R signaling strength is below its survival threshold, a cell may undergo apoptosis. When the signaling strength is above the survival threshold, but below the proliferation threshold, the cell survives but does not divide. Signaling strength above the proliferation threshold enables entry into cell cycle. Assuming that individual cell thresholds are log-normally distributed, we derive population-average rates for apoptosis and entry into cell cycle. We have analyzed the adiabatic change in homeostasis as thymic output decreases. With a parameter set representative of a healthy individual, the model predicts a unique equilibrium number of T cells. In a parameter range representative of persistent viral or bacterial infection, where naive T cell cycle progression is impaired, a decrease in thymic output may result in the collapse of the naive T cell repertoire.

  4. Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

    PubMed

    Cai, Haogang; Muller, James; Depoil, David; Mayya, Viveka; Sheetz, Michael P; Dustin, Michael L; Wind, Shalom J

    2018-04-30

    Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

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

    PubMed Central

    2015-01-01

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

  6. ERK-dependent T cell receptor threshold calibration in rheumatoid arthritis.

    PubMed

    Singh, Karnail; Deshpande, Pratima; Pryshchep, Sergey; Colmegna, Inés; Liarski, Vladimir; Weyand, Cornelia M; Goronzy, Jörg J

    2009-12-15

    Immune responses to citrullinated neoantigens and clinical efficacy of costimulation blockade indicate a general defect in maintaining T cell tolerance in rheumatoid arthritis (RA). To examine whether TCR threshold calibration contributes to disease pathogenesis, signaling in RA T cells was quantified. RA patients had a selective increase in ERK phosphorylation compared with demographically matched controls due to a mechanism distal of Ras activation. Increased ERK responses included naive and memory CD4 and CD8 T cells and did not correlate with disease activity. The augmented ERK activity delayed SHP-1 recruitment to the TCR synapse and sustained TCR-induced Zap70 and NF-kappaB signaling, facilitating responses to suboptimal stimulation. Increased responsiveness of the ERK pathway was also a characteristic finding in the SKG mouse model of RA where it preceded clinical symptoms. Treatment with subtherapeutic doses of a MEK-1/2 inhibitor delayed arthritis onset and reduced severity, suggesting that increased ERK phosphorylation predisposes for autoimmunity and can be targeted to prevent disease.

  7. Antigen-Specific T-Cell Activation Independently of the MHC: Chimeric Antigen Receptor-Redirected T Cells

    PubMed Central

    Chmielewski, Markus; Hombach, Andreas A.; Abken, Hinrich

    2013-01-01

    Adoptive T-cell therapy has recently shown promise in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T-cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC) expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient’s T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR) which consists in the extracellular part of an antibody-derived domain for binding with a “tumor-associated antigen” and in the intracellular part of a T-cell receptor (TCR)-derived signaling moiety for T-cell activation. The specificity of CAR-mediated T-cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T-cell targeting by an engineered CAR in comparison to TCR modified T cells and the impact of the CAR activation threshold on redirected T-cell activation. Finally we review most significant progress recently made in early stage clinical trials to treat cancer. PMID:24273543

  8. Antigen-Specific T-Cell Activation Independently of the MHC: Chimeric Antigen Receptor-Redirected T Cells.

    PubMed

    Chmielewski, Markus; Hombach, Andreas A; Abken, Hinrich

    2013-01-01

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

  9. T cell costimulation by chemokine receptors.

    PubMed

    Molon, Barbara; Gri, Giorgia; Bettella, Monica; Gómez-Moutón, Concepción; Lanzavecchia, Antonio; Martínez-A, Carlos; Mañes, Santos; Viola, Antonella

    2005-05-01

    Signals mediated by chemokine receptors may compete with T cell receptor stop signals and determine the duration of T cell-antigen-presenting cell interactions. Here we show that during T cell stimulation by antigen-presenting cells, T cell chemokine receptors coupled to G(q) and/or G(11) protein were recruited to the immunological synapse by a G(i)-independent mechanism. When chemokine receptors were sequestered at the immunological synapse, T cells became insensitive to chemotactic gradients, formed more stable conjugates and finally responded with enhanced proliferation and cytokine production. We suggest that chemokine receptor trapping at the immunological synapse enhances T cell activation by improving T cell-antigen-presenting cell attraction and impeding the 'distraction' of successfully engaged T cells by other chemokine sources.

  10. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    PubMed

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-05-01

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  11. Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

    PubMed

    Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

    2010-04-07

    Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

  12. Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

    PubMed Central

    Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

    2010-01-01

    Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. PMID:20150895

  13. Expression of NK cell receptors on decidual T cells in human pregnancy.

    PubMed

    Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

    2009-06-01

    Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

  14. T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

    PubMed

    Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

    2014-05-01

    Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

  15. Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen

    PubMed Central

    Rafiq, S; Purdon, TJ; Daniyan, AF; Koneru, M; Dao, T; Liu, C; Scheinberg, DA; Brentjens, RJ

    2017-01-01

    CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy. PMID:27924074

  16. 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

  17. Tumor-targeting domains for chimeric antigen receptor T cells.

    PubMed

    Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

    2017-01-01

    Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

  18. Expression of activating natural killer-cell receptors is a hallmark of the innate-like T-cell neoplasm in peripheral T-cell lymphomas.

    PubMed

    Uemura, Yu; Isobe, Yasushi; Uchida, Akiko; Asano, Junko; Nishio, Yuji; Sakai, Hirotaka; Hoshikawa, Masahiro; Takagi, Masayuki; Nakamura, Naoya; Miura, Ikuo

    2018-04-01

    Peripheral T- or natural killer (NK)-cell lymphomas are rare and difficult-to-recognize diseases. It remains arduous to distinguish between NK cell- and cytotoxic T-lymphocyte-derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK-cell receptors and examined the expression using immunohistochemistry in 22 cases with T- and NK-cell neoplasms comprising angioimmunoblastic T-cell lymphoma, anaplastic lymphoma kinase (ALK)-positive and -negative anaplastic large-cell lymphomas, extranodal NK/T-cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T-cell lymphoma, aggressive NK-cell leukemia, and other peripheral T-cell lymphomas. Inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK-cell receptors were expressed predominantly in TIA-1-positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK-cell neoplasms and cytotoxic T-lymphocyte-derived lymphomas including monomorphic epitheliotropic intestinal T-cell lymphoma. One Epstein-Barr virus-harboring cytotoxic T-lymphocyte-derived lymphoma mimicking extranodal NK/T-cell lymphoma, nasal type lacked these NK-cell receptors, indicating different cell origin from NK and innate-like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log-rank test, P = .024). We propose that the presence of activating NK-cell receptors may provide new insights into understanding peripheral T-cell lymphomas and characterizing them as innate-like T-cell neoplasm. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on

  19. c-MPL provides tumor-targeted T-cell receptor-transgenic T cells with costimulation and cytokine signals.

    PubMed

    Nishimura, Christopher D; Brenner, Daniel A; Mukherjee, Malini; Hirsch, Rachel A; Ott, Leah; Wu, Meng-Fen; Liu, Hao; Dakhova, Olga; Orange, Jordan S; Brenner, Malcolm K; Lin, Charles Y; Arber, Caroline

    2017-12-21

    Adoptively transferred T-cell receptor (TCR)-engineered T cells depend on host-derived costimulation and cytokine signals for their full and sustained activation. However, in patients with cancer, both signals are frequently impaired. Hence, we developed a novel strategy that combines both essential signals in 1 transgene by expressing the nonlymphoid hematopoietic growth factor receptor c-MPL (myeloproliferative leukemia), the receptor for thrombopoietin (TPO), in T cells. c-MPL signaling activates pathways shared with conventional costimulatory and cytokine receptor signaling. Thus, we hypothesized that host-derived TPO, present in the tumor microenvironment, or pharmacological c-MPL agonists approved by the US Food and Drug Administration could deliver both signals to c-MPL-engineered TCR-transgenic T cells. We found that c-MPL + polyclonal T cells expand and proliferate in response to TPO, and persist longer after adoptive transfer in immunodeficient human TPO-transgenic mice. In TCR-transgenic T cells, c-MPL activation enhances antitumor function, T-cell expansion, and cytokine production and preserves a central memory phenotype. c-MPL signaling also enables sequential tumor cell killing, enhances the formation of effective immune synapses, and improves antileukemic activity in vivo in a leukemia xenograft model. We identify the type 1 interferon pathway as a molecular mechanism by which c-MPL mediates immune stimulation in T cells. In conclusion, we present a novel immunotherapeutic strategy using c-MPL-enhanced transgenic T cells responding to either endogenously produced TPO (a microenvironment factor in hematologic malignancies) or c-MPL-targeted pharmacological agents. © 2017 by The American Society of Hematology.

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

    PubMed

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

    2013-04-18

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

  1. T Cell Calcium Signaling Regulation by the Co-Receptor CD5

    PubMed Central

    Freitas, Claudia M. Tellez

    2018-01-01

    Calcium influx is critical for T cell effector function and fate. T cells are activated when T cell receptors (TCRs) engage peptides presented by antigen-presenting cells (APC), causing an increase of intracellular calcium (Ca2+) concentration. Co-receptors stabilize interactions between the TCR and its ligand, the peptide-major histocompatibility complex (pMHC), and enhance Ca2+ signaling and T cell activation. Conversely, some co-receptors can dampen Ca2+ signaling and inhibit T cell activation. Immune checkpoint therapies block inhibitory co-receptors, such as cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed death 1 (PD-1), to increase T cell Ca2+ signaling and promote T cell survival. Similar to CTLA-4 and PD-1, the co-receptor CD5 has been known to act as a negative regulator of T cell activation and to alter Ca2+ signaling and T cell function. Though much is known about the role of CD5 in B cells, recent research has expanded our understanding of CD5 function in T cells. Here we review these recent findings and discuss how our improved understanding of CD5 Ca2+ signaling regulation could be useful for basic and clinical research. PMID:29701673

  2. 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.

  3. Naive T-cell receptor transgenic T cells help memory B cells produce antibody

    PubMed Central

    Duffy, Darragh; Yang, Chun-Ping; Heath, Andrew; Garside, Paul; Bell, Eric B

    2006-01-01

    Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high-affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response – the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen-specific T-cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin-2-deficient mice we showed that interleukin-2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen-primed and naive CD4 T cells. PMID:17067314

  4. How chimeric antigen receptor design affects adoptive T cell therapy

    PubMed Central

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

    2016-01-01

    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. PMID:27163336

  5. Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses.

    PubMed

    Tay, Szun Szun; Wong, Yik Chun; McDonald, David M; Wood, Nicole A W; Roediger, Ben; Sierro, Frederic; Mcguffog, Claire; Alexander, Ian E; Bishop, G Alex; Gamble, Jennifer R; Weninger, Wolfgang; McCaughan, Geoffrey W; Bertolino, Patrick; Bowen, David G

    2014-06-24

    CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

  6. 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. © 2016 Wiley Periodicals, Inc.

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

    PubMed

    Fujiwara, Hiroshi

    2014-02-01

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

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

    PubMed

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

    2017-03-29

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

  9. 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.

  10. T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

    PubMed Central

    Patas, Kostas; Willing, Anne; Demiralay, Cüneyt; Engler, Jan Broder; Lupu, Andreea; Ramien, Caren; Schäfer, Tobias; Gach, Christian; Stumm, Laura; Chan, Kenneth; Vignali, Marissa; Arck, Petra C.; Friese, Manuel A.; Pless, Ole; Wiedemann, Klaus; Agorastos, Agorastos; Gold, Stefan M.

    2018-01-01

    While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD) is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR) repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20), who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20). T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder. PMID:29515587

  11. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma.

    PubMed

    Harrer, Dennis C; Simon, Bianca; Fujii, Shin-Ichiro; Shimizu, Kanako; Uslu, Ugur; Schuler, Gerold; Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

    2017-08-17

    Adoptive T-cell therapy relying on conventional T cells transduced with T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has caused substantial tumor regression in several clinical trials. However, genetically engineered T cells have been associated with serious side-effects due to off-target toxicities and massive cytokine release. To obviate these concerns, we established a protocol adaptable to GMP to expand and transiently transfect γ/δ T cells with mRNA. PBMC from healthy donors were stimulated using zoledronic-acid or OKT3 to expand γ/δ T cells and bulk T cells, respectively. Additionally, CD8 + T cells and γ/δ T cells were MACS-isolated from PBMC and expanded with OKT3. Next, these four populations were electroporated with RNA encoding a gp100/HLA-A2-specific TCR or a CAR specific for MCSP. Thereafter, receptor expression, antigen-specific cytokine secretion, specific cytotoxicity, and killing of the endogenous γ/δ T cell-target Daudi were analyzed. Using zoledronic-acid in average 6 million of γ/δ T cells with a purity of 85% were generated from one million PBMC. MACS-isolation and OKT3-mediated expansion of γ/δ T cells yielded approximately ten times less cells. OKT3-expanded and CD8 + MACS-isolated conventional T cells behaved correspondingly similar. All employed T cells were efficiently transfected with the TCR or the CAR. Upon respective stimulation, γ/δ T cells produced IFNγ and TNF, but little IL-2 and the zoledronic-acid expanded T cells exceeded MACS-γ/δ T cells in antigen-specific cytokine secretion. While the cytokine production of γ/δ T cells was in general lower than that of conventional T cells, specific cytotoxicity against melanoma cell lines was similar. In contrast to OKT3-expanded and MACS-CD8 + T cells, mock-electroporated γ/δ T cells also lysed tumor cells reflecting the γ/δ T cell-intrinsic anti-tumor activity. After transfection, γ/δ T cells were still able to kill MHC-deficient Daudi cells. We

  13. 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

  14. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin.

    PubMed

    Levite, M; Cahalon, L; Hershkoviz, R; Steinman, L; Lider, O

    1998-01-15

    The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the beta1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1-4 N-terminal and its 4-11, 5-11, and 6-11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation.

  15. T-cell receptor signaling activates an ITK/NF-κB/GATA-3 axis in T-cell lymphomas facilitating resistance to chemotherapy

    PubMed Central

    Wang, Tianjiao; Lu, Ye; Polk, Avery; Chowdhury, Pinki; Zamalloa, Carlos Murga; Fujiwara, Hiroshi; Suemori, Koichiro; Beyersdorf, Niklas; Hristov, Alexandra C.; Lim, Megan S.; Bailey, Nathanael G.; Wilcox, Ryan A.

    2016-01-01

    Purpose T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T-cell specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR’s role in mediating resistance to chemotherapy. Experimental Design Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following T-cell receptor (TCR) engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results Here we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3, and promotes chemotherapy resistance. Conclusions These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented activation of this signaling axis and overcame chemotherapy resistance. PMID:27780854

  16. 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. © 2015 by The American Society of Hematology.

  17. Integration of T Cell Receptor, Notch and Cytokine Signals Programs in Mouse γδ T Cell Effector Differentiation.

    PubMed

    Zarin, Payam; In, Tracy S H; Chen, Edward L Y; Singh, Jastaranpreet; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Anderson, Michele K; Zúñiga-Pflücker, Juan Carlos

    2018-05-13

    γδ T-cells perform a wide range of tissue and disease specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon-γ (IFNγ) and interleukin-17 (IL-17) producing γδ T-cells remain unknown. Here, we define the cues involved in the functional programming of γδ T-cells, by examining the roles of T-cell receptor (TCR), Notch, and cytokine-receptor signaling. KN6 γδTCR-transduced Rag2 -/- T-cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL-17 producing γδ T cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL-1β, IL-21 and IL-23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL-17 producing γδ T-cell subsets. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. Chimeric antigen receptor T cells targeting Fc μ receptor selectively eliminate CLL cells while sparing healthy B cells.

    PubMed

    Faitschuk, Elena; Hombach, Andreas A; Frenzel, Lukas P; Wendtner, Clemens-Martin; Abken, Hinrich

    2016-09-29

    Adoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)-modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged "on-target off-tumor" toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcμR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcμR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcμR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcμR CAR T cells did not attack healthy B cells. T cells with anti-FcμR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcμR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcμR CAR T cells for the treatment of CLL. © 2016 by The American Society of Hematology.

  19. The growth threshold conjecture: a theoretical framework for understanding T-cell tolerance.

    PubMed

    Arias, Clemente F; Herrero, Miguel A; Cuesta, José A; Acosta, Francisco J; Fernández-Arias, Cristina

    2015-07-01

    Adaptive immune responses depend on the capacity of T cells to target specific antigens. As similar antigens can be expressed by pathogens and host cells, the question naturally arises of how can T cells discriminate friends from foes. In this work, we suggest that T cells tolerate cells whose proliferation rates remain below a permitted threshold. Our proposal relies on well-established facts about T-cell dynamics during acute infections: T-cell populations are elastic (they expand and contract) and they display inertia (contraction is delayed relative to antigen removal). By modelling inertia and elasticity, we show that tolerance to slow-growing populations can emerge as a population-scale feature of T cells. This result suggests a theoretical framework to understand immune tolerance that goes beyond the self versus non-self dichotomy. It also accounts for currently unexplained observations, such as the paradoxical tolerance to slow-growing pathogens or the presence of self-reactive T cells in the organism.

  20. Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy.

    PubMed

    Brown, Christine E; Alizadeh, Darya; Starr, Renate; Weng, Lihong; Wagner, Jamie R; Naranjo, Araceli; Ostberg, Julie R; Blanchard, M Suzette; Kilpatrick, Julie; Simpson, Jennifer; Kurien, Anita; Priceman, Saul J; Wang, Xiuli; Harshbarger, Todd L; D'Apuzzo, Massimo; Ressler, Julie A; Jensen, Michael C; Barish, Michael E; Chen, Mike; Portnow, Jana; Forman, Stephen J; Badie, Behnam

    2016-12-29

    A patient with recurrent multifocal glioblastoma received chimeric antigen receptor (CAR)-engineered T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2). Multiple infusions of CAR T cells were administered over 220 days through two intracranial delivery routes - infusions into the resected tumor cavity followed by infusions into the ventricular system. Intracranial infusions of IL13Rα2-targeted CAR T cells were not associated with any toxic effects of grade 3 or higher. After CAR T-cell treatment, regression of all intracranial and spinal tumors was observed, along with corresponding increases in levels of cytokines and immune cells in the cerebrospinal fluid. This clinical response continued for 7.5 months after the initiation of CAR T-cell therapy. (Funded by Gateway for Cancer Research and others; ClinicalTrials.gov number, NCT02208362 .).

  1. Chimeric Antigen Receptor T-Cell Therapy for the Community Oncologist

    PubMed Central

    Levine, Bruce L.

    2016-01-01

    The field of cancer immunotherapy has rapidly progressed in the past decade as several therapeutic modalities have entered into the clinic. One such immunotherapy that has shown promise in the treatment of cancer is the use of chimeric antigen receptor (CAR)-modified T lymphocytes. CARs are engineered receptors constructed from antigen recognition regions of antibodies fused to T-cell signaling and costimulatory domains that can be used to reprogram a patient’s T cells to specifically target tumor cells. CAR T-cell therapy has demonstrated sustained complete responses for some patients with advanced leukemia, and a number of CAR therapies are being evaluated in clinical studies. CAR T-cell therapy-associated toxicities, including cytokine release syndrome, macrophage activation syndrome, and tumor lysis syndrome, have been observed and effectively managed in the clinic. In patients with significant clinical responses, sustained B-cell aplasia has also been observed and is a marker of CAR T-cell persistence that might provide long-term disease control. Education on CAR T-cell therapy efficacy and safety management is critical for clinicians and patients who are considering this novel type of treatment. In the present report, the current landscape of CAR T-cell therapy, the effective management of patients undergoing treatment, and which patients are the most suitable candidates for current trials are discussed. Implications for Practice: The present report describes the current status of chimeric antigen receptor (CAR) T lymphocytes as an immunotherapy for patients with relapsed or refractory B-cell malignancies. CAR T cells targeting CD19, a protein expressed on many B-cell malignancies, typically induce high complete response rates in patients with B-cell leukemia or lymphoma who have very limited therapeutic options. Recent clinical trial results of CD19 CAR T-cell therapies and the management of CAR T-cell-associated adverse events are discussed. The present

  2. HIV Envelope gp120 Alters T Cell Receptor Mobilization in the Immunological Synapse of Uninfected CD4 T Cells and Augments T Cell Activation

    PubMed Central

    Deng, Jing; Mitsuki, Yu-ya; Shen, Guomiao; Ray, Jocelyn C.; Cicala, Claudia; Arthos, James; Dustin, Michael L.

    2016-01-01

    ABSTRACT HIV is transmitted most efficiently from cell to cell, and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied to supported lipid bilayers that were reconstituted with HIV Env gp120, anti-T cell receptor (anti-TCR) monoclonal antibody, and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse and affected synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, interleukin-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells but also for boosting the T cell activation state, thereby increasing its likelihood of undergoing productive replication in targeted CD4 T cells. IMPORTANCE There are about two million new HIV infections every year. A better understanding of how HIV is transmitted to susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the

  3. Bayesian multivariate Poisson abundance models for T-cell receptor data.

    PubMed

    Greene, Joshua; Birtwistle, Marc R; Ignatowicz, Leszek; Rempala, Grzegorz A

    2013-06-07

    A major feature of an adaptive immune system is its ability to generate B- and T-cell clones capable of recognizing and neutralizing specific antigens. These clones recognize antigens with the help of the surface molecules, called antigen receptors, acquired individually during the clonal development process. In order to ensure a response to a broad range of antigens, the number of different receptor molecules is extremely large, resulting in a huge clonal diversity of both B- and T-cell receptor populations and making their experimental comparisons statistically challenging. To facilitate such comparisons, we propose a flexible parametric model of multivariate count data and illustrate its use in a simultaneous analysis of multiple antigen receptor populations derived from mammalian T-cells. The model relies on a representation of the observed receptor counts as a multivariate Poisson abundance mixture (m PAM). A Bayesian parameter fitting procedure is proposed, based on the complete posterior likelihood, rather than the conditional one used typically in similar settings. The new procedure is shown to be considerably more efficient than its conditional counterpart (as measured by the Fisher information) in the regions of m PAM parameter space relevant to model T-cell data. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

    PubMed Central

    Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

    2017-01-01

    Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

  5. Genetic engineering with T cell receptors.

    PubMed

    Zhang, Ling; Morgan, Richard A

    2012-06-01

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

  6. Adoptive T-cell immunotherapy of cancer using chimeric antigen receptor-grafted T cells.

    PubMed

    Davies, David Marc; Maher, John

    2010-06-01

    Harnessing the power of the immune system to target cancer has long been a goal of tumor immunologists. One avenue under investigation is the modification of T cells to express a chimeric antigen receptor (CAR). Expression of such a receptor enables T-cell specificity to be redirected against a chosen tumor antigen. Substantial research in this field has been carried out, incorporating a wide variety of malignancies and tumor-associated antigens. Ongoing investigations will ensure this area continues to expand at a rapid pace. This review will explain the evolution of CAR technology over the last two decades in addition to detailing the associated benefits and disadvantages. The outcome of recent phase I clinical trials and the impact that these have had upon the direction of future research in this field will also be addressed.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. T-cell receptor revision: friend or foe?

    PubMed Central

    Hale, J Scott; Fink, Pamela J

    2010-01-01

    T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue. PMID:20201984

  9. T-cell receptor revision: friend or foe?

    PubMed

    Hale, J Scott; Fink, Pamela J

    2010-04-01

    T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.

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

    PubMed

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

    2017-04-01

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

  11. Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel

    PubMed Central

    Irving, Melita; Vuillefroy de Silly, Romain; Scholten, Kirsten; Dilek, Nahzli; Coukos, George

    2017-01-01

    T-cells play a critical role in tumor immunity. Indeed, the presence of tumor-infiltrating lymphocytes is a predictor of favorable patient prognosis for many indications and is a requirement for responsiveness to immune checkpoint blockade therapy targeting programmed cell death 1. For tumors lacking immune infiltrate, or for which antigen processing and/or presentation has been downregulated, a promising immunotherapeutic approach is chimeric antigen receptor (CAR) T-cell therapy. CARs are hybrid receptors that link the tumor antigen specificity and affinity of an antibody-derived single-chain variable fragment with signaling endodomains associated with T-cell activation. CAR therapy targeting CD19 has yielded extraordinary clinical responses against some hematological tumors. Solid tumors, however, remain an important challenge to CAR T-cells due to issues of homing, tumor vasculature and stromal barriers, and a range of obstacles in the tumor bed. Protumoral immune infiltrate including T regulatory cells and myeloid-derived suppressor cells have been well characterized for their ability to upregulate inhibitory receptors and molecules that hinder effector T-cells. A critical role for metabolic barriers in the tumor microenvironment (TME) is emerging. High glucose consumption and competition for key amino acids by tumor cells can leave T-cells with insufficient energy and biosynthetic precursors to support activities such as cytokine secretion and lead to a phenotypic state of anergy or exhaustion. CAR T-cell expansion protocols that promote a less differentiated phenotype, combined with optimal receptor design and coengineering strategies, along with immunomodulatory therapies that also promote endogenous immunity, offer great promise in surmounting immunometabolic barriers in the TME and curing solid tumors. PMID:28421069

  12. Premature cell senescence and T cell receptor-independent activation of CD8T cells in Juvenile Idiopathic Arthritis*

    PubMed Central

    Dvergsten, Jeffrey A.; Mueller, Robert G.; Griffin, Patricia; Abedin, Sameem; Pishko, Allyson; Michel, Joshua J.; Rosenkranz, Margalit E.; Reed, Ann M.; Kietz, Daniel A.; Vallejo, Abbe N.

    2013-01-01

    Objectives CD8T cells lacking CD28 were originally reported by Wedderburn and colleagues as a characteristic feature of JIA, but the relevance of these unusual cells to JIA remains to be elucidated. Because of recent evidence that CD28 loss is typical of terminally differentiated lymphocytes, we examined for functional subsets of CD8T cells in JIA. Methods Following informed consent/assent, blood and/or waste synovial fluid were collected from children with definite diagnosis of JIA (n = 98). De-identified blood (n = 33) and cord blood (n = 13) samples from healthy donors were also collected. CD8T and CD4T cells were screened for novel receptors, and where indicated, bioassays were performed to determine functional relevance of the identified receptor. Results Patients had a naïve T cell compartment with shortened telomeres, and their entire T cell pool had reduced proliferative capacity. They had an over abundance of CD31+CD28null CD8T cells, which was a significant feature of oligoarticular JIA (n = 62) compared to polyarticular JIA (n = 36). CD31+CD28null CD8T cells had limited mitotic capacity, and expressed high levels of the senescence antigens γH2Ax and/or p16. Ligation of CD31, independent of the TCR, sufficiently induced tyrosine phosphorylation, vesicle exocytosis, and production of IFN-γ and IL-10. Conclusion These data provide the first evidence for cell senescence, represented by CD31+CD28null CD8T cells, in the pathophysiology of JIA. Activation of these unusual cells in a TCR-independent manner suggests they are maladaptive, and could be potential targets for immunotherapy. PMID:23686519

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

    PubMed Central

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

    2015-01-01

    The mammalian T cell receptor (TCR) orchestrates immunity by responding to many billions of different ligands that it has never encountered before and cannot adapt to at the protein sequence level. This remarkable receptor exists in two main heterodimeric isoforms: αβ TCR and γδ TCR. The αβ TCR is expressed on the majority of peripheral T cells. Most αβ T cells recognize peptides, derived from degraded proteins, presented at the cell surface in molecular cradles called major histocompatibility complex (MHC) molecules. Recent reports have described other αβ T cell subsets. These ‘unconventional’ T cells bear TCRs that are capable of recognizing lipid ligands presented in the context of the MHC-like CD1 protein family or bacterial metabolites bound to the MHC-related protein 1 (MR1). γδ T cells constitute a minority of the T cell pool in human blood, but can represent up to half of total T cells in tissues such as the gut and skin. The identity of the preferred ligands for γδ T cells remains obscure, but it is now known that this receptor can also functionally engage CD1-lipid, or immunoglobulin (Ig) superfamily proteins called butyrophilins in the presence of pyrophosphate intermediates of bacterial lipid biosynthesis. Interactions between TCRs and these ligands allow the host to discriminate between self and non-self and co-ordinate an attack on the latter. Here, we describe how cells of the T lymphocyte lineage and their antigen receptors are generated and discuss the various modes of antigen recognition by these extraordinarily versatile receptors. PMID:25753381

  14. 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

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

    PubMed

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

    2015-12-01

    The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion. ©2015 American Association for Cancer Research.

  16. T-cell receptor gene rearrangement in Epstein-Barr virus infectious mononucleosis.

    PubMed

    Marbello, L; Riva, M; Veronese, S; Nosari, A M; Ravano, E; Colosimo, A; Paris, L; Morra, E

    2012-09-01

    This report describes the case of a previously healthy young man who presented with fever, pharyngitis, cervical lymphadenopathy, lymphocytosis, and severe thrombocytopenia. Serological tests for Epstein-Barr virus were diagnostic of a primary Epstein-Barr virus infectious mononucleosis but severe thrombocytopenia aroused the suspicion of a lymphoproliferative disease. T-cell receptor gene analysis performed on peripheral and bone marrow blood revealed a T-cell receptor γ-chain rearrangement without the evidence of malignancy using standard histologic and immunophenotype studies. Signs and symptoms of the infectious disease, blood count, and T-cell receptor gene rearrangement resolved with observation without the evidence of emergence of a lymphoproliferative disease. In the contest of a suspected lymphoproliferative disease, molecular results should be integrated with all available data for an appropriate diagnosis.

  17. T-cell Receptor Signaling Activates an ITK/NF-κB/GATA-3 axis in T-cell Lymphomas Facilitating Resistance to Chemotherapy.

    PubMed

    Wang, Tianjiao; Lu, Ye; Polk, Avery; Chowdhury, Pinki; Zamalloa, Carlos Murga; Fujiwara, Hiroshi; Suemori, Koichiro; Beyersdorf, Niklas; Hristov, Alexandra C; Lim, Megan S; Bailey, Nathanael G; Wilcox, Ryan A

    2017-05-15

    Purpose: T-cell lymphomas are a molecularly heterogeneous group of non-Hodgkin lymphomas (NHL) that account for a disproportionate number of NHL disease-related deaths due to their inherent and acquired resistance to standard multiagent chemotherapy regimens. Despite their molecular heterogeneity and frequent loss of various T cell-specific receptors, the T-cell antigen receptor is retained in the majority of these lymphomas. As T-cell receptor (TCR) engagement activates a number of signaling pathways and transcription factors that regulate T-cell growth and survival, we examined the TCR's role in mediating resistance to chemotherapy. Experimental Design: Genetic and pharmacologic strategies were utilized to determine the contribution of tyrosine kinases and transcription factors activated in conventional T cells following TCR engagement in acquired chemotherapy resistance in primary T-cell lymphoma cells and patient-derived cell lines. Results: Here, we report that TCR signaling activates a signaling axis that includes ITK, NF-κB, and GATA-3 and promotes chemotherapy resistance. Conclusions: These observations have significant therapeutic implications, as pharmacologic inhibition of ITK prevented the activation of this signaling axis and overcame chemotherapy resistance. Clin Cancer Res; 23(10); 2506-15. ©2016 AACR . ©2016 American Association for Cancer Research.

  18. Effects of GABA receptor antagonists on thresholds of P23H rat retinal ganglion cells to electrical stimulation of the retina

    NASA Astrophysics Data System (ADS)

    Jensen, Ralph J.; Rizzo, Joseph F., III

    2011-06-01

    An electronic retinal prosthesis may provide useful vision for patients suffering from retinitis pigmentosa (RP). In animal models of RP, the amount of current needed to activate retinal ganglion cells (RGCs) is higher than in normal, healthy retinas. In this study, we sought to reduce the stimulation thresholds of RGCs in a degenerate rat model (P23H-line 1) by blocking GABA receptor mediated inhibition in the retina. We examined the effects of TPMPA, a GABAC receptor antagonist, and SR95531, a GABAA receptor antagonist, on the electrically evoked responses of RGCs to biphasic current pulses delivered to the subretinal surface through a 400 µm diameter electrode. Both TPMPA and SR95531 reduced the stimulation thresholds of ON-center RGCs on average by 15% and 20% respectively. Co-application of the two GABA receptor antagonists had the greatest effect, on average reducing stimulation thresholds by 32%. In addition, co-application of the two GABA receptor antagonists increased the magnitude of the electrically evoked responses on average three-fold. Neither TPMPA nor SR95531, applied alone or in combination, had consistent effects on the stimulation thresholds of OFF-center RGCs. We suggest that the effects of the GABA receptor antagonists on ON-center RGCs may be attributable to blockage of GABA receptors on the axon terminals of ON bipolar cells.

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

    PubMed Central

    Brudno, Jennifer N.

    2016-01-01

    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

  20. 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. © The Author 2016. Published by Oxford University Press.

  1. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

  2. Glucose-Sensing Receptor T1R3: A New Signaling Receptor Activated by Glucose in Pancreatic β-Cells.

    PubMed

    Kojima, Itaru; Nakagawa, Yuko; Hamano, Kunihisa; Medina, Johan; Li, Longfei; Nagasawa, Masahiro

    2015-01-01

    Subunits of the sweet taste receptors T1R2 and T1R3 are expressed in pancreatic β-cells. Compared with T1R3, mRNA expression of T1R2 is considerably lower. At the protein level, expression of T1R2 is undetectable in β-cells. Accordingly, a major component of the sweet taste-sensing receptor in β-cells may be a homodimer of T1R3 rather than a heterodimer of T1R2/T1R3. Inhibition of this receptor by gurmarin or deletion of the T1R3 gene attenuates glucose-induced insulin secretion from β-cells. Hence the T1R3 homodimer functions as a glucose-sensing receptor (GSR) in pancreatic β-cells. When GSR is activated by the T1R3 agonist sucralose, elevation of intracellular ATP concentration ([ATP]i) is observed. Sucralose increases [ATP]i even in the absence of ambient glucose, indicating that sucralose increases [ATP]i not simply by activating glucokinase, a rate-limiting enzyme in the glycolytic pathway. In addition, sucralose augments elevation of [ATP]i induced by methylsuccinate, suggesting that sucralose activates mitochondrial metabolism. Nonmetabolizable 3-O-methylglucose also increases [ATP]i and knockdown of T1R3 attenuates elevation of [ATP]i induced by high concentration of glucose. Collectively, these results indicate that the T1R3 homodimer functions as a GSR; this receptor is involved in glucose-induced insulin secretion by activating glucose metabolism probably in mitochondria.

  3. How to train your T cell: genetically engineered chimeric antigen receptor T cells versus bispecific T-cell engagers to target CD19 in B acute lymphoblastic leukemia.

    PubMed

    Ruella, Marco; Gill, Saar

    2015-06-01

    Antigen-specific T cell-based immunotherapy is getting its day in the sun. The contemporaneous development of two potent CD19-specific immunotherapeutic modalities for the treatment of B-cell malignancies provides exciting opportunities for patients, physicians and scientists alike. Patients with relapsed, refractory or poor-risk B-cell acute lymphoblastic leukemia (ALL) previously had few therapeutic options and now have two potential new lifelines. Physicians will have the choice between two powerful modalities and indeed could potentially enroll some patients on trials exploring both modalities if needed. For scientists interested in tumor immunology, the advent of chimeric antigen receptor T-cell therapy and of bispecific T-cell engagers (BiTEs) provides unprecedented opportunities to explore the promise and limitations of antigen-specific T-cell therapy in the context of human leukemia. In this article, we compare chimeric antigen receptor T cells and BiTEs targeting CD19 in B-cell ALL in the setting of the available clinical literature.

  4. A new insight in chimeric antigen receptor-engineered T cells for cancer immunotherapy.

    PubMed

    Zhang, Erhao; Xu, Hanmei

    2017-01-03

    Adoptive cell therapy using chimeric antigen receptor (CAR)-engineered T cells has emerged as a very promising approach to combating cancer. Despite its ability to eliminate tumors shown in some clinical trials, CAR-T cell therapy involves some significant safety challenges, such as cytokine release syndrome (CRS) and "on-target, off-tumor" toxicity, which is related to poor control of the dose, location, and timing of T cell activity. In the past few years, some strategies to avoid the side effects of CAR-T cell therapy have been reported, including suicide gene, inhibitory CAR, dual-antigen receptor, and the use of exogenous molecules as switches to control the CAR-T cell functions. Because of the advances of the CAR paradigm and other forms of cancer immunotherapy, the most effective means of defeating the cancer has become the integration therapy with the combinatorial control system of switchable dual-receptor CAR-T cell and immune checkpoint blockade.

  5. Chimeric antigen receptor T-cell therapy for glioblastoma.

    PubMed

    Rodriguez, Analiz; Brown, Christine; Badie, Behnam

    2017-09-01

    Chimeric antigen receptor (CAR) T-cell therapy has shown great promise in the treatment of hematological disease, and its utility for treatment of solid tumors is beginning to unfold. Glioblastoma continues to portend a grim prognosis and immunotherapeutic approaches are being explored as a potential treatment strategy. Identification of appropriate glioma-associated antigens, barriers to cell delivery, and presence of an immunosuppressive microenvironment are factors that make CAR T-cell therapy for glioblastoma particularly challenging. However, insights gained from preclinical studies and ongoing clinical trials indicate that CAR T-cell therapy will continue to evolve and likely become integrated with current therapeutic strategies for malignant glioma. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Chimeric Antigen Receptor (CAR) T Cell Therapy for Malignant Pleural Mesothelioma (MPM)

    PubMed Central

    Klampatsa, Astero; Haas, Andrew R.; Moon, Edmund K.; Albelda, Steven M.

    2017-01-01

    Cancer immunotherapy has now become a recognized approach to treating cancers. In addition to checkpoint blockade, adoptive T cell transfer (ACT) using chimeric antigen receptors (CARs) has shown impressive clinical outcomes in leukemias and is now being explored in solid tumors. CARs are engineered receptors, stably or transiently transduced into T cells, that aim to enhance T cell effector function by recognizing and binding to a specific tumor-associated antigen. In this review, we provide a summary of CAR T cell preclinical studies and clinical trials for malignant pleural mesothelioma (MPM), a rare, locally invasive pleural cancer with poor prognosis. We list other attractive potential targets for CAR T cell therapy for MPM, and discuss augmentation strategies of CAR T cell therapy with other forms of immunotherapy in this disease. PMID:28862644

  7. Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

    PubMed

    Taupin, J L; Anderson, P

    1994-12-01

    The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

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

    PubMed Central

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

    2017-01-01

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

  9. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

    PubMed

    Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

    2018-02-27

    Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

  10. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity

    PubMed Central

    Davenport, A. J.; Cross, R. S.; Watson, K. A.; Liao, Y.; Shi, W.; Prince, H. M.; Beavis, P. A.; Trapani, J. A.; Kershaw, M. H.; Ritchie, D. S.; Darcy, P. K.; Jenkins, M. R.

    2018-01-01

    Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. PMID:29440406

  11. Engineering Chimeric Antigen Receptor T cells to Treat Glioblastoma.

    PubMed

    Choi, Bryan D; O'Rourke, Donald M; Maus, Marcela V

    2017-08-01

    Immunotherapy has emerged as a promising strategy for glioblastoma (GBM), a disease that remains universally fatal despite currently available standard-of-care. Adoptive T cell therapy has been shown to produce potent antitumor immunity while obviating the need for traditional antigen presentation and primary immune responses. Chimeric antigen receptors (CARs) are specialized molecules that can be expressed on the surface of T cells allowing for redirected cytotoxicity against tumor antigens of interest. To date, the application of CAR T cells for GBM has been relatively limited, in large part due to a dearth of well-described tumor specific antigens that are both homogenously and frequently expressed. A mutated version of the epidermal growth factor receptor, EGFRvIII, is a constitutively activated tyrosine kinase that is expressed on the surface of GBM and other common neoplasms, but completely absent from all normal tissues. We have recently generated CAR T cells directed against EGFRvIII and reported results from a Phase I clinical trial investigating this platform in patients with EGFRvIII-expressing GBM. Our study showed that despite conventional notions of central nervous system "immune-privilege," EGFRvIII CAR T cells trafficked to intracerebral tumors, leading to successful targeting and eradication of this antigen in the brain. Here, we review our experience with EGFRvIII CAR T cells and highlight important considerations for the clinical translation of this therapy in patients with GBM.

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

    PubMed Central

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

    2016-01-01

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

  13. A Novel Bcl-x Isoform Connected to the T Cell Receptor Regulates Apoptosis in T Cells

    PubMed Central

    Yang, Xiao-Feng; Weber, Georg F.

    2014-01-01

    Summary We define a novel Bcl-x isoform, Bcl-xγ, that is generated by alternative splicing and characterized by a unique 47 amino acid C-terminus. Bcl-xγ is expressed primarily in thymocytes, where it may depend on an interaction between the TCR and host MHC products, and in mature T cells, where its expression is associated with ligation of the T cell receptor. Overexpression of Bcl-xγ in T cells inhibits activation-induced apoptosis; inhibition of Bcl-xγ, after stable expression of Bcl-xγ antisense cDNA, enhances activation-induced apoptosis. In contrast to other Bcl-x isoforms, cells that fail to express Bcl-xγ after CD3 ligation undergo programmed cell death, while activated T cells that express Bcl-xγ are spared. Identification of Bcl-xγ helps provide amolecular explanation of T cell activation and death after antigen engagement. PMID:9390687

  14. Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells.

    PubMed

    Jensen, Michael C; Riddell, Stanley R

    2014-01-01

    A major advance in adoptive T-cell therapy (ACT) is the ability to efficiently endow patient's T cells with reactivity for tumor antigens through the stable or regulated introduction of genes that encode high affinity tumor-targeting T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). Case reports and small series of patients treated with TCR- or CAR-modified T cells have shown durable responses in a subset of patients, particularly with B-cell malignancies treated with T cells modified to express a CAR that targets the CD19 molecule. However, many patients do not respond to therapy and serious on and off-target toxicities have been observed with TCR- and CAR-modified T cells. Thus, challenges remain to make ACT with gene-modified T cells a reproducibly effective and safe therapy and to expand the breadth of patients that can be treated to include those with common epithelial malignancies. This review discusses research topics in our laboratories that focus on the design and implementation of ACT with CAR-modified T cells. These include cell intrinsic properties of distinct T-cell subsets that may facilitate preparing therapeutic T-cell products of defined composition for reproducible efficacy and safety, the design of tumor targeting receptors that optimize signaling of T-cell effector functions and facilitate tracking of migration of CAR-modified T cells in vivo, and novel CAR designs that have alternative ligand binding domains or confer regulated function and/or survival of transduced T cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    PubMed Central

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

    2016-01-01

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

  16. 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

    PubMed Central

    Shores, Elizabeth W.; Ono, Masao; Kawabe, Tsutomo; Sommers, Connie L.; Tran, Tom; Lui, Kin; Udey, Mark C.; Ravetch, Jeffrey; Love, Paul E.

    1998-01-01

    The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η−/− and ζ/η−/−–Fcγ−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations. PMID:9529325

  18. Chimeric-antigen receptor T (CAR-T) cell therapy for solid tumors: challenges and opportunities

    PubMed Central

    Xia, An-Liang; Wang, Xiao-Chen; Lu, Yi-Jun; Lu, Xiao-Jie; Sun, Beicheng

    2017-01-01

    Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells. However, CAR T-cell therapy for solid tumors currently is faced with numerous challenges such as physical barriers, the immunosuppressive tumor microenvironment and the specificity and safety. The clinical results in solid tumors have been much less encouraging, with multiple cases of toxicity and a lack of therapeutic response. In this review, we will discuss the current stats and challenges of CAR-T cell therapy for solid tumors, and propose possibl e solutions and future perspectives. PMID:29163850

  19. Chimeric switch receptor: switching for improved adoptive T-cell therapy against cancers.

    PubMed

    Tay, Johan Ck; Zha, Shijun; Wang, Shu

    2017-12-01

    Adoptive T-lymphocyte transfer-based immunotherapy for cancers has seen huge leaps with both CARs and engineered TCRs. Despite this, issues relating to safety and efficacy persist. To address this, chimeric switch receptors have been created to reverse the outcomes of their original signaling pathways in order to confer immune cells with the ability to overcome the immunosuppressive tumor microenvironment and to allow them to have greater in vivo persistence. Activating switch receptors exploit the inhibitory molecules expressed by cancer cells to further stimulate the tumor antigen-specific T lymphocytes. On the other hand, inhibitory switch receptors inhibit the effects of tumor-reactive T lymphocytes on unintended targets. This paper reviews the switch receptors reported thus far, and lists out potential improvements and future works.

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

    PubMed Central

    Mamonkin, Maksim; Rouce, Rayne H.; Tashiro, Haruko

    2015-01-01

    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

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

    PubMed Central

    Gill, Saar

    2016-01-01

    The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T-cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML. PMID:27890255

  2. In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

    PubMed Central

    Carbone, Catherine B.; Fernandes, Ricardo A.; Hui, Enfu; Su, Xiaolei; Garcia, K. Christopher; Vale, Ronald D.

    2017-01-01

    T cell signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR–pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase–phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR–pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor–ligand pairs using purified proteins on model membranes. Using a model receptor–ligand pair (FRB–FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR–pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR–pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor–ligand pairs on the T cell are sufficient to create spatial organization at membrane–membrane interfaces. PMID:29042512

  3. Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

    PubMed

    Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

    2017-11-01

    T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

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

    PubMed

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

    2015-11-01

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

  5. H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

    PubMed Central

    Sakamoto, Soichiro; Kawabata, Hiroshi; Masuda, Taro; Uchiyama, Tatsuki; Mizumoto, Chisaki; Ohmori, Katsuyuki; Koeffler, H. Phillip; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi

    2015-01-01

    Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. PMID:26441243

  6. CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

    PubMed Central

    Bridgeman, J S; Ladell, K; Sheard, V E; Miners, K; Hawkins, R E; Price, D A; Gilham, D E

    2014-01-01

    Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions. PMID:24116999

  7. TRAIL Death Receptor-4, Decoy Receptor-1 and Decoy Receptor-2 Expression on CD8+ T Cells Correlate with the Disease Severity in Patients with Rheumatoid Arthritis

    PubMed Central

    2010-01-01

    Background Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL) expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. Methods The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28) using Spearman Rho Analysis. Results While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4) and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. Conclusions Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis. PMID:20799941

  8. Receptor revision in CD4 T cells is influenced by follicular helper T cell formation and germinal-center interactions.

    PubMed

    Higdon, Lauren E; Deets, Katherine A; Friesen, Travis J; Sze, Kai-Yin; Fink, Pamela J

    2014-04-15

    Peripheral CD4 T cells in Vβ5 transgenic (Tg) C57BL/6J mice undergo tolerance to an endogenous superantigen encoded by mouse mammary tumor virus 8 (Mtv-8) by either deletion or T-cell receptor (TCR) revision. Revision is a process by which surface expression of the Vβ5(+) TCR is down-regulated in response to Mtv-8 and recombination activating genes are expressed to drive rearrangement of the endogenous TCRβ locus, effecting cell rescue through the expression of a newly generated, non-self-reactive TCR. In an effort to identify the microenvironment in which revision takes place, we show here that the proportion of T follicular helper cells (Tfh) and production of high-affinity antibody during a primary response are increased in Vβ5 Tg mice in an Mtv-8-dependent manner. Revising T cells have a Tfh-like surface phenotype and transcription factor profile, with elevated expression of B-cell leukemia/lymphoma 6 (Bcl-6), CXC chemokine receptor 5, programmed death-1, and other Tfh-associated markers. Efficient revision requires Bcl-6 and is inhibited by B lymphocyte-induced maturation protein-1. Revision completes less efficiently in the absence of signaling lymphocytic activation molecule-associated protein although initiation proceeds normally. These data indicate that Tfh formation is required for the initiation of revision and germinal-center interactions for its completion. The germinal center is known to provide a confined space in which B-cell antigen receptors undergo selection. Our data extend the impact of this selective microenvironment into the arena of T cells, suggesting that this fluid structure also provides a regulatory environment in which TCR revision can safely take place.

  9. Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

    PubMed

    Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

    2010-06-22

    Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

  10. Decreased diacylglycerol metabolism enhances ERK activation and augments CD8+ T cell functional responses.

    PubMed

    Riese, Matthew J; Grewal, Jashanpreet; Das, Jayajit; Zou, Tao; Patil, Vineet; Chakraborty, Arup K; Koretzky, Gary A

    2011-02-18

    Modulation of T cell receptor signal transduction in CD8(+) T cells represents a novel strategy toward enhancing the immune response to tumor. Recently, levels of guanine exchange factors, RasGRP and SOS, within T cells have been shown to represent a key determinant in the regulation of the analog to the digital activation threshold of Ras. One important for regulating activation levels of RasGRP is diacylglycerol (DAG), and its levels are influenced by diacylglycerol kinase-ζ (DGKζ), which metabolizes DAG into phosphatidic acid, terminating DAG-mediated Ras signaling. We sought to determine whether DGKζ-deficient CD8(+) T cells demonstrated enhanced in vitro responses in a manner predicted by the current model of Ras activation and to evaluate whether targeting this threshold confers enhanced CD8(+) T cell responsiveness to tumor. We observed that DGKζ-deficient CD8(+) T cells conform to most predictions of the current model of how RasGRP levels influence Ras activation. But our results differ in that the EC(50) value of stimulation is not altered for any T cell receptor stimulus, a finding that suggests a further degree of complexity to how DGKζ deficiency affects signals important for Ras and ERK activation. Additionally, we found that DGKζ-deficient CD8(+) T cells demonstrate enhanced responsiveness in a subcutaneous lymphoma model, implicating the analog to a digital conversion threshold as a novel target for potential therapeutic manipulation.

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

    PubMed

    Jindal, Vishal; Arora, Ena; Gupta, Sorab

    2018-05-05

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

  12. 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.

  13. Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL.

    PubMed

    Blanco, Gonzalo; Vardi, Anna; Puiggros, Anna; Gómez-Llonín, Andrea; Muro, Manuel; Rodríguez-Rivera, María; Stalika, Evangelia; Abella, Eugenia; Gimeno, Eva; López-Sánchez, Manuela; Senín, Alicia; Calvo, Xavier; Abrisqueta, Pau; Bosch, Francesc; Ferrer, Ana; Stamatopoulos, Kostas; Espinet, Blanca

    2018-01-01

    Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4 + and CD8 + T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4 + T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.

  14. K-RAS GTPase- and B-RAF kinase-mediated T-cell tolerance defects in rheumatoid arthritis.

    PubMed

    Singh, Karnail; Deshpande, Pratima; Li, Guangjin; Yu, Mingcan; Pryshchep, Sergey; Cavanagh, Mary; Weyand, Cornelia M; Goronzy, Jörg J

    2012-06-19

    Autoantibodies to common autoantigens and neoantigens, such as IgG Fc and citrullinated peptides, are immunological hallmarks of rheumatoid arthritis (RA). We examined whether a failure in maintaining tolerance is mediated by defects in T-cell receptor activation threshold settings. RA T cells responded to stimulation with significantly higher ERK phosphorylation (P < 0.001). Gene expression arrays of ERK pathway members suggested a higher expression of KRAS and BRAF, which was confirmed by quantitative PCR (P = 0.003), Western blot, and flow cytometry (P < 0.01). Partial silencing of KRAS and BRAF lowered activation-induced phosphorylated ERK levels (P < 0.01). In individual cells, levels of these signaling molecules correlated with ERK phosphorylation, attesting that their concentrations are functionally important. In confocal studies, B-RAF/K-RAS clustering was increased in RA T cells 2 min after T-cell receptor stimulation (P < 0.001). Overexpression of B-RAF and K-RAS in normal CD4 T cells amplified polyclonal T-cell proliferation and facilitated responses to citrullinated peptides. We propose that increased expression of B-RAF and K-RAS lowers T-cell activation thresholds in RA T cells, enabling responses to autoantigens.

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

    PubMed

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

    2017-01-01

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

  16. Mechanosensing drives acuity of αβ T-cell recognition

    PubMed Central

    Feng, Yinnian; Brazin, Kristine N.; Kobayashi, Eiji; Mallis, Robert J.; Reinherz, Ellis L.; Lang, Matthew J.

    2017-01-01

    T lymphocytes use surface αβ T-cell receptors (TCRs) to recognize peptides bound to MHC molecules (pMHCs) on antigen-presenting cells (APCs). How the exquisite specificity of high-avidity T cells is achieved is unknown but essential, given the paucity of foreign pMHC ligands relative to the ubiquitous self-pMHC array on an APC. Using optical traps, we determine physicochemical triggering thresholds based on load and force direction. Strikingly, chemical thresholds in the absence of external load require orders of magnitude higher pMHC numbers than observed physiologically. In contrast, force applied in the shear direction (∼10 pN per TCR molecule) triggers T-cell Ca2+ flux with as few as two pMHC molecules at the interacting surface interface with rapid positional relaxation associated with similarly directed motor-dependent transport via ∼8-nm steps, behaviors inconsistent with serial engagement during initial TCR triggering. These synergistic directional forces generated during cell motility are essential for adaptive T-cell immunity against infectious pathogens and cancers. PMID:28811364

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

    PubMed Central

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

    2010-01-01

    CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells. PMID:20467460

  18. Identification of a G protein coupled receptor induced in activated T cells.

    PubMed

    Kaplan, M H; Smith, D I; Sundick, R S

    1993-07-15

    Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

  19. GARP: a key receptor controlling FOXP3 in human regulatory T cells.

    PubMed

    Probst-Kepper, M; Geffers, R; Kröger, A; Viegas, N; Erck, C; Hecht, H-J; Lünsdorf, H; Roubin, R; Moharregh-Khiabani, D; Wagner, K; Ocklenburg, F; Jeron, A; Garritsen, H; Arstila, T P; Kekäläinen, E; Balling, R; Hauser, H; Buer, J; Weiss, S

    2009-09-01

    Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

  20. Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

    PubMed Central

    Pyo, Suhkneung; Kang, Chung Hyo; Lee, Chong Ock; Lee, Heung Kyoung; Choi, Sang Un; Park, Chi Hoon

    2018-01-01

    Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. PMID:29874279

  1. A T-Cell Receptor Breaks the Rules | Center for Cancer Research

    Cancer.gov

    Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their

  2. Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

    PubMed

    Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

    2013-05-01

    Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

  3. Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling

    PubMed Central

    Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

    2009-01-01

    Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve αCD3/CD28-stimulated CD8 cells. Consequently, αCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs. PMID:19740334

  4. Adenosine regulates CD8 T-cell priming by inhibition of membrane-proximal T-cell receptor signalling.

    PubMed

    Linnemann, Carsten; Schildberg, Frank A; Schurich, Anna; Diehl, Linda; Hegenbarth, Silke I; Endl, Elmar; Lacher, Svenja; Müller, Christa E; Frey, Jürgen; Simeoni, Luca; Schraven, Burkhart; Stabenow, Dirk; Knolle, Percy A

    2009-09-01

    Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after alphaCD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naïve alphaCD3/CD28-stimulated CD8 cells. Consequently, alphaCD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

  5. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells

    PubMed Central

    Tone, Masahide; Tone, Yukiko; Adams, Elizabeth; Yates, Stephen F.; Frewin, Mark R.; Cobbold, Stephen P.; Waldmann, Herman

    2003-01-01

    Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+ regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+ T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences. PMID:14608036

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

    PubMed

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

    2015-01-01

    Small chemicals like drugs tend to bind to proteins via noncovalent bonds, e.g. hydrogen bonds, salt bridges or electrostatic interactions. Some chemicals interact with other molecules than the actual target ligand, representing so-called 'off-target' activities of drugs. Such interactions are a main cause of adverse side effects to drugs and are normally classified as predictable type A reactions. Detailed analysis of drug-induced immune reactions revealed that off-target activities also affect immune receptors, such as highly polymorphic human leukocyte antigens (HLA) or T cell receptors (TCR). Such drug interactions with immune receptors may lead to T cell stimulation, resulting in clinical symptoms of delayed-type hypersensitivity. They are assigned the 'pharmacological interaction with immune receptors' (p-i) concept. Analysis of p-i has revealed that drugs bind preferentially or exclusively to distinct HLA molecules (p-i HLA) or to distinct TCR (p-i TCR). P-i reactions differ from 'conventional' off-target drug reactions as the outcome is not due to the effect on the drug-modified cells themselves, but is the consequence of reactive T cells. Hence, the complex and diverse clinical manifestations of delayed-type hypersensitivity are caused by the functional heterogeneity of T cells. In the abacavir model of p-i HLA, the drug binding to HLA may result in alteration of the presenting peptides. More importantly, the drug binding to HLA generates a drug-modified HLA, which stimulates T cells directly, like an allo-HLA. In the sulfamethoxazole model of p-i TCR, responsive T cells likely require costimulation for full T cell activation. These findings may explain the similarity of delayed-type hypersensitivity reactions to graft-versus-host disease, and how systemic viral infections increase the risk of delayed-type hypersensitivity reactions. © 2015 The Author(s) Published by S. Karger AG, Basel.

  7. T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

    NASA Technical Reports Server (NTRS)

    Adams, C. L.; Sams, C. F.

    2000-01-01

    Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

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

    PubMed

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

    2015-06-16

    Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition.

    PubMed

    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 targeting CD19 on B-cell malignancies. The clinical trials of CD19 chimeric antigen receptor therapy have thus far not attempted to select defined subsets before 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 using adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.

  10. 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

  11. Effects of cryopreservation on chimeric antigen receptor T cell functions.

    PubMed

    Xu, Hao; Cao, Wenyue; Huang, Liang; Xiao, Min; Cao, Yang; Zhao, Lei; Wang, Na; Zhou, Jianfeng

    2018-06-14

    Chimeric antigen receptor T (CART) cell therapy has emerged as a potentially curative "drug" for cancer treatment. Cryopreservation of CART cells is necessary for their clinical application. Systematic studies on the effects of cryopreservation on the antitumor function of CART cells are lacking. Therefore, we compared the phenotypes and functions of CART cells that were cryopreserved during ex vivo expansion with those of freshly isolated populations. T cells expressing an anti-B-cell-maturation-antigen (BCMA) chimeric antigen receptor (CAR) were expanded in vitro for 10 days and then cryopreserved. After one month, the cells were resuscitated, and their transduction rates, apoptosis rates and cell subsets were examined via flow cytometry. The results indicated no significant changes in transduction rates or cell subsets, and the survival rate of the resuscitated cells was approximately 90% Furthermore, similar tumoricidal effects and degranulation functions of the resuscitated cells compared with normally cultured cells were verified by calcein release and CD107a assays. A NOD/SCID mouse model was used to estimate the differences in the in vivo antitumor effects of the cryopreserved and normally cultured T cells, but no significant differences were observed. Following co-culture with several target cell types, the cytokines released by the cryopreserved and normally cultured T cells were measured via enzyme-linked immunosorbent assays (ELISAs). The results revealed that the release of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was significantly decreased. These data demonstrated that with the exception of a decrease in cytokine release, the cryopreserved CART cells retained their antitumor functions. Copyright © 2018. Published by Elsevier Inc.

  12. Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

    PubMed

    Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

    2017-07-25

    Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

  13. Expression of inhibitory receptors and polyfunctional responses of T cells are linked to the risk of congenital transmission of T. cruzi

    PubMed Central

    Thomas, María Carmen; Carrilero, Bartolomé; González, John Mario; Cuéllar, Adriana; Segovia, Manuel; Puerta, Concepción Judith

    2017-01-01

    Congenital T. cruzi infections involve multiple factors in which complex interactions between the parasite and the immune system of pregnant women play important roles. In this study, we used an experimental murine model of chronic infection with T. cruzi to evaluate the changes in the expression of inhibitory receptors and the polyfunctionality of T cells during gestation and their association with congenital transmission rate of T. cruzi infection. The results showed that pregnant naïve mice had a higher percentage of CD4+ and CD8+ T cells that expressed inhibitory receptors than cells from non-pregnant naïve mice. However, in mice chronically infected with T. cruzi, gestation induced a significant decrease in the frequency of T cells that expressed or co-expressed inhibitory receptors, as well as an increase in the frequency of polyfunctional CD4+ and CD8+ T cells. This different behavior may be due to the breakdown in the infected mice of the gestation-induced immune homeostasis, probably to control the parasite load. Remarkably, it was observed that the mothers that transmitted the parasite had a higher frequency of T cells that expressed and co-expressed inhibitory receptors as well as a lower frequency of polyfunctional parasite-specific T cells than those that did not transmit it, even though the parasitemia load was similar in both groups. All together these data suggest that the maternal immune profile of the CD4+ and CD8+ T cells could be a determining factor in the congenital transmission of T. cruzi. PMID:28598971

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

    PubMed Central

    Maus, Marcela V.; June, Carl H.

    2016-01-01

    Chimeric antigen receptors (CARs) 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 ≈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 mechanisms regulating the persistence of CAR T cells. Additionally, 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. PMID:27084741

  15. Cytokine Release Syndrome After Chimeric Antigen Receptor T Cell Therapy for Acute Lymphoblastic Leukemia.

    PubMed

    Fitzgerald, Julie C; Weiss, Scott L; Maude, Shannon L; Barrett, David M; Lacey, Simon F; Melenhorst, J Joseph; Shaw, Pamela; Berg, Robert A; June, Carl H; Porter, David L; Frey, Noelle V; Grupp, Stephan A; Teachey, David T

    2017-02-01

    Initial success with chimeric antigen receptor-modified T cell therapy for relapsed/refractory acute lymphoblastic leukemia is leading to expanded use through multicenter trials. Cytokine release syndrome, the most severe toxicity, presents a novel critical illness syndrome with limited data regarding diagnosis, prognosis, and therapy. We sought to characterize the timing, severity, and intensive care management of cytokine release syndrome after chimeric antigen receptor-modified T cell therapy. Retrospective cohort study. Academic children's hospital. Thirty-nine subjects with relapsed/refractory acute lymphoblastic leukemia treated with chimeric antigen receptor-modified T cell therapy on a phase I/IIa clinical trial (ClinicalTrials.gov number NCT01626495). All subjects received chimeric antigen receptor-modified T cell therapy. Thirteen subjects with cardiovascular dysfunction were treated with the interleukin-6 receptor antibody tocilizumab. Eighteen subjects (46%) developed grade 3-4 cytokine release syndrome, with prolonged fever (median, 6.5 d), hyperferritinemia (median peak ferritin, 60,214 ng/mL), and organ dysfunction. Fourteen (36%) developed cardiovascular dysfunction treated with vasoactive infusions a median of 5 days after T cell therapy. Six (15%) developed acute respiratory failure treated with invasive mechanical ventilation a median of 6 days after T cell therapy; five met criteria for acute respiratory distress syndrome. Encephalopathy, hepatic, and renal dysfunction manifested later than cardiovascular and respiratory dysfunction. Subjects had a median of 15 organ dysfunction days (interquartile range, 8-20). Treatment with tocilizumab in 13 subjects resulted in rapid defervescence (median, 4 hr) and clinical improvement. Grade 3-4 cytokine release syndrome occurred in 46% of patients following T cell therapy for relapsed/refractory acute lymphoblastic leukemia. Clinicians should be aware of expanding use of this breakthrough therapy and

  16. Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells.

    PubMed

    Park, Jae H; Brentjens, Renier J

    2010-04-01

    Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

  17. Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

    PubMed

    Benvenuto, Federica; Voci, Adriana; Carminati, Enrico; Gualandi, Francesca; Mancardi, Gianluigi; Uccelli, Antonio; Vergani, Laura

    2015-12-10

    Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

  18. 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

  19. 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. Copyright © 2015, American Association for the Advancement of Science.

  20. Scavenger receptor WC1 contributes to the γδ T cell response to Leptospira.

    PubMed

    Wang, Fei; Herzig, Carolyn T A; Chen, Chuang; Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2011-03-01

    WC1 molecules are exclusively expressed on the surface of γδ T cells. They belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. WC1 molecules have been grouped on the basis of antibody reactivity. The expression of WC1 molecules from these serologically defined groups is correlated with differences in γδ T cell responses. The expression of receptors within the WC1.1 group correlates with the capacity of γδ T cells to respond to Leptospira antigen. In this study, we used RNA interference to directly investigate the role of WC1 expression in the response to Leptospira borgpetersenii. We found that when three out of thirteen WC1 gene products were downregulated by RNA interference, γδ T cell proliferation and IFN-γ production in response to Leptospira antigen was significantly reduced. Our data demonstrate that specific receptors in the WC1 family directly participate in Leptospira recognition and/or activation of γδ T cells. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen

    DOE PAGES

    Visperas, Patrick R.; Wilson, Christopher G.; Winger, Jonathan A.; ...

    2016-12-13

    ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. Finally, the screen is based on a fluorescence polarizationmore » assay for peptide binding to ZAP-70.« less

  2. Chimeric antigen receptor T cells: power tools to wipe out leukemia and lymphoma.

    PubMed

    Riet, Tobias; Abken, Hinrich

    2015-08-01

    Adoptive cell therapy for malignant diseases is showing promise in recent early-phase trials in the treatment of B cell leukemia/lymphoma. Genetically engineered with a tumor-specific chimeric antigen receptor, patient's T cells produce lasting and complete leukemia regression. However, treatment is associated with some toxicity which needs our attention and the field still faces some hurdles at the scientific, technologic and clinical levels. Surmounting these obstacles will establish chimeric antigen receptor T cell therapy as a powerful approach to cure hematologic malignancies, paving the way for the treatment of other common types of cancer in the future.

  3. Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

    PubMed Central

    Imberg, Keren; Mercer, Frances; Zhong, Shi; Krogsgaard, Michelle; Unutmaz, Derya

    2013-01-01

    Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8+ and CD4+ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression. PMID:23437112

  4. Adoptive immunotherapy for hematological malignancies: Current status and new insights in chimeric antigen receptor T cells.

    PubMed

    Allegra, Alessandro; Innao, Vanessa; Gerace, Demetrio; Vaddinelli, Doriana; Musolino, Caterina

    2016-11-01

    Hematological malignancies frequently express cancer-associated antigens that are shared with normal cells. Such tumor cells elude the host immune system because several T cells targeted against self-antigens are removed during thymic development, and those that persist are eliminated by a regulatory population of T cells. Chimeric antigen receptor-modified T cells (CAR-Ts) have emerged as a novel modality for tumor immunotherapy due to their powerful efficacy against tumor cells. These cells are created by transducing genes-coding fusion proteins of tumor antigen-recognition single-chain Fv connected to the intracellular signaling domains of T cell receptors, and are classed as first-, second- and third-generation, differing on the intracellular signaling domain number of T cell receptors. CAR-T treatment has emerged as a promising approach for patients with hematological malignancies, and there are several works reporting clinical trials of the use of CAR-modified T-cells in acute lymphoblastic leukemia, chronic lymphoblastic leukemia, multiple myeloma, lymphoma, and in acute myeloid leukemia by targeting different antigens. This review reports the history of adoptive immunotherapy using CAR-Ts, the CAR-T manufacturing process, and T cell therapies in development for hematological malignancies. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    Di, Shengmeng; Li, Zonghai

    2016-04-01

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

  6. T Cell Receptor-Major Histocompatibility Complex Interaction Strength Defines Trafficking and CD103+ Memory Status of CD8 T Cells in the Brain.

    PubMed

    Sanecka, Anna; Yoshida, Nagisa; Kolawole, Elizabeth Motunrayo; Patel, Harshil; Evavold, Brian D; Frickel, Eva-Maria

    2018-01-01

    T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii- infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103 + ) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-20

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

  9. Thy-1+ dendritic epidermal cells express T3 antigen and the T-cell receptor gamma chain.

    PubMed Central

    Stingl, G; Koning, F; Yamada, H; Yokoyama, W M; Tschachler, E; Bluestone, J A; Steiner, G; Samelson, L E; Lew, A M; Coligan, J E

    1987-01-01

    The murine epidermis is a heterogeneous epithelium composed of keratinocytes, melanocytes, Langerhans cells, and a recently described subpopulation (2-3%) of bone-marrow-derived leukocytes with a dendritic morphology and the cell surface phenotype Thy-1+, L3T4-, Lyt-2-. Previous studies have demonstrated that cell lines derived from freshly explanted Thy-1+ dendritic epidermal cells (DEC) have abundant mRNA for rearranged T-cell receptor (TCR) gamma-chain genes. Analysis of Thy-1+ DEC in situ, freshly isolated cell suspensions of Thy-1+ DEC, and long-term Thy-1+ DEC lines demonstrated that 100% of the Thy-1+ DEC reacted with a monoclonal antibody to the epsilon chain of the murine T3 complex and that 40-60% of resident Thy-1+ DEC were also reactive with an antiserum to the TCR gamma chain. Two Thy-1+ DEC lines expressed a disulfide-linked 70-kDa molecule that could be precipitated with an anti-gamma-chain antiserum and could be coprecipitated with an antiserum to the T3 delta chain; the molecule appeared as a single 34-kDa band under reducing conditions. The phenotype of Thy-1+ DEC (T3+, L3T4-, Lyt-2-, TCR gamma chain+) thus resembles that of the recently described subpopulation of murine and human lymphocytes that have been identified in the thymus, peripheral blood, and fetal blood. Images PMID:2885839

  10. Specificity, Privacy, and Degeneracy in the CD4 T Cell Receptor Repertoire Following Immunization

    PubMed Central

    Sun, Yuxin; Best, Katharine; Cinelli, Mattia; Heather, James M.; Reich-Zeliger, Shlomit; Shifrut, Eric; Friedman, Nir; Shawe-Taylor, John; Chain, Benny

    2017-01-01

    T cells recognize antigen using a large and diverse set of antigen-specific receptors created by a complex process of imprecise somatic cell gene rearrangements. In response to antigen-/receptor-binding-specific T cells then divide to form memory and effector populations. We apply high-throughput sequencing to investigate the global changes in T cell receptor sequences following immunization with ovalbumin (OVA) and adjuvant, to understand how adaptive immunity achieves specificity. Each immunized mouse contained a predominantly private but related set of expanded CDR3β sequences. We used machine learning to identify common patterns which distinguished repertoires from mice immunized with adjuvant with and without OVA. The CDR3β sequences were deconstructed into sets of overlapping contiguous amino acid triplets. The frequencies of these motifs were used to train the linear programming boosting (LPBoost) algorithm LPBoost to classify between TCR repertoires. LPBoost could distinguish between the two classes of repertoire with accuracies above 80%, using a small subset of triplet sequences present at defined positions along the CDR3. The results suggest a model in which such motifs confer degenerate antigen specificity in the context of a highly diverse and largely private set of T cell receptors. PMID:28450864

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

    PubMed

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

    2006-09-01

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

  12. Adoptive transfer of murine T cells expressing a chimeric-PD1-Dap10 receptor as an immunotherapy for lymphoma.

    PubMed

    Lynch, Adam; Hawk, William; Nylen, Emily; Ober, Sean; Autin, Pierre; Barber, Amorette

    2017-11-01

    Adoptive transfer of T cells is a promising cancer therapy and expression of chimeric antigen receptors can enhance tumour recognition and T-cell effector functions. The programmed death protein 1 (PD1) receptor is a prospective target for a chimeric antigen receptor because PD1 ligands are expressed on many cancer types, including lymphoma. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 extracellular domain fused to the cytoplasmic domain of CD3ζ. Additionally, chimeric antigen receptor therapies use various co-stimulatory domains to enhance efficacy. Hence, the inclusion of a Dap10 or CD28 co-stimulatory domain in the chPD1 receptor was compared to determine which domain induced optimal anti-tumour immunity in a mouse model of lymphoma. The chPD1 T cells secreted pro-inflammatory cytokines and lysed RMA lymphoma cells. Adoptive transfer of chPD1 T cells significantly reduced established tumours and led to tumour-free survival in lymphoma-bearing mice. When comparing chPD1 receptors containing a Dap10 or CD28 domain, both receptors induced secretion of pro-inflammatory cytokines; however, chPD1-CD28 T cells also secreted anti-inflammatory cytokines whereas chPD1-Dap10 T cells did not. Additionally, chPD1-Dap10 induced a central memory T-cell phenotype compared with chPD1-CD28, which induced an effector memory phenotype. The chPD1-Dap10 T cells also had enhanced in vivo persistence and anti-tumour efficacy compared with chPD1-CD28 T cells. Therefore, adoptive transfer of chPD1 T cells could be a novel therapy for lymphoma and inclusion of the Dap10 co-stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T-cell differentiation phenotype for anti-tumour therapies. © 2017 John Wiley & Sons Ltd.

  13. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    PubMed

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Copyright© Ferrata Storti Foundation.

  14. CMV drives the expansion of highly functional memory T cells expressing NK-cell receptors in renal transplant recipients.

    PubMed

    Makwana, Nandini; Foley, Bree; Fernandez, Sonia; Lee, Silvia; Irish, Ashley; Pircher, Hanspeter; Price, Patricia

    2017-08-01

    Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post-transplant. We describe how active and latent CMV affect T-cell subsets in RTRs who are stable on maintenance therapy. T-cell responses to CMV were assessed in RTRs (n = 54) >2 years post-transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8 + T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T-cell (T EM ), terminally differentiated T-cell (T EMRA ) and CD57 + T EMRA cell populations. Expression of NK-cell receptors, LIR-1 and KLRG1 on CD4 + and CD8 + CD57 + T EM and T EMRA cells correlated with elevated interferon-γ and cytotoxic responses to anti-CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8 + T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) -1 peptides. The data show that latent and active CMV infection can alter T-cell subsets in RTRs many years after transplantation, and up-regulate T-cell expression of NK-cell receptors. This may enhance effector responses of CD4 + and CD8 + T cells against CMV. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Fujiwara, Hiroshi

    2014-12-15

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

  16. Engineered chimeric antigen receptor-expressing T cells for the treatment of pancreatic ductal adenocarcinoma

    PubMed Central

    Beatty, Gregory L

    2014-01-01

    Adoptive cell therapy with chimeric antigen receptor (CAR)-engineered T cells is under investigation as an approach to restore productive T cell immunosurveillance in patients with pancreatic ductal adenocarcinoma. Early findings demonstrate safety of this cell-based therapy and the capacity of CAR-expressing T cells to mediate anti-tumor activity as well as induce endogeneous antitumoral immune responses. PMID:25050204

  17. Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

    PubMed

    Kochenderfer, James N; Rosenberg, Steven A

    2013-05-01

    Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

  18. T-cell receptor transfer for boosting HIV-1-specific T-cell immunity in HIV-1-infected patients.

    PubMed

    Mummert, Christiane; Hofmann, Christian; Hückelhoven, Angela G; Bergmann, Silke; Mueller-Schmucker, Sandra M; Harrer, Ellen G; Dörrie, Jan; Schaft, Niels; Harrer, Thomas

    2016-09-10

    Strategies to cure HIV-1 infection require the eradication of viral reservoirs. An innovative approach for boosting the cytotoxic T-lymphocyte response is the transfer of T-cell receptors (TCRs). Previously, we have shown that electroporation of TCR-encoding mRNA is able to reprogram CD8 T cells derived from healthy donors. So far, it is unknown whether the transfer of HIV-1-specific TCRs is capable to reprogram CD8 T cells of HIV-1-infected patients. To assess the efficiency of TCR-transfer by mRNA electroporation and the functionality of reprogramed T cells in HIV-1-infected patients, we performed an in-vitro analysis of TCR-transfer into T cells from HIV-1-infected patients in various stages of disease and from healthy controls. Peripheral blood mononuclear cells from 16 HIV-1-infected patients (nine HLA-A02-positive, seven HLA-A02-negative) and from five healthy controls were electroporated with mRNA-constructs encoding TCRs specific for the HLA-A02/HIV-1-gag p17 epitope SLYNTVATL (SL9). Functionality of the TCRs was measured by γIFN-ELISpot assays. SL9/TCR transfection into peripheral blood mononuclear cells from both HLA-A02-positive and HLA-A02-negative HIV-1-infected patients and from healthy blood donors reprogramed T cells for recognition of SL9-presenting HLA-A02-positive cells in γIFN-ELISpot assays. SL9/TCR-transfer into T cells from an immunodeficient AIDS patient could induce recognition of SL9-expressing target cells only after reversion of T-cell dysfunction by antiretroviral therapy. The transfer of HIV-1-p17-specific TCRs into T cells is functional both in HIV-1-infected patients as well as in healthy blood donors. TCR-transfer is a promising method to boost the immune system against HIV-1.

  19. 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.

  20. Heparan Sulfate Modification of the Transmembrane Receptor CD47 Is Necessary for Inhibition of T Cell Receptor Signaling by Thrombospondin-1*

    PubMed Central

    Kaur, Sukhbir; Kuznetsova, Svetlana A.; Pendrak, Michael L.; Sipes, John M.; Romeo, Martin J.; Li, Zhuqing; Zhang, Lijuan; Roberts, David D.

    2011-01-01

    Cell surface proteoglycans on T cells contribute to retroviral infection, binding of chemokines and other proteins, and are necessary for some T cell responses to the matricellular glycoprotein thrombospondin-1. The major cell surface proteoglycans expressed by primary T cells and Jurkat T cells have an apparent Mr > 200,000 and are modified with chondroitin sulfate and heparan sulfate chains. Thrombospondin-1 bound in a heparin-inhibitable manner to this proteoglycan and to a soluble form released into the medium. Based on mass spectrometry, knockdown, and immunochemical analyses, the proteoglycan contains two major core proteins as follows: amyloid precursor-like protein-2 (APLP2, apparent Mr 230,000) and CD47 (apparent Mr > 250,000). CD47 is a known thrombospondin-1 receptor but was not previously reported to be a proteoglycan. This proteoglycan isoform of CD47 is widely expressed on vascular cells. Mutagenesis identified glycosaminoglycan modification of CD47 at Ser64 and Ser79. Inhibition of T cell receptor signaling by thrombospondin-1 was lost in CD47-deficient T cells that express the proteoglycan isoform of APLP2, indicating that binding to APLP2 is not sufficient. Inhibition of CD69 induction was restored in CD47-deficient cells by re-expressing CD47 or an S79A mutant but not by the S64A mutant. Therefore, inhibition of T cell receptor signaling by thrombospondin-1 is mediated by CD47 and requires its modification at Ser64. PMID:21343308

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

    PubMed

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

    2015-08-01

    The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT. Copyright © 2015 by The American Association of Immunologists, Inc.

  2. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice.

    PubMed

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J

    2013-05-01

    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases. Published by Elsevier B.V.

  3. Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

    PubMed Central

    Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

    2014-01-01

    The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

  4. 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

  5. Force generation upon T cell receptor engagement.

    PubMed

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

    2011-05-10

    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 Ca(2+) concentration ([Ca(2+)](i)) was monitored simultaneously to verify the cell commitment in the activation process. [Ca(2+)](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

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

    PubMed

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

    2014-03-06

    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. 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

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

    PubMed Central

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

    2016-01-01

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

  9. Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells

    PubMed Central

    Berger, Carolina; Sommermeyer, Daniel; Hudecek, Michael; Berger, Michael; Balakrishnan, Ashwini; Paszkiewicz, Paulina J.; Kosasih, Paula L.; Rader, Christoph; Riddell, Stanley R.

    2014-01-01

    Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) since it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors, and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1-expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates. PMID:25355068

  10. T Lymphocyte Activation Threshold is Increased in Reduced Gravity

    NASA Technical Reports Server (NTRS)

    Adams, Charley L.; Gonzalez, M.; Sams, C. F.

    2000-01-01

    There have been substantial advances in molecular and cellular biology that have provided new insight into the biochemical and genetic basis of lymphocyte recognition, activation and expression of distinct functional phenotypes. It has now become evident that for both T and B cells, stimuli delivered through their receptors can result in either clonal expansion or apoptosis. In the case of T cells, clonal expansion of helper cells is accompanied by differentiation into two major functional subsets which regulate the immune response. The pathways between the membrane and the nucleus and their molecular components are an area of very active investigation. This meeting will draw together scientists working on diverse aspects of this problem, including receptor ligand interactions, intracellular pathways that transmit receptor mediated signals and the effect of such signal transduction pathways on gene regulation. The aim of this meeting is to integrate the information from these various experimental approaches into a new synthesis and molecular explanation of T cell activation, differentiation and death.

  11. Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming

    PubMed Central

    Banchereau, Jacques; Zurawski, Sandra; Thompson-Snipes, LuAnn; Blanck, Jean-Philippe; Clayton, Sandra; Munk, Adiel; Cao, Yanying; Wang, Zhiqing; Khandelwal, Sunaina; Hu, Jiancheng; McCoy, William H.; Palucka, Karolina A.; Reiter, Yoram; Fremont, Daved H.; Zurawski, Gerard; Colonna, Marco; Shaw, Andrey S.; Klechevsky, Eynav

    2012-01-01

    Human Langerhans cells (LCs) are highly efficient at priming cytolytic CD8+ T cells compared with dermal CD14+ dendritic cells (DCs). Here we show that dermal CD14+ DCs instead prime a fraction of naïve CD8+ T cells into cells sharing the properties of type 2 cytokine-secreting CD8+ T cells (TC2). Differential expression of the CD8-antagonist receptors on dermal CD14+ DCs, the Ig-like transcript (ILT) inhibitory receptors, explains the difference between the two types of DCs. Inhibition of CD8 function on LCs inhibited cytotoxic T lymphocytes (CTLs) and enhanced TC2 generation. In addition, blocking ILT2 or ILT4 on dermal CD14+ DCs enhanced the generation of CTLs and inhibited TC2 cytokine production. Lastly, addition of soluble ILT2 and ILT4 receptors inhibited CTL priming by LCs. Thus, ILT receptor expression explains the polarization of CD8+ T-cell responses by LCs vs. dermal CD14+ DCs. PMID:23112154

  12. Cutaneous T-cell lymphoma (CTCL): Current practices in blood assessment and the utility of T-cell receptor (TCR)-Vβ chain restriction.

    PubMed

    Gibson, Juliet F; Huang, Jing; Liu, Kristina J; Carlson, Kacie R; Foss, Francine; Choi, Jaehyuk; Edelson, Richard; Hussong, Jerry W; Mohl, Ramsey; Hill, Sally; Girardi, Michael

    2016-05-01

    Accurate quantification of malignant cells in the peripheral blood of patients with cutaneous T-cell lymphoma is important for early detection, prognosis, and monitoring disease burden. We sought to determine the spectrum of current clinical practices; critically evaluate elements of current International Society for Cutaneous Lymphomas (ISCL) B1 and B2 staging criteria; and assess the potential role of T-cell receptor-Vβ analysis by flow cytometry. We assessed current clinical practices by survey, and performed a retrospective analysis of 161 patients evaluated at Yale (2011-2014) to compare the sensitivity, specificity, positive predictive value, and negative predictive value of parameters for ISCL B2 staging. There was heterogeneity in clinical practices among institutions. ISCL B1 criteria did not capture 5 Yale cohort cases with immunophenotypic abnormalities that later progressed. T-cell receptor-Vβ testing was more specific than polymerase chain reaction and aided diagnosis in detecting clonality, but was of limited benefit in quantification of tumor burden. Because of limited follow-up involving a single center, further investigation will be necessary to conclude whether our proposed diagnostic algorithm is of general clinical benefit. We propose further study of modified B1 criteria: CD4/CD8 ratio 5 or greater, %CD4(+) CD26(-) 20% or greater, or %CD4(+) CD7(-) 20% or greater, with evidence of clonality. T-cell receptor-Vβ testing should be considered in future diagnostic and staging algorithms. Copyright © 2015 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  14. Chimeric Antigen Receptor-Redirected T cells return to the bench

    PubMed Central

    Geldres, Claudia; Savoldo, Barbara; Dotti, Gianpietro

    2016-01-01

    While the clinical progress of chimeric antigen receptor T cell (CAR-T) immunotherapy has garnered attention to the field, our understanding of the biology of these chimeric molecules is still emerging. Our aim within this review is to bring to light the mechanistic understanding of these multi-modular receptors and how these individual components confer particular properties to CAR-Ts. In addition, we will discuss extrinsic factors that can be manipulated to influence CAR-T performance such as choice of cellular population, culturing conditions and additional modifications that enhance their activity particularly in solid tumors. Finally, we will also consider the emerging toxicity associated with CAR-Ts. By breaking apart the CAR and examining the role of each piece, we can build a better functioning cellular vehicle for optimized treatment of cancer patients. PMID:26797495

  15. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Variability and repertoire size of T-cell receptor V alpha gene segments.

    PubMed

    Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

    The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

  17. Anti-EGFRvIII Chimeric Antigen Receptor-Modified T Cells for Adoptive Cell Therapy of Glioblastoma

    PubMed Central

    Ren, Pei-pei; Li, Ming; Li, Tian-fang; Han, Shuang-yin

    2017-01-01

    Glioblastoma (GBM) is one of the most devastating brain tumors with poor prognosis and high mortality. Although radical surgical treatment with subsequent radiation and chemotherapy can improve the survival, the efficacy of such regimens is insufficient because the GBM cells can spread and destroy normal brain structures. Moreover, these non-specific treatments may damage adjacent healthy brain tissue. It is thus imperative to develop novel therapies to precisely target invasive tumor cells without damaging normal tissues. Immunotherapy is a promising approach due to its capability to suppress the growth of various tumors in preclinical model and clinical trials. Adoptive cell therapy (ACT) using T cells engineered with chimeric antigen receptor (CAR) targeting an ideal molecular marker in GBM, e.g. epidermal growth factor receptor type III (EGFRvIII) has demonstrated a satisfactory efficacy in treating malignant brain tumors. Here we summarize the recent progresses in immunotherapeutic strategy using CAR-modified T cells oriented to EGFRvIII against GBM. PMID:28302023

  18. Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

    PubMed Central

    Yates, Kathleen B.; Bi, Kevin; Darko, Samuel; Godec, Jernej; Gerdemann, Ulrike; Swadling, Leo; Douek, Daniel C.; Klenerman, Paul; Barnes, Eleanor J.; Sharpe, Arlene H.

    2017-01-01

    Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV. PMID:28934479

  19. Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

    PubMed Central

    Boulassel, Mohamed-Rachid; Galal, Ahmed

    2012-01-01

    Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

  20. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    PubMed

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  1. T-cell Receptor Specificity Maintained by Altered Thermodynamics*

    PubMed Central

    Madura, Florian; Rizkallah, Pierre J.; Miles, Kim M.; Holland, Christopher J.; Bulek, Anna M.; Fuller, Anna; Schauenburg, Andrea J. A.; Miles, John J.; Liddy, Nathaniel; Sami, Malkit; Li, Yi; Hossain, Moushumi; Baker, Brian M.; Jakobsen, Bent K.; Sewell, Andrew K.; Cole, David K.

    2013-01-01

    The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition. PMID:23698002

  2. 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

  3. 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

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

    PubMed

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

    2017-05-12

    Hypertension remains to be a global public health burden and demands novel intervention strategies such as targeting T cells and T-cell-derived cytokines. Mineralocorticoid receptor (MR) antagonists have been clinically used to treat hypertension. However, the function of T-cell MR in blood pressure (BP) regulation has not been elucidated. We aim to determine the role of T-cell MR in BP regulation and to explore the mechanism. Using T-cell MR knockout mouse in combination with angiotensin II-induced hypertensive mouse model, we demonstrated that MR deficiency in T cells strikingly decreased both systolic and diastolic BP and attenuated renal and vascular damage. Flow cytometric analysis showed that T-cell MR knockout mitigated angiotensin II-induced accumulation of interferon-gamma (IFN-γ)-producing T cells, particularly CD8 + population, in both kidneys and aortas. Similarly, eplerenone attenuated angiotensin II-induced elevation of BP and accumulation of IFN-γ-producing T cells in wild-type mice. In cultured CD8 + T cells, T-cell MR knockout suppressed IFN-γ expression whereas T-cell MR overexpression and aldosterone both enhanced IFN-γ expression. At the molecular level, MR interacted with NFAT1 (nuclear factor of activated T-cells 1) and activator protein-1 in T cells. Finally, T-cell MR overexpressing mice manifested more elevated BP compared with control mice after angiotensin II infusion and such difference was abolished by IFN-γ-neutralizing antibodies. MR may interact with NFAT1 and activator protein-1 to control IFN-γ in T cells and to regulate target organ damage and ultimately BP. Targeting MR in T cells specifically may be an effective novel approach for hypertension treatment. © 2017 American Heart Association, Inc.

  5. Co-ordination of incoming and outgoing traffic in antigen-presenting cells by pattern recognition receptors and T cells.

    PubMed

    Nair, Priyanka; Amsen, Derk; Blander, J Magarian

    2011-12-01

    Dendritic cells are innate sentinels of the immune system and potent activators of naÏve T cells. Mechanisms must exist to enable these cells to achieve maximal activation of T cells specific for microbial antigens, while avoiding activation of T cells specific for self-antigens. Here we discuss how a combination of signals from pattern recognition receptors and T cells co-ordinates subcellular trafficking of antigen with both major histocompatibility complex class I and class II molecules and T-cell costimulatory molecules, resulting in the preferential presentation of microbial peptides within a stimulatory context. © 2011 John Wiley & Sons A/S.

  6. Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus.

    PubMed

    Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F; Criscitiello, Michael F

    2018-04-17

    Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. © 2018, Ott et al.

  7. Somatic hypermutation of T cell receptor α chain contributes to selection in nurse shark thymus

    PubMed Central

    Ott, Jeannine A; Castro, Caitlin D; Deiss, Thaddeus C; Ohta, Yuko; Flajnik, Martin F

    2018-01-01

    Since the discovery of the T cell receptor (TcR), immunologists have assigned somatic hypermutation (SHM) as a mechanism employed solely by B cells to diversify their antigen receptors. Remarkably, we found SHM acting in the thymus on α chain locus of shark TcR. SHM in developing shark T cells likely is catalyzed by activation-induced cytidine deaminase (AID) and results in both point and tandem mutations that accumulate non-conservative amino acid replacements within complementarity-determining regions (CDRs). Mutation frequency at TcRα was as high as that seen at B cell receptor loci (BcR) in sharks and mammals, and the mechanism of SHM shares unique characteristics first detected at shark BcR loci. Additionally, fluorescence in situ hybridization showed the strongest AID expression in thymic corticomedullary junction and medulla. We suggest that TcRα utilizes SHM to broaden diversification of the primary αβ T cell repertoire in sharks, the first reported use in vertebrates. PMID:29664399

  8. Lymphocyte apheresis for chimeric antigen receptor T-cell manufacturing in children and young adults with leukemia and neuroblastoma.

    PubMed

    Ceppi, Francesco; Rivers, Julie; Annesley, Colleen; Pinto, Navin; Park, Julie R; Lindgren, Catherine; Mgebroff, Stephanie; Linn, Naomi; Delaney, Meghan; Gardner, Rebecca A

    2018-06-01

    The first step in the production of chimeric antigen receptor T cells is the collection of autologous T cells using apheresis technology. The procedure is technically challenging, because patients often have low leukocyte counts and are heavily pretreated with multiple lines of chemotherapy, marrow transplantation, and/or radiotherapy. Here, we report our experience of collecting T lymphocytes for chimeric antigen receptor T-cell manufacturing in pediatric and young adult patients with leukemia, non-Hodgkin lymphoma, or neuroblastoma. Apheresis procedures were performed on a COBE Spectra machine using the mononuclear cell program, with a collection target of 1 × 10 9 total mononuclear cells per kilogram. Data were collected regarding preapheresis and postapheresis blood counts, apheresis parameters, products, and adverse events. Ninety-nine patients (ages 1.3-25.7 years) and 102 apheresis events were available for analysis. Patients underwent apheresis at a variety of absolute lymphocyte cell counts, with a median absolute lymphocyte count of 944 cells/μL (range, 142-6944 cells/μL). Twenty-two patients (21.6%) had absolute lymphocyte counts less than 500 cells/μL. The mononuclear cell target was obtained in 100% of all apheresis harvests, and chimeric antigen receptor T-cell production was possible from the majority of collections (94%). Mononuclear cell collection efficiency was 65.4%, and T-lymphocyte collection efficiency was 83.4%. Ten patients (9.8%) presented with minor adverse events during the 102 apheresis procedures, with one exception of a severe allergy. Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts. © 2018 AABB.

  9. Characterization of avian T-cell receptor γ genes

    PubMed Central

    Six, Adrien; Rast, Jonathan P.; McCormack, Wayne T.; Dunon, Dominique; Courtois, David; Li, Yue; Chen, Chen-lo H.; Cooper, Max D.

    1996-01-01

    In birds and mammals T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the γδ T-cell lineage, the present studies sought to define the chicken TCRγ locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR γ chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCRγ cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a γδ T-cell line, but not in B or αβ T-cell lines. Three multimember Vγ subfamilies, three Jγ gene segments, and a single constant region Cγ gene were identified in the avian TCRγ locus. Members of each of the three Vγ subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCRγ repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-Jγ recombination, nuclease activity, and P- and N-nucleotide additions to generate a diverse repertoire of avian TCRγ genes early in ontogeny. PMID:8986811

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

    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 ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

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

    PubMed

    Borroto, Aldo; Abia, David; Alarcón, Balbino

    2014-09-01

    Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 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

  13. Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells.

    PubMed

    Noyan, Kajsa; Nguyen, Son; Betts, Michael R; Sönnerborg, Anders; Buggert, Marcus

    2018-01-01

    Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a "healthy" state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status.

  14. delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

    PubMed

    Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

    2006-02-01

    Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

  15. Early and Definitive Diagnosis of Toxic Shock Syndrome by Detection of Marked Expansion of T-Cell-Receptor Vβ2-Positive T Cells

    PubMed Central

    Kato, Hidehito; Yamada, Ritsuko; Okano, Hiroya; Ohta, Hiroaki; Imanishi, Ken’ichi; Kikuchi, Ken; Totsuka, Kyouichi; Uchiyama, Takehiko

    2003-01-01

    We describe two cases of early toxic shock syndrome, caused by the superantigen produced from methicillin-resistant Staphylococcus aureus and diagnosed on the basis of an expansion of T-cell-receptor Vβ2-positive T cells. One case-patient showed atypical symptoms. Our results indicate that diagnostic systems incorporating laboratory techniques are essential for rapid, definitive diagnosis of toxic shock syndrome. PMID:12643839

  16. Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function.

    PubMed

    Bubeck Wardenburg, J; Fu, C; Jackman, J K; Flotow, H; Wilkinson, S E; Williams, D H; Johnson, R; Kong, G; Chan, A C; Findell, P R

    1996-08-16

    Two families of tyrosine kinases, the Src and Syk families, are required for T-cell receptor activation. While the Src kinases are responsible for phosphorylation of receptor-encoded signaling motifs and for up-regulation of ZAP-70 activity, the downstream substrates of ZAP-70 are unknown. Evidence is presented herein that the Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is a substrate of ZAP-70. Phosphorylation of SLP-76 is diminished in T cells that express a catalytically inactive ZAP-70. Moreover, SLP-76 is preferentially phosphorylated by ZAP-70 in vitro and in heterologous cellular systems. In T cells, overexpression of wild-type SLP-76 results in a hyperactive receptor, while expression of a SLP-76 molecule that is unable to be tyrosine-phosphorylated attenuates receptor function. In addition, the SH2 domain of SLP-76 is required for T-cell receptor function, although its role is independent of the ability of SLP-76 to undergo tyrosine phosphorylation. As SLP-76 interacts with both Grb2 and phospholipase C-gamma1, these data indicate that phosphorylation of SLP-76 by ZAP-70 provides an important functional link between the T-cell receptor and activation of ras and calcium pathways.

  17. Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

    PubMed

    Spriggs, M K; Lioubin, P J; Slack, J; Dower, S K; Jonas, U; Cosman, D; Sims, J E; Bauer, J

    1990-12-25

    Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.

  18. Chimeric Antigen Receptor T-Cells (CAR T-Cells) for Cancer Immunotherapy - Moving Target for Industry?

    PubMed

    Salmikangas, Paula; Kinsella, Niamh; Chamberlain, Paul

    2018-05-31

    The first CD19 CAR T-cell products, Kymriah and Yescarta, are entering the US market and also being evaluated for marketing authorization in the EU. This breakthrough has expanded the interest and also investments towards novel chimeric antigen receptor (CAR) designs, both for hematological malignancies and solid tumors. At the same time, there is active development in moving from autologous products to allogeneic, off-the-shelf -products. New manufacturing technologies are also emerging for production of these complex genetically-modified cells and even decentralized manufacturing in hospitals is under consideration. However, the high potency of CAR T-cells is associated with toxicity and not all patients respond to the treatment. In addition, the number of patient and product variables impacting the clinical outcome is high. The race towards novel CAR T treatment options for cancer patients has begun, but without careful design of the constructs and overall understanding of the factors that impact the ultimate outcome in each case, the road towards commercial success may be long and winding. This review discusses the product- and patient-related variables that may pose challenges for the industry and developers both from the scientific and regulatory perspective.

  19. αβ T cell receptors as predictors of health and disease

    PubMed Central

    Attaf, Meriem; Huseby, Eric; Sewell, Andrew K

    2015-01-01

    The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR ‘signatures' raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome. PMID:25619506

  20. 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

  1. Lack of T-cell receptor-induced signaling is crucial for CD95 ligand up-regulation and protects cutaneous T-cell lymphoma cells from activation-induced cell death.

    PubMed

    Klemke, Claus-Detlev; Brenner, Dirk; Weiss, Eva-Maria; Schmidt, Marc; Leverkus, Martin; Gülow, Karsten; Krammer, Peter H

    2009-05-15

    Restimulation of previously activated T cells via the T-cell receptor (TCR) leads to activation-induced cell death (AICD), which is, at least in part, dependent on the death receptor CD95 (APO-1, FAS) and its natural ligand (CD95L). Here, we characterize cutaneous T-cell lymphoma (CTCL) cells (CTCL tumor cell lines and primary CTCL tumor cells from CTCL patients) as AICD resistant. We show that CTCL cells have elevated levels of the CD95-inhibitory protein cFLIP. However, cFLIP is not responsible for CTCL AICD resistance. Instead, our data suggest that reduced TCR-proximal signaling in CTCL cells is responsible for the observed AICD resistance. CTCL cells exhibit no PLC-gamma1 activity, resulting in an impaired Ca(2+)release and reduced generation of reactive oxygen species upon TCR stimulation. Ca(2+) and ROS production are crucial for up-regulation of CD95L and reconstitution of both signals resulted in AICD sensitivity of CTCL cells. In accordance with these data, CTCL tumor cells from patients with Sézary syndrome do not up-regulate CD95L upon TCR-stimulation and are therefore resistant to AICD. These results show a novel mechanism of AICD resistance in CTCL that could have future therapeutic implications to overcome apoptosis resistance in CTCL patients.

  2. T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome

    PubMed Central

    Gaballa, Ahmed; Sundin, Mikael; Stikvoort, Arwen; Abumaree, Muhamed; Uzunel, Mehmet; Sairafi, Darius; Uhlin, Michael

    2016-01-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up. PMID:27727179

  3. The mediator subunit Med23 contributes to controlling T-cell activation and prevents autoimmunity.

    PubMed

    Sun, Yang; Zhu, Xiaoyan; Chen, Xufeng; Liu, Haifeng; Xu, Yu; Chu, Yajing; Wang, Gang; Liu, Xiaolong

    2014-10-10

    T-cell activation is critical for successful immune responses and is controlled at multiple levels. Although many changes of T-cell receptor-associated signalling molecules affect T-cell activation, the transcriptional mechanisms that control this process remain largely unknown. Here we find that T cell-specific deletion of the mediator subunit Med23 leads to hyperactivation of T cells and aged Med23-deficient mice exhibit an autoimmune syndrome. Med23 specifically and consistently promotes the transcription of multiple negative regulators of T-cell activation. In the absence of Med23, the T-cell activation threshold is lower, which results in enhanced antitumour T-cell function. Cumulatively, our data suggest that Med23 contributes to controlling T-cell activation at the transcriptional level and prevents the development of autoimmunity.

  4. Genetic tracing of the gustatory and trigeminal neural pathways originating from T1R3-expressing taste receptor cells and solitary chemoreceptor cells.

    PubMed

    Ohmoto, Makoto; Matsumoto, Ichiro; Yasuoka, Akihito; Yoshihara, Yoshihiro; Abe, Keiko

    2008-08-01

    We established transgenic mouse lines expressing a transneuronal tracer, wheat germ agglutinin (WGA), under the control of mouse T1R3 gene promoter/enhancer. In the taste buds, WGA transgene was faithfully expressed in T1R3-positive sweet/umami taste receptor cells. WGA protein was transferred not laterally to the synapse-bearing, sour-responsive type III cells in the taste buds but directly to a subset of neurons in the geniculate and nodose/petrosal ganglia, and further conveyed to a rostro-central region of the nucleus of solitary tract. In addition, WGA was expressed in solitary chemoreceptor cells in the nasal epithelium and transferred along the trigeminal sensory pathway to the brainstem neurons. The solitary chemoreceptor cells endogenously expressed T1R3 together with bitter taste receptors T2Rs. This result shows an exceptional signature of receptor expression. Thus, the t1r3-WGA transgenic mice revealed the sweet/umami gustatory pathways from taste receptor cells and the trigeminal neural pathway from solitary chemoreceptor cells.

  5. A T-Cell Receptor Breaks the Rules | Center for Cancer Research

    Cancer.gov

    Most mature T cells function immunologically when a T-cell receptor (TCR) located on the cell surface encounters and engages its ligand, a major histocompatability complex (MHC), which displays a specific part of a target protein called an antigen. This antigen-presenting complex is assembled from one of the dozen or so MHC molecules that every person inherits from their parents; and the antigen fragment, called a peptide epitope, is excised from one of thousands of possible proteins—originally part of an invading pathogen or a cancer cell—that T cells are capable of identifying and attacking. The framework of an MHC molecule holding a centrally displayed or “presented” peptide is what engages the TCR and triggers T-cell action. This role of MHC molecules presenting antigens to the TCR is a central tenet of immunology, with the fit between a TCR and the MHC framework actually “hardwired” into their three-dimensional structures.

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

    PubMed

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

    2015-06-01

    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 with 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 toward a Th17 phenotype by lepr-deficient T cells was attributed to reduced activation of the STAT3 and its downstream targets. This study establishes cell-intrinsic roles for LepR signaling in the immune system and suggests that leptin signaling during T cell differentiation plays a crucial role in T cell peripheral effector function. Copyright © 2015 by The American Association of Immunologists, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    1986-08-01

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

  8. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

    PubMed

    Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

    2017-06-01

    Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Anti-EGFRvIII Chimeric Antigen Receptor-Modified T Cells for Adoptive Cell Therapy of Glioblastoma.

    PubMed

    Ren, Pei-Pei; Li, Ming; Li, Tian-Fang; Han, Shuang-Yin

    2017-01-01

    Glioblastoma (GBM) is one of the most devastating brain tumors with poor prognosis and high mortality. Although radical surgical treatment with subsequent radiation and chemotherapy can improve the survival, the efficacy of such regimens is insufficient because the GBM cells can spread and destroy normal brain structures. Moreover, these non-specific treatments may damage adjacent healthy brain tissue. It is thus imperative to develop novel therapies to precisely target invasive tumor cells without damaging normal tissues. Immunotherapy is a promising approach due to its capability to suppress the growth of various tumors in preclinical model and clinical trials. Adoptive cell therapy (ACT) using T cells engineered with chimeric antigen receptor (CAR) targeting an ideal molecular marker in GBM, e.g. epidermal growth factor receptor type III (EGFRvIII) has demonstrated a satisfactory efficacy in treating malignant brain tumors. Here we summarize the recent progresses in immunotherapeutic strategy using CAR-modified T cells oriented to EGFRvIII against GBM. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Generating and Expanding Autologous Chimeric Antigen Receptor T Cells from Patients with Acute Myeloid Leukemia.

    PubMed

    Kenderian, Saad S; June, Carl H; Gill, Saar

    2017-01-01

    Adoptive transfer of genetically engineered T cells can lead to profound and durable responses in patients with hematologic malignancies, generating enormous enthusiasm among scientists, clinicians, patients, and biotechnology companies. The success of adoptive cellular immunotherapy depends upon the ability to manufacture good quality T cells. We discuss here the methodologies and reagents that are used to generate T cells for the preclinical study of chimeric antigen receptor T cell therapy for acute myeloid leukemia (AML).

  11. T-cell receptor gene therapy: critical parameters for clinical success.

    PubMed

    Linnemann, Carsten; Schumacher, Ton N M; Bendle, Gavin M

    2011-09-01

    T-cell receptor (TCR) gene therapy aims to induce immune reactivity against tumors by introducing genes encoding a tumor-reactive TCR into patient T cells. This approach has been extensively tested in preclinical mouse models, and initial clinical trials have demonstrated the feasibility and potential of TCR gene therapy as a cancer treatment. However, data obtained from preclinical and clinical studies suggest that both the therapeutic efficacy and the safety of TCR gene therapy can be and needs to be further enhanced. This review highlights those strategies that can be followed to develop TCR gene therapy into a clinically relevant treatment option for cancer patients.

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

    PubMed Central

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

    2016-01-01

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

  13. Angiotensin II type 1 receptor blocker telmisartan induces apoptosis and autophagy in adult T-cell leukemia cells.

    PubMed

    Kozako, Tomohiro; Soeda, Shuhei; Yoshimitsu, Makoto; Arima, Naomichi; Kuroki, Ayako; Hirata, Shinya; Tanaka, Hiroaki; Imakyure, Osamu; Tone, Nanako; Honda, Shin-Ichiro; Soeda, Shinji

    2016-05-01

    Adult T-cell leukemia/lymphoma (ATL), an aggressive T-cell malignancy that develops after long-term infection with human T-cell leukemia virus (HTLV-1), requires new treatments. Drug repositioning, reuse of a drug previously approved for the treatment of another condition to treat ATL, offers the possibility of reduced time and risk. Among clinically available angiotensin II receptor blockers, telmisartan is well known for its unique ability to activate peroxisome proliferator-activated receptor-γ, which plays various roles in lipid metabolism, cellular differentiation, and apoptosis. Here, telmisartan reduced cell viability and enhanced apoptotic cells via caspase activation in ex vivo peripheral blood monocytes from asymptomatic HTLV-1 carriers (ACs) or via caspase-independent cell death in acute-type ATL, which has a poor prognosis. Telmisartan also induced significant growth inhibition and apoptosis in leukemia cell lines via caspase activation, whereas other angiotensin II receptor blockers did not induce cell death. Interestingly, telmisartan increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation and autophagy. Thus, telmisartan simultaneously caused caspase activation and autophagy. A hypertension medication with antiproliferation effects on primary and leukemia cells is intriguing. Patients with an early diagnosis of ATL are generally monitored until the disease progresses; thus, suppression of progression from AC and indolent ATL to acute ATL is important. Our results suggest that telmisartan is highly effective against primary cells and leukemia cell lines in caspase-dependent and -independent manners, and its clinical use may suppress acute transformation and improve prognosis of patients with this mortal disease. This is the first report demonstrating a cell growth-inhibitory effect of telmisartan in fresh peripheral blood mononuclear cells from leukemia patients.

  14. A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells.

    PubMed

    Moeller, Maria; Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Tanner, Jane E; Cerutti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

    2004-05-01

    T cells engineered to express single-chain antibody receptors that incorporate TCR-zeta and cluster designation (CD)28 signaling domains (scFv-alpha-erbB2-CD28-zeta) can be redirected in vivo to cancer cells that lack triggering costimulatory molecules. To assess the contribution of CD28 signaling to the function of the scFv-CD28-zeta receptor, we expressed a series of mutated scFv-CD28-zeta receptors directed against erbB2. Residues known to be critical for CD28 signaling were mutated from tyrosine to phenylalanine at position 170 or proline to alanine at positions 187 and 190. Primary mouse T cells expressing either of the mutant receptors demonstrated impaired cytokine (IFN-gamma and GM-CSF) production and decreased proliferation after antigen ligation in vitro and decreased antitumor efficacy in vivo compared with T cells expressing the wild-type scFv-CD28-zeta receptor, suggesting a key signaling role for the CD28 component of the scFv-CD28-zeta receptor. Importantly, cell surface expression, binding capacity and cytolytic activity mediated by the scFv-CD28-zeta receptor were not diminished by either mutation. Overall, this study has definitively demonstrated a functional role for the CD28 component of the scFv-CD28-zeta receptor and has shown that incorporation of costimulatory activity in chimeric scFv receptors is a powerful approach for improving adoptive cancer immunotherapy.

  15. Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

    PubMed

    Ridley, Anna; Hatano, Hiroko; Wong-Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K; Al-Mossawi, Hussein; Ladell, Kristin; Price, David A; Bowness, Paul; Kollnberger, Simon

    2016-04-01

    In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA. © 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.

  16. Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

    1994-09-01

    An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

  17. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    PubMed

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  18. Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices

    NASA Astrophysics Data System (ADS)

    Singha, Santiswarup; Shao, Kun; Yang, Yang; Clemente-Casares, Xavier; Solé, Patricia; Clemente, Antonio; Blanco, Jesús; Dai, Qin; Song, Fayi; Liu, Shang Wan; Yamanouchi, Jun; Umeshappa, Channakeshava Sokke; Nanjundappa, Roopa Hebbandi; Detampel, Pascal; Amrein, Matthias; Fandos, César; Tanguay, Robert; Newbigging, Susan; Serra, Pau; Khadra, Anmar; Chan, Warren C. W.; Santamaria, Pere

    2017-07-01

    We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

  19. Expansion of natural (NK1+) T cells that express alpha beta T cell receptors in transporters associated with antigen presentation-1 null and thymus leukemia antigen positive mice

    PubMed Central

    1996-01-01

    Thymic selection of natural killer-1+ natural T cells that express alpha beta T cell receptors requires a conserved beta 2-microglobulin- associated molecule, presumably CD1d, displayed by CD4+8+ thymocytes. Here we demonstrate that positive selection of natural T cells occurs independent of transporters associated with antigen presentation-1 (TAP- 1) function. Moreover, natural T cells in TAP-1o/o mice are numerically expanded. Several H-2 class Ib molecules function in a TAP-independent manner, suggesting that if expressed in TAP-1o/o thymocytes, they could play a role in natural T cell development. Of these class Ib molecules, H-2TL is expressed by TAP-1o/o thymocytes. Moreover, we find that thymi of TL+ mice congenic or transgenic for H-2T18 also have a numerically expanded natural T cell repertoire compared with TL- mice. This expansion, as in TAP-1o/o thymi, is evident in each of the limited T cell receptor V beta chains expressed by natural T cells, suggesting that TL and CD1d impact similar repertoires. Thus TL, in addition to CD1d, plays a role in natural T cell development. PMID:8879233

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

    PubMed Central

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

    2016-01-01

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

  1. Chimeric Antigen Receptor T cells for B Cell Neoplasms: Choose the Right CAR for You.

    PubMed

    Ruella, Marco; June, Carl H

    2016-10-01

    Genetic redirection of T lymphocytes allows us to unleash these potent cellular immune effectors against cancer. Chimeric antigen receptor (CAR) T cells are the best-in-class example that genetic engineering of T cells can lead to deep and durable responses, as has been shown in several clinical trials for CD19+ B cell malignancies. As a consequence, in the last few years, several academic institutions and commercial partners have started developing anti-CD19 CAR T cell products. Although most of these T cell products are highly effective in vivo, basic differences among them can generate different performance characteristics and thereby impact their long-term clinical outcome. Several strategies are being implemented in order to solve the current open issues of CART19 therapy: (i) increasing efficacy against indolent B cell leukemias and lymphomas, (ii) avoiding or preventing antigen-loss relapses, (iii) reducing and managing toxicity, and (iv) bringing this CART therapy to routine clinical practice. The field of CART therapies is thriving, and exciting new avenues are opening for both scientists and patients.

  2. T-Cell Receptor- and CD28-induced Vav1 activity is required for the accumulation of primed T cells into antigenic tissue

    PubMed Central

    David, Rachel; Ma, Liang; Ivetic, Aleksandar; Takesono, Aya; Ridley, Anne J.; Chai, Jian-Guo; Tybulewicz, Victor; Marelli-Berg, Federica M.

    2016-01-01

    Localization of primed T cells to antigenic tissue is essential for the development of effective immunity. Together with tissue-selective homing molecules, T-cell receptor (TCR)- and CD28-mediated signals have been shown to promote transendothelial migration of specific T cells into non-lymphoid antigen-rich tissue tissue. However, the cellular and molecular requirements for T-cell accumulation to target tissue following their recruitment are largely undefined. The guanine nucleotide exchange factor (GEF) Vav1 has an integral role in coupling TCR and CD28 to signalling pathways that regulate T cell activation and migration. Here, we have investigated the contribution of TCR- and CD28-induced Vav1 activity to the trafficking and localization of primed HY-specific CD4+ T cells to antigenic sites. Severe migratory defects displayed by Vav1-/- T cells in vitro were fully compensated by a combination of shear flow and chemokines, leading to normal recruitment of Vav1-/- T cells in vivo. In contrast, Vav1-/- T-cell retention into antigen-rich tissue was severely impaired, reflecting their inability to engage in sustained TCR- and CD28-mediated interactions with tissue-resident antigen-presenting cells (APCs). This novel function of APC-induced, TCR- and CD28-mediated Vav1 activity in the regulation of effector T-cell immunity highlights its potential as a therapeutic target in T-cell-mediated tissue damage. PMID:19060239

  3. Chimeric Antigen Receptors T Cell Therapy in Solid Tumor: Challenges and Clinical Applications.

    PubMed

    Mirzaei, Hamid R; Rodriguez, Analiz; Shepphird, Jennifer; Brown, Christine E; Badie, Behnam

    2017-01-01

    Adoptive cellular immunotherapy (ACT) employing engineered T lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated promising antitumor effects in advanced hematologic cancers, such as relapsed or refractory acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma, supporting the translation of ACT to non-hematological malignancies. Although CAR T cell therapy has made remarkable strides in the treatment of patients with certain hematological cancers, in solid tumors success has been limited likely due to heterogeneous antigen expression, immunosuppressive networks in the tumor microenvironment limiting CAR T cell function and persistence, and suboptimal trafficking to solid tumors. Here, we outline specific approaches to overcome barriers to CAR T cell effectiveness in the context of the tumor microenvironment and offer our perspective on how expanding the use of CAR T cells in solid tumors may require modifications in CAR T cell design. We anticipate these modifications will further expand CAR T cell therapy in clinical practice.

  4. T-cell receptor repertoire variation may be associated with type 2 diabetes mellitus in humans.

    PubMed

    Frankl, Joseph A; Thearle, Marie S; Desmarais, Cindy; Bogardus, Clifton; Krakoff, Jonathan

    2016-03-01

    Recent work in Pima Indians, a population with high rates of obesity and type 2 diabetes mellitus (T2DM), demonstrated that human leukocyte antigen haplotype DRB1*02 carriers have an increased acute insulin response and decreased risk for the development of T2DM, implicating loss of self-tolerance in the pathogenesis of T2DM. Advances in genomic sequencing have made T-cell receptor repertoire analysis a practical mode of investigation. High-throughput sequencing of T-cell receptor complementarity-determining region 3 was carried out in male Pima Indians with normal glucose regulation (n = 11; age = 31 ± 8 years; %fat = 30.2 ± 8.7%) and the protective DRB1*02 haplotype versus those with T2DM without DRB1*02 (n = 7; age = 34 ± 8 years; %fat = 31.2 ± 4.7%). Findings were partially replicated in another cohort by assessing the predictive ability of T-cell receptor variation on risk of T2DM in Pima Indian men (n = 27; age = 28.9 ± 7.1 years; %fat = 28.8 ± 7.1%) and women (n = 20; age = 29 ± 7.0 years; %fat = 37.1 ± 6.8%) with baseline normal glucose regulation but without the protective haplotype who were invited to follow-up examinations as frequently as every 2 years where diabetes status was assessed by a 75-g oral glucose tolerance test. Of these subjects, 13 developed diabetes. T-cell receptor complementarity-determining region 3 length was shorter in those with T2DM, and a one-nucleotide decrease in complementarity-determining region 3 length was associated with a nearly threefold increase in risk for future diabetes. The frequency of one variable gene, TRBV7-8, was higher in those with T2DM. A 1% increase in TRBV7-8 frequency was associated with a greater than threefold increase in diabetes risk. These results indicate that T-cell autoimmunity may be an important component in progression to T2DM in Pima Indians. Copyright © 2015 John Wiley & Sons, Ltd.

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

    PubMed Central

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

    2015-01-01

    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

  6. Antigen-Specific Immune Modulation Targets mTORC1 Function To Drive Chemokine Receptor-Mediated T Cell Tolerance.

    PubMed

    Chen, Weirong; Wan, Xiaoxiao; Ukah, Tobechukwu K; Miller, Mindy M; Barik, Subhasis; Cattin-Roy, Alexis N; Zaghouani, Habib

    2016-11-01

    To contain autoimmunity, pathogenic T cells must be eliminated or diverted from reaching the target organ. Recently, we defined a novel form of T cell tolerance whereby treatment with Ag downregulates expression of the chemokine receptor CXCR3 and prevents diabetogenic Th1 cells from reaching the pancreas, leading to suppression of type 1 diabetes (T1D). This report defines the signaling events underlying Ag-induced chemokine receptor-mediated tolerance. Specifically, we show that the mammalian target of rapamycin complex 1 (mTORC1) is a major target for induction of CXCR3 downregulation and crippling of Th1 cells. Indeed, Ag administration induces upregulation of programmed death-ligand 1 on dendritic cells in a T cell-dependent manner. In return, programmed death-ligand 1 interacts with the constitutively expressed programmed death-1 on the target T cells and stimulates docking of Src homology 2 domain-containing tyrosine phosphatase 2 phosphatase to the cytoplasmic tail of programmed death-1. Active Src homology 2 domain-containing tyrosine phosphatase 2 impairs the signaling function of the PI3K/protein kinase B (AKT) pathway, leading to functional defect of mTORC1, downregulation of CXCR3 expression, and suppression of T1D. Thus, mTORC1 component of the metabolic pathway serves as a target for chemokine receptor-mediated T cell tolerance and suppression of T1D. Copyright © 2016 by The American Association of Immunologists, Inc.

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

    PubMed Central

    Geyer, Mark B.; Brentjens, Renier J.

    2016-01-01

    The past several years have been marked by extraordinary advances in clinical applications of immunotherapy. In particular, adoptive cellular therapy utilizing chimeric antigen receptor (CAR) modified T cells targeted to CD19 has demonstrated substantial clinical efficacy in children and adults with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL), and durable clinical benefit in a smaller subset of patients with relapsed or refractory chronic lymphocytic leukemia (CLL) or B cell non-Hodgkin lymphoma (B-NHL). Early phase clinical trials are presently assessing CAR T cell safety and efficacy in additional malignancies. Herein, we discuss clinical results from the largest series to date investigating CD19-targeted CAR T cells in B-ALL, CLL, and B-NHL, including discussion of differences in CAR T cell design and production and treatment approach, as well as clinical efficacy, nature of severe cytokine release syndrome and neurologic toxicities, and CAR T cell expansion and persistence. We additionally review the current and forthcoming use of CAR T cells in multiple myeloma and several solid tumors, and highlight challenges and opportunities afforded by the current state of CAR T cell therapies, including strategies to overcome inhibitory aspects of the tumor microenvironment and enhance antitumor efficacy. PMID:27592405

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

    PubMed

    Geyer, Mark B; Brentjens, Renier J

    2016-11-01

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

  9. Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

    PubMed

    Meghraoui-Kheddar, Aïda; Pierre, Alexandre; Sellami, Mehdi; Audonnet, Sandra; Lemaire, Flora; Le Naour, Richard

    2017-09-01

    Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4 + and CD8 + T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response. Copyright © 2017 the American Physiological Society.

  10. CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.

    PubMed

    Gollmer, Kathrin; Asperti-Boursin, François; Tanaka, Yoshihiko; Okkenhaug, Klaus; Vanhaesebroeck, Bart; Peterson, Jeffrey R; Fukui, Yoshinori; Donnadieu, Emmanuel; Stein, Jens V

    2009-07-16

    CD4(+) T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4(+) T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3Kdelta(D910A/D910A) or PI3Kgamma-deficient TCR-tg CD4(+) T cells showed similar responsiveness to CCL21 costimulation as control CD4(+) T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4(+) T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca(2+) signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

  11. Methods for quantifying T cell receptor binding affinities and thermodynamics

    PubMed Central

    Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

  12. Chimeric antigen receptor T-cell immunotherapy for glioblastoma: practical insights for neurosurgeons.

    PubMed

    Choi, Bryan D; Curry, William T; Carter, Bob S; Maus, Marcela V

    2018-06-01

    The prognosis for glioblastoma (GBM) remains exceedingly poor despite state-of-the-art multimodal therapy. Immunotherapy, particularly with cytotoxic T cells, represents a promising alternative. Perhaps the most prominent T-cell technology is the chimeric antigen receptor (CAR), which in 2017 received accelerated approval from the Food and Drug Administration for the treatment of hematological malignancies. Several CARs for GBM have been recently tested in clinical trials with exciting results. The authors review these clinical data and discuss areas of ongoing research.

  13. Advancing chimeric antigen receptor T cell therapy with CRISPR/Cas9.

    PubMed

    Ren, Jiangtao; Zhao, Yangbing

    2017-09-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system, an RNA-guided DNA targeting technology, is triggering a revolution in the field of biology. CRISPR/Cas9 has demonstrated great potential for genetic manipulation. In this review, we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications, especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy. Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared. The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored. We also address the safety concerns associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy. As an integration-free gene insertion method, CRISPR/Cas9 holds great promise as an efficient gene knock-in platform. Given the tremendous progress that has been made in the past few years, we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.

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

    PubMed Central

    Champagne, Eric

    2011-01-01

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

  15. γδ T cell receptor ligands and modes of antigen recognition.

    PubMed

    Champagne, Eric

    2011-04-01

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

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

    PubMed

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

    2012-10-02

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

  17. A caspase 8-based suicide switch induces apoptosis in nanobody-directed chimeric receptor expressing T cells.

    PubMed

    Khaleghi, Sepideh; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J; Pognonec, Philippe

    2012-04-01

    In accordance with the two-step hypothesis of T cell activation and the observation that stimulation through the T cell receptor (TCR) alone may lead to anergy, we focused on the introduction of co-stimulatory signaling to this type of receptors to achieve optimal activation. Enhanced mRNA and cell surface receptor expression via the co-stimulatory gene fragment (OX40) was confirmed by RT-PCR and flow cytometry. Inclusion of the OX40 co-stimulatory signaling region in series with the TCR led to enhanced antigen-induced IL-2 production after stimulation by MUC1-expressing cancer cell lines as compared to the chimeric receptor without OX40. Moreover, with the aim of maintaining high efficiency, while providing a means of controlling any possible unwanted proliferation in vivo, a regulation system was used. This controls the dimerization of a membrane-bound caspase 8 protein. Toward that goal, pFKC8 and CAR constructs were co-transfected into Jurkat cells, and the level of apoptosis was measured. 24 h after addition of the dimerizer, a 91% decrease in transfected cells was observed.

  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

    2016-03-03

    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. © 2016 by The American Society of Hematology.

  19. Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells.

    PubMed

    Stroncek, David F; Ren, Jiaqiang; Lee, Daniel W; Tran, Minh; Frodigh, Sue Ellen; Sabatino, Marianna; Khuu, Hanh; Merchant, Melinda S; Mackall, Crystal L

    2016-07-01

    Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability. CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation. A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 10(6) vs 1502 ± 1066 × 10(6); P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 10(6) transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients. Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes. Published by Elsevier Inc.

  20. Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors

    PubMed Central

    Wang, Qiong J.; Yu, Zhiya; Griffith, Kayla; Hanada, Ken-ichi; Restifo, Nicholas P.; Yang, James C.

    2015-01-01

    KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60–70% of pancreatic cancers and 20–30% of colorectal cancers. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCRs) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBLs) transduced with these TCRs could recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types. PMID:26701267

  1. 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. ©2015 American Association for Cancer Research.

  2. The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

    PubMed

    Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

    1998-12-01

    To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

  3. Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

    PubMed Central

    Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

    2013-01-01

    Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer

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

    PubMed Central

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

    2017-01-01

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

  5. Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor.

    PubMed

    Matos, Tiago R; de Rie, Menno A; Teunissen, Marcel B M

    2017-06-01

    High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

    PubMed Central

    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

  7. Lactisole inhibits the glucose-sensing receptor T1R3 expressed in mouse pancreatic β-cells.

    PubMed

    Hamano, Kunihisa; Nakagawa, Yuko; Ohtsu, Yoshiaki; Li, Longfei; Medina, Johan; Tanaka, Yuji; Masuda, Katsuyoshi; Komatsu, Mitsuhisa; Kojima, Itaru

    2015-07-01

    Glucose activates the glucose-sensing receptor T1R3 and facilitates its own metabolism in pancreatic β-cells. An inhibitor of this receptor would be helpful in elucidating the physiological function of the glucose-sensing receptor. The present study was conducted to examine whether or not lactisole can be used as an inhibitor of the glucose-sensing receptor. In MIN6 cells, in a dose-dependent manner, lactisole inhibited insulin secretion induced by sweeteners, acesulfame-K, sucralose and glycyrrhizin. The IC50 was ∼4 mmol/l. Lactisole attenuated the elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) evoked by sucralose and acesulfame-K but did not affect the elevation of intracellular cAMP concentration ([cAMP]c) induced by these sweeteners. Lactisole also inhibited the action of glucose in MIN6 cells. Thus, lactisole significantly reduced elevations of intracellular [NADH] and intracellular [ATP] induced by glucose, and also inhibited glucose-induced insulin secretion. To further examine the effect of lactisole on T1R3, we prepared HEK293 cells stably expressing mouse T1R3. In these cells, sucralose elevated both [Ca2+]c and [cAMP]c. Lactisole attenuated the sucralose-induced increase in [Ca2+]c but did not affect the elevation of [cAMP]c. Finally, lactisole inhibited insulin secretion induced by a high concentration of glucose in mouse islets. These results indicate that the mouse glucose-sensing receptor was inhibited by lactisole. Lactisole may be useful in assessing the role of the glucose-sensing receptor in mouse pancreatic β-cells. © 2015 Society for Endocrinology.

  8. Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells.

    PubMed

    Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A

    2018-05-01

    B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

  9. Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

    PubMed

    Karim, Ahmad Faisal; Reba, Scott M; Li, Qing; Boom, W Henry; Rojas, Roxana E

    2017-09-01

    We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4 + T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4 + T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4 + T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4 + T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4 + T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4 + T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. CD8+ memory T-cell inflation renders compromised CD4+ T-cell-dependent CD8+ T-cell immunity via naïve T-cell anergy.

    PubMed

    Xu, Aizhang; Freywald, Andrew; Xie, Yufeng; Li, Zejun; Xiang, Jim

    2017-01-01

    Whether inflation of CD8 + memory T (mT) cells, which is often derived from repeated prime-boost vaccinations or chronic viral infections in the elderly, would affect late CD8 + T-cell immunity is a long-standing paradox. We have previously established an animal model with mT-cell inflation by transferring ConA-stimulated monoclonal CD8 + T cells derived from Ova-specific T-cell-receptor transgenic OTI mice into irradiation-induced lymphopenic B6 mice. In this study, we also established another two animal models with mT-cell inflation by transferring, 1) ConA-stimulated monoclonal CD8 + T cells derived from lymphocytic choriomeningitis virus glycoprotein-specific T-cell-receptor transgenic P14 mice, and 2) ConA-stimulated polyclonal CD8 + T cells derived from B6.1 mice into B6 mice with irradiation-induced lymphopenia. We vaccinated these mice with recombinant Ova-expressing Listeria monocytogenes and Ova-pulsed dendritic cells, which stimulated CD4 + T cell-independent and CD4 + T-cell-dependent CD8 + T-cell responses, respectively, and assessed Ova-specific CD8 + T-cell responses by flow cytometry. We found that Ova-specific CD8 + T-cell responses derived from the latter but not the former vaccination were significantly reduced in mice with CD8 + mT-cell inflation compared to wild-type B6 mice. We determined that naïve CD8 + T cells purified from splenocytes of mice with mT-cell inflation had defects in cell proliferation upon stimulation in vitro and in vivo and upregulated T-cell anergy-associated Itch and GRAIL molecules. Taken together, our data reveal that CD8 + mT-cell inflation renders compromised CD4 + T-cell-dependent CD8 + T-cell immunity via naïve T-cell anergy, and thus show promise for the design of efficient vaccines for elderly patients with CD8 + mT-cell inflation.

  13. Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Tomuleasa, Ciprian; Fuji, Shigeo; Berce, Cristian; Onaciu, Anca; Chira, Sergiu; Petrushev, Bobe; Micu, Wilhelm-Thomas; Moisoiu, Vlad; Osan, Ciprian; Constantinescu, Catalin; Pasca, Sergiu; Jurj, Ancuta; Pop, Laura; Berindan-Neagoe, Ioana; Dima, Delia; Kitano, Shigehisa

    2018-01-01

    Chimeric antigen receptor (CAR) T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects. PMID:29515572

  14. Chimeric Antigen Receptor T-Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia.

    PubMed

    Tomuleasa, Ciprian; Fuji, Shigeo; Berce, Cristian; Onaciu, Anca; Chira, Sergiu; Petrushev, Bobe; Micu, Wilhelm-Thomas; Moisoiu, Vlad; Osan, Ciprian; Constantinescu, Catalin; Pasca, Sergiu; Jurj, Ancuta; Pop, Laura; Berindan-Neagoe, Ioana; Dima, Delia; Kitano, Shigehisa

    2018-01-01

    Chimeric antigen receptor (CAR) T-cell technology has seen a rapid development over the last decade mostly due to the potential that these cells may have in treating malignant diseases. It is a generally accepted principle that very few therapeutic compounds deliver a clinical response without treatment-related toxicity, and studies have shown that CAR T-cells are not an exception to this rule. While large multinational drug companies are currently investigating the potential role of CAR T-cells in hematological oncology, the potential of such cellular therapies are being recognized worldwide as they are expected to expand in the patient to support the establishment of the immune memory, provide a continuous surveillance to prevent and/or treat a relapse, and keep the targeted malignant cell subpopulation in check. In this article, we present the possible advantages of using CAR T-cells in treating acute lymphoblastic leukemia, presenting the technology and the current knowledge in their preclinical and early clinical trial use. Thus, this article first presents the main present-day knowledge on the standard of care for acute lymphoblastic leukemia. Afterward, current knowledge is presented about the use of CAR T-cells in cancer immunotherapy, describing their design, the molecular constructs, and the preclinical data on murine models to properly explain the background for their clinical use. Last, but certainly not least, this article presents the use of CAR T-cells for the immunotherapy of B-cell acute lymphoblastic leukemia, describing both their potential clinical advantages and the possible side effects.

  15. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, bothmore » DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.« less

  16. Activation‐Induced Killer Cell Immunoglobulin‐like Receptor 3DL2 Binding to HLA–B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis

    PubMed Central

    Ridley, Anna; Hatano, Hiroko; Wong‐Baeza, Isabel; Shaw, Jacqueline; Matthews, Katherine K.; Al‐Mossawi, Hussein; Ladell, Kristin; Price, David A.; Bowness, Paul

    2016-01-01

    Objective In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin‐like receptor 3DL2 (KIR‐3DL2). The aim of this study was to determine the factors that induce KIR‐3DL2 expression, and to characterize the relationship between HLA–B27 and the phenotype and function of KIR‐3DL2–expressing CD4+ T cells in SpA. Methods In total, 34 B27+ patients with SpA, 28 age‐ and sex‐matched healthy controls (20 B27− and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template‐switch anchored reverse transcription–polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme‐linked immunosorbent assay. Results Cellular activation induced KIR‐3DL2 expression on both naive and effector CD4+ T cells. KIR‐3DL2 binding to B27+ cells promoted expression of KIR‐3DL2, the Th17‐specific transcription factor retinoic acid receptor–related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR‐3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen‐presenting cells, KIR‐3DL2+CD4+ T cells produced less interleukin‐2 (IL‐2) but more IL‐17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR‐3DL2 to B27 heavy chains. Conclusion KIR‐3DL2 binding to HLA–B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA–B27–KIR‐3DL2 interactions for the treatment of B27+ patients with SpA. PMID:26841353

  17. Bet v 1-specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1-specific T-cell effector function in an activation-dependent manner.

    PubMed

    Schmetterer, Klaus G; Haiderer, Daniela; Leb-Reichl, Victoria M; Neunkirchner, Alina; Jahn-Schmid, Beatrice; Küng, Hans J; Schuch, Karina; Steinberger, Peter; Bohle, Barbara; Pickl, Winfried F

    2011-01-01

    Regulatory T (Treg) cells establish and maintain tolerance to self-antigens and many foreign antigens, such as allergens, by suppressing effector T-cell proliferation and function. We have previously shown that human T-cell receptor (TCR) αβ-chains specific for allergen-derived epitopes confer allergen specificity on peripheral blood T cells of individuals with and without allergy. To study the feasibility of generating allergen-specific human Treg cells by retroviral transduction of a transcription unit encoding forkhead box protein 3 (FOXP3) and allergen-specific TCR αβ-chains. cDNAs encoding the α and β-chains of a Bet v 1(142-153)-specific TCR (TCR alpha variable region 6/TCR beta variable region 20) and human FOXP3 were linked via picornaviral 2A sequences and expressed as single translational unit from an internal ribosomal entry site-green fluorescence protein-containing retroviral vector. Retrovirally transduced peripheral blood T cells were tested for expression of transgenes, Treg phenotype, and regulatory capacity toward allergen-specific effector T cells. Transduced T cells displayed a Treg phenotype with clear-cut upregulation of CD25, CD39, and cytotoxic T-lymphocyte antigen 4. The transduced cells were hyporesponsive in cytokine production and secretion and, like naturally occurring Treg cells, did not proliferate after antigen-specific or antigen-mimetic stimulation. However, proliferation was inducible upon exposure to exogenous IL-2. In coculture experiments, TRAV6(+)TRBV20(+)FOXP3(+) transgenic T cells, unlike FOXP3(+) single transgenic T cells or naturally occurring Treg cells, highly significantly suppressed T cell cytokine production and proliferation of corresponding allergen-specific effector T cells in an allergen-specific, dose-dependent manner. We demonstrate a transgenic approach to engineer human allergen-specific Treg cells that exert their regulatory function in an activation-dependent manner. Customized Treg cells might become

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

    PubMed

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

    2016-01-01

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

  19. Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment.

    PubMed

    Wang, Zhenguang; Guo, Yelei; Han, Weidong

    2017-12-01

    Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

  20. Tailored chemokine receptor modification improves homing of adoptive therapy T cells in a spontaneous tumor model.

    PubMed

    Garetto, Stefano; Sardi, Claudia; Martini, Elisa; Roselli, Giuliana; Morone, Diego; Angioni, Roberta; Cianciotti, Beatrice Claudia; Trovato, Anna Elisa; Franchina, Davide Giuseppe; Castino, Giovanni Francesco; Vignali, Debora; Erreni, Marco; Marchesi, Federica; Rumio, Cristiano; Kallikourdis, Marinos

    2016-07-12

    In recent years, tumor Adoptive Cell Therapy (ACT), using administration of ex vivo-enhanced T cells from the cancer patient, has become a promising therapeutic strategy. However, efficient homing of the anti-tumoral T cells to the tumor or metastatic site still remains a substantial hurdle. Yet the tumor site itself attracts both tumor-promoting and anti-tumoral immune cell populations through the secretion of chemokines. We attempted to identify these chemokines in a model of spontaneous metastasis, in order to "hijack" their function by expressing matching chemokine receptors on the cytotoxic T cells used in ACT, thus allowing us to enhance the recruitment of these therapeutic cells. Here we show that this enabled the modified T cells to preferentially home into spontaneous lymph node metastases in the TRAMP model, as well as in an inducible tumor model, E.G7-OVA. Due to the improved homing, the modified CD8+ T cells displayed an enhanced in vivo protective effect, as seen by a significant delay in E.G7-OVA tumor growth. These results offer a proof of principle for the tailored application of chemokine receptor modification as a means of improving T cell homing to the target tumor, thus enhancing ACT efficacy. Surprisingly, we also uncover that the formation of the peri-tumoral fibrotic capsule, which has been shown to impede T cell access to tumor, is partially dependent on host T cell presence. This finding, which would be impossible to observe in immunodeficient model studies, highlights possible conflicting roles that T cells may play in a therapeutic context.

  1. Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells.

    PubMed

    Shum, Thomas; Omer, Bilal; Tashiro, Haruko; Kruse, Robert L; Wagner, Dimitrios L; Parikh, Kathan; Yi, Zhongzhen; Sauer, Tim; Liu, Daofeng; Parihar, Robin; Castillo, Paul; Liu, Hao; Brenner, Malcolm K; Metelitsa, Leonid S; Gottschalk, Stephen; Rooney, Cliona M

    2017-11-01

    Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer. Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201 . ©2017 American Association for Cancer Research.

  2. GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

    PubMed Central

    Ku, Chia-Jui; Sekiguchi, JoAnn M.; Panwar, Bharat; Guan, Yuanfang; Takahashi, Satoru; Yoh, Keigyou; Maillard, Ivan; Hosoya, Tomonori

    2017-01-01

    ABSTRACT Allelic exclusion describes the essential immunological process by which feedback repression of sequential DNA rearrangements ensures that only one autosome expresses a functional T or B cell receptor. In wild-type mammals, approximately 60% of cells have recombined the DNA of one T cell receptor β (TCRβ) V-to-DJ-joined allele in a functional configuration, while the second allele has recombined only the DJ sequences; the other 40% of cells have recombined the V to the DJ segments on both alleles, with only one of the two alleles predicting a functional TCRβ protein. Here we report that the transgenic overexpression of GATA3 leads predominantly to biallelic TCRβ gene (Tcrb) recombination. We also found that wild-type immature thymocytes can be separated into distinct populations based on intracellular GATA3 expression and that GATA3LO cells had almost exclusively recombined only one Tcrb locus (that predicted a functional receptor sequence), while GATA3HI cells had uniformly recombined both Tcrb alleles (one predicting a functional and the other predicting a nonfunctional rearrangement). These data show that GATA3 abundance regulates the recombination propensity at the Tcrb locus and provide new mechanistic insight into the historic immunological conundrum for how Tcrb allelic exclusion is mediated. PMID:28320875

  3. Inhibition of endothelial receptor expression and of T-cell ligand activity by mycophenolate mofetil.

    PubMed

    Blaheta, R A; Leckel, K; Wittig, B; Zenker, D; Oppermann, E; Harder, S; Scholz, M; Weber, S; Schuldes, H; Encke, A; Markus, B H

    1998-12-01

    The novel immunosuppressive drug mycophenolate mofetil (CellCept, MMF) blocks DNA-synthesis by the inhibition of the enzyme inosine monophosphate dehydrogenase (IMDH). IMDH is also involved in the synthesis of adhesion receptors which are known to play an important role in the regulation of cell-cell contacts. Therefore, application of MMF might lead to a reduction of cellular infiltrates in the course of transplant rejection. To evaluate the therapeutic value of MMF, we investigated to what extent MMF blocks T-lymphocyte infiltration in vitro with regard to (a) adhesion to endothelial cells, (b) horizontal migration along these cells and (c) penetration through the endothelial cells. The results demonstrated a strong inhibition of both CD4+ and CD8+ T-cell adhesion and penetration by MMF. The ID50 value for CD4+ T-cell adhesion was calculated to be 0.03 microM and the ID50 value for CD4+ T-cell penetration 1.21 microM. MMF did not significantly influence the horizontal migration of T-lymphocytes along the human vascular endothelial cell (HUVEC) borders. FACS-analysis revealed a diminished E-selectin and P-selectin expression on endothelial cell membranes in the presence of MMF. Although MMF did not interfere with the synthesis of T-cell adhesion ligands, the binding activity of lymphocytic leucocyte function associated antigen 1 (LFA-1), very late antigen 4 (VLA-4) and PSGL-1 (P-selectin glycoprotein ligand 1) to immobilized intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin was impaired. Moreover, MMF prevented VLA-4 and PSGL-1 receptor accumulation on the membranes of T-cell pseudopodia. It can be concluded that MMF possesses potent infiltration blocking properties. MMF evoked down-regulation of specific endothelial membrane molecules and the loss of protein localization in the lymphocyte protrusions might be predominantly responsible for the observed blockade of cell adhesion and penetration.

  4. Incorporation of Immune Checkpoint Blockade into Chimeric Antigen Receptor T Cells (CAR-Ts): Combination or Built-In CAR-T

    PubMed Central

    Yoon, Dok Hyun; Osborn, Mark J.; Tolar, Jakub; Kim, Chong Jai

    2018-01-01

    Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade. PMID:29364163

  5. Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

    PubMed

    Liang, Dongchun; Woo, Jeong-Im; Shao, Hui; Born, Willi K; O'Brien, Rebecca L; Kaplan, Henry J; Sun, Deming

    2018-01-01

    Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

  6. Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

    PubMed Central

    Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

    2014-01-01

    T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

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

    PubMed Central

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

    2013-01-01

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

  8. The effect of inhibitory signals on the priming of drug-hapten-specific T-cells that express distinct Vβ receptors

    PubMed Central

    Gibson, Andrew; Faulkner, Lee; Lichtenfels, Maike; Ogese, Monday; Al-Attar, Zaid; Alfirevic, Ana; Esser, Philipp R.; Martin, Stefan F.; Pirmohamed, Munir; Park, B. Kevin; Naisbitt, Dean J.

    2017-01-01

    Drug hypersensitivity involves the activation of T-cells in an HLA allele-restricted manner. Since the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T-cell response. Thus, we have utilized a T-cell priming assay and nitroso sulfamethoxazole (SMX-NO) as a model antigen to investigate (1) the activation of specific T-cell receptor (TCR)Vβ subtypes, (2) the impact of PD-1, CTLA4 and TIM-3 co-inhibitory signalling on activation of naïve and memory T-cells and (3) the ability of Tregs to prevent responses. An expansion of the TCR repertoire was observed for nine different Vβ subtypes, while spectratyping revealed that SMX-NO-specific T-cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vβ-restricted antigen-specific T-cell responses is governed by regulatory signals. Blockade of PDL-1/CTLA4 signalling dampened activation of SMX-NO-specific naïve and memory T-cells, while blockade of TIM-3 produced no effect. PD-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T-cell activation and expression of each receptor was enhanced on dividing T-cells. As these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T-cell activation was investigated. Tregs significantly dampened the priming of T-cells. In conclusion, our findings demonstrate that distinct TCR Vβ subtypes, dysregulation of co-inhibitory signalling pathways and dysfunctional Tregs may influence predisposition to hypersensitivity. PMID:28687658

  9. The signaling symphony: T cell receptor tunes cytokine-mediated T cell differentiation

    PubMed Central

    Huang, Weishan; August, Avery

    2015-01-01

    T cell development, differentiation, and maintenance are orchestrated by 2 key signaling axes: the antigen-specific TCR and cytokine-mediated signals. The TCR signals the recognition of self- and foreign antigens to control T cell homeostasis for immune tolerance and immunity, which is regulated by a variety of cytokines to determine T cell subset homeostasis and differentiation. TCR signaling can synergize with or antagonize cytokine-mediated signaling to fine tune T cell fate; however, the latter is less investigated. Murine models with attenuated TCR signaling strength have revealed that TCR signaling can function as regulatory feedback machinery for T cell homeostasis and differentiation in differential cytokine milieus, such as IL-2-mediated Treg development; IL-7-mediated, naïve CD8+ T cell homeostasis; and IL-4-induced innate memory CD8+ T cell development. In this review, we discuss the symphonic cross-talk between TCR and cytokine-mediated responses that differentially control T cell behavior, with a focus on the negative tuning by TCR activation on the cytokine effects. PMID:25525115

  10. Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells.

    PubMed

    Wu, S Vincent; Rozengurt, Nora; Yang, Moon; Young, Steven H; Sinnett-Smith, James; Rozengurt, Enrique

    2002-02-19

    Although a role for the gastric and intestinal mucosa in molecular sensing has been known for decades, the initial molecular recognition events that sense the chemical composition of the luminal contents has remained elusive. Here we identified putative taste receptor gene transcripts in the gastrointestinal tract. Our results, using reverse transcriptase-PCR, demonstrate the presence of transcripts corresponding to multiple members of the T2R family of bitter taste receptors in the antral and fundic gastric mucosa as well as in the lining of the duodenum. In addition, cDNA clones of T2R receptors were detected in a rat gastric endocrine cell cDNA library, suggesting that these receptors are expressed, at least partly, in enteroendocrine cells. Accordingly, expression of multiple T2R receptors also was found in STC-1 cells, an enteroendocrine cell line. The expression of alpha subunits of G proteins implicated in intracellular taste signal transduction, namely Galpha(gust), and Galpha(t)-(2), also was demonstrated in the gastrointestinal mucosa as well as in STC-1 cells, as revealed by reverse transcriptase-PCR and DNA sequencing, immunohistochemistry, and Western blotting. Furthermore, addition of compounds widely used in bitter taste signaling (e.g., denatonium, phenylthiocarbamide, 6-n-propil-2-thiouracil, and cycloheximide) to STC-1 cells promoted a rapid increase in intracellular Ca(2+) concentration. These results demonstrate the expression of bitter taste receptors of the T2R family in the mouse and rat gastrointestinal tract.

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

    PubMed

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

    2017-07-01

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

  12. Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

    PubMed Central

    Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

    2007-01-01

    Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

  13. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    PubMed

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

  14. Chimeric Antigen Receptor-Modified T Cells Redirected to EphA2 for the Immunotherapy of Non-Small Cell Lung Cancer.

    PubMed

    Li, Ning; Liu, Shaohui; Sun, Mingjiao; Chen, Wei; Xu, Xiaogang; Zeng, Zhu; Tang, Yemin; Dong, Yongquan; Chang, Alex H; Zhao, Qiong

    2018-02-01

    Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is overexpressed in more than 90% of non-small cell lung cancer (NSCLC) but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR)-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Activation-induced Modification in the CD3 Complex of the γδ T Cell Receptor

    PubMed Central

    Hayes, Sandra M.; Laky, Karen; El-Khoury, Dalal; Kappes, Dietmar J.; Fowlkes, B.J.; Love, Paul E.

    2002-01-01

    The T cell antigen receptor complexes expressed on αβ and γδ T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The γδ T cell receptors (TCRs) expressed on ex vivo γδ T cells lack CD3δ, whereas αβ TCRs contain CD3δ. While this result correlates with the phenotype of CD3δ−/− mice, in which γδ T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3δ is a component of the γδ TCR. Since earlier studies examined the subunit composition of γδ TCRs expressed on activated and expanded peripheral γδ T cells or γδ TCR+ intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the γδ TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3δ, in the γδ TCR. Further analyses revealed that this protein is not CD3δ, but instead is a differentially glycosylated form of CD3γ. These results provide further evidence for a major difference in the subunit composition of αβ- and γδ TCR complexes and raise the possibility that modification of CD3γ may have important functional consequences in activated γδ T cells. PMID:12438426

  16. Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

    PubMed Central

    Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

    2018-01-01

    Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

  17. The phosphatase JKAP/DUSP22 inhibits T-cell receptor signalling and autoimmunity by inactivating Lck.

    PubMed

    Li, Ju-Pi; Yang, Chia-Yu; Chuang, Huai-Chia; Lan, Joung-Liang; Chen, Der-Yuan; Chen, Yi-Ming; Wang, Xiaohong; Chen, Alice J; Belmont, John W; Tan, Tse-Hua

    2014-04-09

    JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knockout T cells display enhanced cell proliferation and cytokine production. JKAP-knockout mice show enhanced T-cell-mediated immune responses and are more susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, the recipient mice that are adoptively transferred with JKAP-knockout T cells show exacerbated EAE symptoms. Aged JKAP-knockout mice spontaneously develop inflammation and autoimmunity. Thus, our results indicate that JKAP is an important phosphatase that inactivates Lck in the TCR signalling turn-off stage, leading to suppression of T-cell-mediated immunity and autoimmunity.

  18. Non-immune cells equipped with T cell receptor-like signaling for cancer cell ablation

    PubMed Central

    Kojima, Ryosuke; Scheller, Leo; Fussenegger, Martin

    2017-01-01

    The ability to engineer custom cell-contact-sensing output devices into human non-immune cells would be useful for extending the applicability of cell-based cancer therapies and avoiding risks associated with engineered immune cells. Here, we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human non-immune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell/target cell interface. We further showed that designer non-immune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to advancement of synthetic biology by extending available design principles to transmit extracellular information to cells. PMID:29131143

  19. Epstein Barr virus–specific cytotoxic T lymphocytes expressing the anti-CD30ζ artificial chimeric T-cell receptor for immunotherapy of Hodgkin disease

    PubMed Central

    Rooney, Cliona M.; Di Stasi, Antonio; Abken, Hinrich; Hombach, Andreas; Foster, Aaron E.; Zhang, Lan; Heslop, Helen E.; Brenner, Malcolm K.; Dotti, Gianpietro

    2007-01-01

    Adoptive transfer of Epstein Barr virus (EBV)–specific cytotoxic T-lymphocytes (EBV-CTLs) has shown that these cells persist in patients with EBV+ Hodgkin lymphoma (HD) to produce complete tumor responses. Treatment failure, however, occurs if a subpopulation of malignant cells in the tumor lacks or loses expression of EBV antigens. We have therefore determined whether we could prepare EBV-CTLs that retained the antitumor activity conferred by their native receptor while expressing a chimeric antigen receptor (CAR) specific for CD30, a molecule highly and consistently expressed on malignant Hodgkin Reed-Sternberg cells. We made a CD30CAR and were able to express it on 26% (± 11%) and 22% (± 5%) of EBV-CTLs generated from healthy donors and HD patients, respectively. These CD30CAR+ CTLs killed both autologous EBV+ cells through their native receptor and EBV−/CD30+ targets through their major histocompatibility complex (MHC)–unrestricted CAR. A subpopulation of activated T cells also express CD30, but the CD30CAR+ CTLs did not impair cellular immune responses, probably because normal T cells express lower levels of the target antigen. In a xenograft model, CD30CAR+ EBV-CTLs could be costimulated by EBV-infected cells and produce antitumor effects even against EBV−/CD30+ tumors. EBV-CTLs expressing both a native and a chimeric antigen receptor may therefore have added value for treatment of HD. PMID:17507664

  20. Targeting Inflammatory T Helper Cells via Retinoic Acid-Related Orphan Receptor Gamma t Is Ineffective to Prevent Allo-Response-Driven Colitis.

    PubMed

    Buchele, Vera; Abendroth, Benjamin; Büttner-Herold, Maike; Vogler, Tina; Rothamer, Johanna; Ghimire, Sakhila; Ullrich, Evelyn; Holler, Ernst; Neurath, Markus F; Hildner, Kai

    2018-01-01

    Intestinal graft-versus-host disease (GvHD) is a life-threatening, inflammatory donor T cell-mediated complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the light of the reported efficacy of interleukin-23 (IL-23)-blockade to mitigate syngeneic intestinal inflammation in inflammatory bowel disease patients, targeting IL-23 and thereby interleukin-17a (IL-17a) producing T helper (Th17) cells as the T cell subset assumed to be mostly regulated by IL-23, has emerged as a putatively general concept to harness immune-mediated mucosal inflammation irrespective of the underlying trigger. However, the role of Th17 cells during allo-response driven colitis remains ambiguous due to a series of studies with inconclusive results. Interestingly, we recently identified granulocyte-macrophage colony-stimulating factor (GM-CSF + ) T cells to be promoted by interleukin-7 (IL-7) signaling and controlled by the activating protein-1 transcription factor family member basic leucine zipper transcription factor ATF-like (BATF) as critical mediators of intestinal GvHD in mice. Given the dual role of BATF, the contribution of IL-23-mediated signaling within donor T cells and bona fide Th17 cells remains to be delineated from the regulation of GM-CSF + T cells in the absence of BATF. Here, we found in a complete MHC class I-mismatched model that genetic inactivation of the IL-23 receptor (IL-23R) or the transcription factor retinoic acid-related orphan receptor gamma t (RORγt) within donor T cells similarly ablated Th17 cell formation in vivo but preserved the T cells' ability to induce intestinal GvHD in a compared to wild-type controls indistinguishable manner. Importantly, RORγt-independent manifestation of intestinal GvHD was completely dependent on BATF-regulated GM-CSF + T cells as BATF/RORγt double-deficient T cells failed to induce colitis and the antibody-mediated blockage of IL-7/IL-7R interaction and GM-CSF significantly diminished signs

  1. NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

    PubMed Central

    Sommermeyer, Daniel; Conrad, Heinke; Krönig, Holger; Gelfort, Haike; Bernhard, Helga; Uckert, Wolfgang

    2013-01-01

    The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy. PMID:22907642

  2. A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

    PubMed Central

    Masubuchi, Yosuke; Nakagawa, Yuko; Ma, Jinhui; Sasaki, Tsutomu; Kitamura, Tadahiro; Yamamoto, Yoritsuna; Kurose, Hitoshi; Kojima, Itaru; Shibata, Hiroshi

    2013-01-01

    Background Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. Conclusions 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism. PMID:23336004

  3. Subcellular distribution of Lck during CD4 T-cell maturation in the thymic medulla regulates the T-cell activation threshold.

    PubMed

    Stephen, Tom Li; Wilson, Bridget S; Laufer, Terri M

    2012-05-08

    Mature peripheral T cells respond to foreign but not to self-antigens. During development in the thymus, deletion of high-affinity self-reactive immature thymocytes contributes to tolerance of mature T cells. However, double-positive thymocytes are positively selected to survive if they respond to self-peptide-MHC complexes; thus, there must be mechanisms to prevent overt reactivity to those same complexes in the periphery. "Developmental tuning" is the active process through which T-cell receptor (TCR)-associated signaling pathways of single-positive (SP) thymocytes are attenuated to respond appropriately to self-peptide-MHC complexes in the periphery. We previously showed that MHC class II expression in the thymic medulla was necessary to tune CD4(+) SP (CD4 SP) thymocytes. CD4 SP thymocytes from mice lacking medullary MHC class II expression had inappropriately enhanced proximal TCR signaling to low-affinity self-ligands that was associated with altered cellular distribution of the tyrosine kinase Lck. Now, we report that activation of both tuned and untuned CD4 SP thymocytes is Lck-dependent. Untuned CD4 SP cells contain a pool of Lck with increased basal phosphorylation that is not associated with the CD4 coreceptor. Phosphorylation of this pool of Lck decreases with tuning. Immunogold transmission electron microscopy of membrane sheets permitted direct visualization of Lck. In the absence of tuning, a significant proportion of Lck and the TCR subunit CD3ζ are expressed on the same protein island; this close association of Lck and the TCR probably explains the enhanced activation of untuned CD4 SP cells. Thus, changes in membrane topography during thymic maturation determine the set point for TCR responsiveness.

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

    PubMed Central

    Maher, John

    2012-01-01

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

  5. Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer.

    PubMed

    Kobold, Sebastian; Steffen, Julius; Chaloupka, Michael; Grassmann, Simon; Henkel, Jonas; Castoldi, Raffaella; Zeng, Yi; Chmielewski, Markus; Schmollinger, Jan C; Schnurr, Max; Rothenfußer, Simon; Schendel, Dolores J; Abken, Hinrich; Sustmann, Claudio; Niederfellner, Gerhard; Klein, Christian; Bourquin, Carole; Endres, Stefan

    2015-01-01

    One bottleneck for adoptive T cell therapy (ACT) is recruitment of T cells into tumors. We hypothesized that combining tumor-specific T cells, modified with a marker antigen and a bispecific antibody (BiAb) that selectively recognizes transduced T cells and tumor cells would improve T cell recruitment to tumors and enhance therapeutic efficacy. SV40 T antigen-specific T cells from T cell receptor (TCR)-I-transgenic mice were transduced with a truncated human epidermal growth factor receptor (EGFR) as a marker protein. Targeting and killing by combined ACT and anti-EGFR-anti-EpCAM BiAb therapy was analyzed in C57Bl/6 mice (n = six to 12 per group) carrying subcutaneous tumors of the murine gastric cancer cell line GC8 (SV40(+) and EpCAM(+)). Anti-EGFR x anti-c-Met BiAb was used for targeting of human tumor-specific T cells to c-Met(+) human tumor cell lines. Differences between experimental conditions were analyzed using the Student's t test, and differences in tumor growth with two-way analysis of variance. Overall survival was analyzed by log-rank test. All statistical tests were two-sided. The BiAb linked EGFR-transduced T cells to tumor cells and enhanced tumor cell lysis. In vivo, the combination of ACT and Biab produced increased T cell infiltration of tumors, retarded tumor growth, and prolonged survival compared with ACT with a control antibody (median survival 95 vs 75 days, P < .001). In human cells, this strategy enhanced recruitment of human EGFR-transduced T cells to immobilized c-Met and recognition of tyrosinase(+) melanoma cells by TCR-, as well as of CEA(+) colon cancer cells by chimeric antigen receptor (CAR)-modified T cells. BiAb recruitment of tumor-specific T cells transduced with a marker antigen to tumor cells may enhance efficacy of ACT. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Germinal Center T Follicular Helper Cell IL-4 Production Is Dependent on Signaling Lymphocytic Activation Molecule Receptor (CD150)

    PubMed Central

    Yusuf, Isharat; Kageyama, Robin; Monticelli, Laurel; Johnston, Robert J.; DiToro, Daniel; Hansen, Kyle; Barnett, Burton; Crotty, Shane

    2010-01-01

    CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (TFH) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect in TFH differentiation, as defined by common TFH surface markers. CXCR5+ TFH cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC TFH) cells can be identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of TFH and GC TFH populations. GC TFH cells are a functionally discrete subset of further polarized TFH cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a TH2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC TFH cell subset and SAP− TFH cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC TFH cells. GC TFH cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in TFH cell and GC TFH cell differentiation. PMID:20525889

  7. Specific T-cell activation in an unspecific T-cell repertoire.

    PubMed

    Van Den Berg, Hugo A; Molina-París, Carmen; Sewell, Andrew K

    2011-01-01

    T-cells are a vital type of white blood cell that circulate around our bodies, scanning for cellular abnormalities and infections. They recognise disease-associated antigens via a surface receptor called the T-cell antigen receptor (TCR). If there were a specific TCR for every single antigen, no mammal could possibly contain all the T-cells it needs. This is clearly absurd and suggests that T-cell recognition must, to the contrary, be highly degenerate. Yet highly promiscuous TCRs would appear to be equally impossible: they are bound to recognise self as well as non-self antigens. We review how contributions from mathematical analysis have helped to resolve the paradox of the promiscuous TCR. Combined experimental and theoretical work shows that TCR degeneracy is essentially dynamical in nature, and that the T-cell can differentially adjust its functional sensitivity to the salient epitope, "tuning up" sensitivity to the antigen associated with disease and "tuning down" sensitivity to antigens associated with healthy conditions. This paradigm of continual modulation affords the TCR repertoire, despite its limited numerical diversity, the flexibility to respond to almost any antigenic challenge while avoiding autoimmunity.

  8. Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

    PubMed Central

    Boursalian, Tamar E.; Fink, Pamela J.

    2003-01-01

    Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

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

    PubMed Central

    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.

    2015-01-01

    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

  10. Chimeric antigen receptor T cell therapy in pancreatic cancer: from research to practice.

    PubMed

    Jindal, Vishal; Arora, Ena; Masab, Muhammad; Gupta, Sorab

    2018-05-04

    Chimeric antigen receptor (CAR) T cell therapy is genetically engineered tumor antigen-specific anticancer immunotherapy, which after showing great success in hematological malignancies is currently being tried in advanced solid tumors like pancreatic cancer. Immunosuppressive tumor microenvironment and dense fibrous stroma are some of the limitation in the success of this novel therapy. However, genetic modifications and combination therapy is the topic of the research to improve its efficacy. In this article, we summarize the current state of knowledge, limitations, and future prospects for CAR T cell therapy in pancreatic cancer.

  11. Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

    PubMed Central

    Chiocchetti, Annalisa; Miglio, Gianluca; Mesturini, Riccardo; Varsaldi, Federica; Mocellin, Marco; Orilieri, Elisabetta; Dianzani, Chiara; Fantozzi, Roberto; Dianzani, Umberto; Lombardi, Grazia

    2006-01-01

    The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 μg ml−1; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). L-Glutamate (1 × 10−8–1 × 10−4 M) significantly (P⩽0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)-ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)-3,5-DHPG; 2.0 × 10−5 M for CHPG. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. L-Glutamate (1 × 10−6 M; 18 h) significantly (P⩽0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms. PMID:16751798

  12. Clonal expansion of T-cell receptor beta gene segment in the retrocochlear lesions of EAE mice.

    PubMed

    Cheng, K C; Lee, K M; Yoo, T J

    1998-01-01

    It has been reported that the T cell receptor V beta 8.2 (TcrbV8.2) gene segment is predominantly expressed in encephalomyelitic T cells responding to myelin basic protein (MBP) in experimental allergic encephalomyelitis (EAE) mice. We have demonstrated retrocochlear hearing loss in EAE mice in previous studies. Administration of a monoclonal antibody specific to the T cell receptor V beta 8 (TcrbV8) subfamily prevented both this type of hearing loss and the central nerve disease. In this study, we examined the role of the TcrbV8.2 gene segment in the retrocochlear lesions of EAE mice. A clonal expression of T cell receptor beta chain gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) was identified in the retrocochlear lesions. The TcrbV8.2 gene segment appears to recombine only with TcrbJ2.1 (32.1%) and TcrbJ2.7 (67.9%) gene segments. The TcrbJ2.7 gene segment has also been previously identified as the dominant TcrbJ gene in the lymph nodes of EAE mice. Only TcrbD2, with a length of 4 amino acids, was observed recombining with these TcrbV8.2 sequences. G and C nucleotides are predominantly expressed at the N regions between the V-D and D-J junctions. This dominant TcrbV gene segment (TcrbV8.2-TcrbD2-TcrbJ2.7) observed in the retrocochlear lesions has been identified in the MBP-specific T cells from the lymph nodes of EAE mice. These results suggest that a small subset of antigen-specific T cells migrate to, and expand at, the retrocochlear lesions, which leads to hearing loss.

  13. CAR-T cells are serial killers.

    PubMed

    Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

    2015-12-01

    Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours.

  14. Understanding clinical development of chimeric antigen receptor T cell therapies.

    PubMed

    de Wilde, Sofieke; Guchelaar, Henk-Jan; Zandvliet, Maarten Laurens; Meij, Pauline

    2017-06-01

    In the past decade, many clinical trials with gene- and cell-based therapies (GCTs) have been performed. Increased interest in the development of these drug products by various stakeholders has become apparent. Despite this growth in clinical studies, the number of therapies receiving marketing authorization approval (MAA) is lagging behind. To enhance the success rate of GCT development, it is essential to better understand the clinical development of these products. Chimeric antigen receptor (CAR) T cells are a GCT product subtype with promising efficacy in cancer treatment which are tested in many clinical trials, but have not yet received MAA. We generated an overview of the characteristics of CAR T-cell clinical development in the United States, Canada and Europe. Subsequently, the characteristics of clinical trials with CAR T-cell products that proceeded to a subsequent clinical trial, used as a proxy for success, were compared with those that did not proceed. From the U.S. and European Union clinical trial databases, 106 CAR T-cell trials were selected, from which 49 were linked to a subsequent trial and 57 were not. The majority of the trials had an academic sponsor from which most did not proceed, whereas most commercially sponsored trials were followed by another clinical trial. Furthermore, trials with a subsequent trial more frequently recruited large patient cohorts and were more often multicenter compared with trials that were not followed up. These characteristics can be used by investigators to better design clinical trials with CAR T cells. We encourage sponsors to plan clinical development ahead for a higher efficiency of product development and thereby achieving a higher success rate of development towards MAA. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  15. Crystal structure of a gammadelta T-cell receptor specific for the human MHC class I homolog MICA.

    PubMed

    Xu, Bin; Pizarro, Juan C; Holmes, Margaret A; McBeth, Christine; Groh, Veronika; Spies, Thomas; Strong, Roland K

    2011-02-08

    γδ T cells play important roles in bridging innate and adaptive immunity, but their recognition mechanisms remain poorly understood. Human γδ T cells of the V(δ)1 subset predominate in intestinal epithelia and respond to MICA and MICB (MHC class I chain-related, A and B; MIC) self-antigens, mediating responses to tumorigenesis or viral infection. The crystal structure of an MIC-reactive V(δ)1 γδ T-cell receptor (TCR) showed expected overall structural homology to antibodies, αβ, and other γδ TCRs, but complementary determining region conformations and conservation of V(δ)1 use revealed an uncharacteristically flat potential binding surface. MIC, likewise, serves as a ligand for the activating immunoreceptor natural killer group 2, D (NKG2D), also expressed on γδ T cells. Although MIC recognition drives both the TCR-dependent stimulatory and NKG2D-dependent costimulatory signals necessary for activation, interaction analyses showed that MIC binding by the two receptors was mutually exclusive. Analysis of relative binding kinetics suggested sequential recognition, defining constraints for the temporal organization of γδ T-cell/target cell interfaces.

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

    PubMed Central

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

    2016-01-01

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

  17. Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

    PubMed Central

    Firor, Amelia E.; Pinz, Kevin G.; Jares, Alexander; Liu, Hua; Salman, Huda; Golightly, Marc; Lan, Fengshuo; Jiang, Xun; Ma, Yupo

    2016-01-01

    Peripheral T-cell lymphomas (PTCLS) comprise a diverse group of difficult to treat, very aggressive non-Hodgkin's lymphomas (NHLS) with poor prognoses and dismal patient outlook. Despite the fact that PTCLs comprise the majority of T-cell malignancies, the standard of care is poorly established. Chimeric antigen receptor (CAR) immunotherapy has shown in B-cell malignancies to be an effective curative option and this extends promise into treating T-cell malignancies. Because PTCLS frequently develop from mature T-cells, CD3 is similarly strongly and uniformly expressed in many PTCL malignancies, with expression specific to the hematological compartment thus making it an attractive target for CAR design. We engineered a robust 3rd generation anti-CD3 CAR construct (CD3CAR) into an NK cell line (NK-92). We found that CD3CAR NK-92 cells specifically and potently lysed diverse CD3+ human PTCL primary samples as well as T-cell leukemia cells lines ex vivo. Furthermore, CD3CAR NK-92 cells effectively controlled and suppressed Jurkat tumor cell growth in vivo and significantly prolonged survival. In this study, we present the CAR directed targeting of a novel target - CD3 using CAR modified NK-92 cells with an emphasis on efficacy, specificity, and potential for new therapeutic approaches that could improve the current standard of care for PTCLs. PMID:27494836

  18. CAR-T cells are serial killers

    PubMed Central

    Davenport, Alexander J; Jenkins, Misty R; Ritchie, David S; Prince, H Miles; Trapani, Joseph A; Kershaw, Michael H; Darcy, Phillip K; Neeson, Paul J

    2015-01-01

    Chimeric antigen receptor (CAR) T cells have enjoyed unprecedented clinical success against haematological malignancies in recent years. However, several aspects of CAR T cell biology remain unknown. We recently compared CAR and T cell receptor (TCR)-based killing in the same effector cell and showed that CAR T cells can not only efficiently kill single tumor targets, they can also kill multiple tumor targets in a sequential manner. Single and serial killing events were not sustained long term due to CAR down-regulation after 20 hours. PMID:26587330

  19. Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor β-DAB2IP signals.

    PubMed

    Yeh, Chiuan-Ren; Ou, Zheng-Yu; Xiao, Guang-Qian; Guancial, Elizabeth; Yeh, Shuyuan

    2015-12-29

    Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERβ), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERβ expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERβ and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERβ expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERβ transcriptional activity. The newly identified relationship between infiltrating T cells/ERβ/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

  20. Integrins in T Cell Physiology

    PubMed Central

    Alabiso, Oscar; Galetto, Alessandra Silvia; Baldanzi, Gianluca

    2018-01-01

    From the thymus to the peripheral lymph nodes, integrin-mediated interactions with neighbor cells and the extracellular matrix tune T cell behavior by organizing cytoskeletal remodeling and modulating receptor signaling. LFA-1 (αLβ2 integrin) and VLA-4 (α4β1 integrin) play a key role throughout the T cell lifecycle from thymocyte differentiation to lymphocyte extravasation and finally play a fundamental role in organizing immune synapse, providing an essential costimulatory signal for the T cell receptor. Apart from tuning T cell signaling, integrins also contribute to homing to specific target organs as exemplified by the importance of α4β7 in maintaining the gut immune system. However, apart from those well-characterized examples, the physiological significance of the other integrin dimers expressed by T cells is far less understood. Thus, integrin-mediated cell-to-cell and cell-to-matrix interactions during the T cell lifespan still represent an open field of research. PMID:29415483

  1. Regulation of T-cell receptor signalling by membrane microdomains

    PubMed Central

    Razzaq, Tahir M; Ozegbe, Patricia; Jury, Elizabeth C; Sembi, Phupinder; Blackwell, Nathan M; Kabouridis, Panagiotis S

    2004-01-01

    There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. PMID:15554919

  2. Binding of hepatitis A virus to its cellular receptor 1 inhibits T-regulatory cell functions in humans.

    PubMed

    Manangeeswaran, Mohanraj; Jacques, Jérôme; Tami, Cecilia; Konduru, Krishnamurthy; Amharref, Nadia; Perrella, Oreste; Casasnovas, Jose M; Umetsu, Dale T; Dekruyff, Rosemarie H; Freeman, Gordon J; Perrella, Alessandro; Kaplan, Gerardo G

    2012-06-01

    CD4+ T-regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-β , which limited leukocyte recruitment and survival, and produced high levels of interleukin-22, which prevented liver damage. Interaction between HAV and its receptor HAVCR1 inhibits Treg-cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection-a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anticancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  3. The Receptor for Advanced Glycation Endproducts Drives T Cell Survival and Inflammation in Type 1 Diabetes Mellitus.

    PubMed

    Durning, Sean P; Preston-Hurlburt, Paula; Clark, Paul R; Xu, Ding; Herold, Kevan C

    2016-10-15

    The ways in which environmental factors participate in the progression of autoimmune diseases are not known. After initiation, it takes years before hyperglycemia develops in patients at risk for type 1 diabetes (T1D). The receptor for advanced glycation endproducts (RAGE) is a scavenger receptor of the Ig family that binds damage-associated molecular patterns and advanced glycated endproducts and can trigger cell activation. We previously found constitutive intracellular RAGE expression in lymphocytes from patients with T1D. In this article, we show that there is increased RAGE expression in T cells from at-risk euglycemic relatives who progress to T1D compared with healthy control subjects, and in the CD8 + T cells in the at-risk relatives who do versus those who do not progress to T1D. Detectable levels of the RAGE ligand high mobility group box 1 were present in serum from at-risk subjects and patients with T1D. Transcriptome analysis of RAGE + versus RAGE - T cells from patients with T1D showed differences in signaling pathways associated with increased cell activation and survival. Additional markers for effector memory cells and inflammatory function were elevated in the RAGE + CD8 + cells of T1D patients and at-risk relatives of patients before disease onset. These studies suggest that expression of RAGE in T cells of subjects progressing to disease predates dysglycemia. These findings imply that RAGE expression enhances the inflammatory function of T cells, and its increased levels observed in T1D patients may account for the chronic autoimmune response when damage-associated molecular patterns are released after cell injury and killing. Copyright © 2016 by The American Association of Immunologists, Inc.

  4. Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

    PubMed

    Schlenker, Ramona; Olguín-Contreras, Luis Felipe; Leisegang, Matthias; Schnappinger, Julia; Disovic, Anja; Rühland, Svenja; Nelson, Peter J; Leonhardt, Heinrich; Harz, Hartmann; Wilde, Susanne; Schendel, Dolores J; Uckert, Wolfgang; Willimsky, Gerald; Noessner, Elfriede

    2017-07-01

    Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. HCV T Cell Receptor Chain Modifications to Enhance Expression, Pairing, and Antigen Recognition in T Cells for Adoptive Transfer.

    PubMed

    Foley, Kendra C; Spear, Timothy T; Murray, David C; Nagato, Kaoru; Garrett-Mayer, Elizabeth; Nishimura, Michael I

    2017-06-16

    T cell receptor (TCR)-gene-modified T cells for adoptive cell transfer can mediate objective clinical responses in melanoma and other malignancies. When introducing a second TCR, mispairing between the endogenous and introduced α and β TCR chains limits expression of the introduced TCR, which can result in impaired efficacy or off-target reactivity and autoimmunity. One approach to promote proper TCR chain pairing involves modifications of the introduced TCR genes: introducing a disulfide bridge, substituting murine for human constant regions, codon optimization, TCR chain leucine zipper fusions, and a single-chain TCR. We have introduced these modifications into our hepatitis C virus (HCV) reactive TCR and utilize a marker gene, CD34t, which allows us to directly compare transduction efficiency with TCR expression and T cell function. Our results reveal that of the TCRs tested, T cells expressing the murine Cβ2 TCR or leucine zipper TCR have the highest levels of expression and the highest percentage of lytic and interferon-γ (IFN-γ)-producing T cells. Our studies give us a better understanding of how TCR modifications impact TCR expression and T cell function that may allow for optimization of TCR-modified T cells for adoptive cell transfer to treat patients with malignancies.

  6. A decrease of regulatory T cells and altered expression of NK receptors are observed in subacute sclerosing panencephalitis.

    PubMed

    Yentur, Sibel P; Gurses, Candan; Demirbilek, Veysi; Adin-Cinar, Suzan; Kuru, Umit; Uysal, Serap; Yapici, Zuhal; Yilmaz, Gülden; Cokar, Ozlem; Onal, Emel; Gökyigit, Aysen; Saruhan-Direskeneli, Güher

    2014-12-01

    Subacute sclerosing panencephalitis (SSPE) is caused by a persistent measles virus infection. Regulatory mechanisms can be responsible for a failure of immunosurveillance in children with SSPE. In this study, peripheral blood cells of 71 patients with SSPE and 57 children with other diseases were compared phenotypically. The proportions of CD4(+), CD8(+) T, and NK cells were homogenous, whereas total CD3(+) T and Treg (CD4(+)CD25(+)CD152(+)) cells were decreased in patients with SSPE. The proportion of CD8(+) T cells expressing the inhibitory NKG2A(+) receptor was also decreased (1.7% ± 1.7% vs. 2.6% ± 1.9%, p = 0.007) in patients with SSPE, whereas the proportion of NK cells expressing activating NKG2C was increased compared with the control group (30.0% ± 17.3% vs. 22.2% ± 17.0%, p = 0.039). The decrease in the number of cells with regulatory phenotype, the lower presence of the inhibitory NK receptors on CD8(+) cells, and higher activating NK receptors on NK cells in SSPE indicate an upregulation of these cell types that favors their response. This state of active immune response may be caused by chronic stimulation of viral antigens leading to altered regulatory pathways.

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

    PubMed

    Kassem, Sahar; Gaud, Guillaume; Bernard, Isabelle; Benamar, Mehdi; Dejean, Anne S; Liblau, Roland; Fournié, Gilbert J; Colacios, Céline; Malissen, Bernard; Saoudi, Abdelhadi

    2016-07-01

    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.

  8. 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

  9. Gammadelta T cells: functional plasticity and heterogeneity.

    PubMed

    Carding, Simon R; Egan, Paul J

    2002-05-01

    Gammadelta T cells remain an enigma. They are capable of generating more unique antigen receptors than alphabeta T cells and B cells combined, yet their repertoire of antigen receptors is dominated by specific subsets that recognize a limited number of antigens. A variety of sometimes conflicting effector functions have been ascribed to them, yet their biological function(s) remains unclear. On the basis of studies of gammadelta T cells in infectious and autoimmune diseases, we argue that gammadelta T cells perform different functions according to their tissue distribution, antigen-receptor structure and local microenvironment; we also discuss how and at what stage of the immune response they become activated.

  10. Decade-Long Safety and Function of Retroviral-Modified Chimeric Antigen Receptor T-cells

    PubMed Central

    Scholler, John; Brady, Troy L.; Binder-Scholl, Gwendolyn; Hwang, Wei-Ting; Plesa, Gabriela; Hege, Kristen M.; Vogel, Ashley N.; Kalos, Michael; Riley, James L.; Deeks, Steven G.; Mitsuyasu, Ronald T.; Bernstein, Wendy B.; Aronson, Naomi E.; Levine, Bruce L.; Bushman, Frederic D.; June, Carl H.

    2015-01-01

    The success of adoptive T cell gene transfer for treatment of cancer and HIV is predicated on generating a response that is both durable and safe. Here we report long term results from three clinical trials to evaluate gammaretroviral vector engineered T-cells for HIV. The vector encoded a chimeric antigen receptor (CAR) comprised of CD4 linked to the CD3-ζ signaling chain (CD4ζ). CAR T-cells were detected in 98% of samples tested for at least 11 years post-infusion at frequencies that exceed average T cell levels after most vaccine approaches. The CD4ζ transgene retained expression and function. There was no evidence of vector-induced immortalization of cells as integration site distributions showed no evidence of persistent clonal expansion or enrichment for integration sites near genes implicated in growth control or transformation. The CD4ζ T cells have stable levels of engraftment, with decay half-lives that exceed 16 years, in marked contrast to previous trials testing engineered T cells. These findings indicate that host immunosuppression prior to T cell transfer is not required in order to achieve long term persistence of gene-modified T cells. Further, our results emphasize the safety of T cells modified by retroviral gene transfer in clinical application, as measured in >500 patient years of follow up. Thus, previous safety issues with integrating viral vectors are hematopoietic stem cell or transgene intrinsic, and not a general feature of retroviral vectors. Engineered T cells are a promising form of synthetic biology for long term delivery of protein based therapeutics. These results provide a framework to guide the therapy of a wide spectrum of human diseases. PMID:22553251

  11. At the Bench: Chimeric antigen receptor (CAR) T cell therapy for the treatment of B cell malignancies.

    PubMed

    Daniyan, Anthony F O; Brentjens, Renier J

    2016-12-01

    The chimeric antigen receptor (CAR) represents the epitome of cellular engineering and is one of the best examples of rational biologic design of a synthetic molecule. The CAR is a single polypeptide with modular domains, consisting of an antibody-derived targeting moiety, fused in line with T cell-derived signaling domains, allowing for T cell activation upon ligand binding. T cells expressing a CAR are able to eradicate selectively antigen-expressing tumor cells in a MHC-independent fashion. CD19, a tumor-associated antigen (TAA) present on normal B cells, as well as most B cell-derived malignancies, was an early target of this technology. Through years of experimental refinement and preclinical optimization, autologously derived CD19-targeting CAR T cells have been successfully, clinically deployed, resulting in dramatic and durable antitumor responses but not without therapy-associated toxicity. As CD19-targeted CAR T cells continue to show clinical success, work at the bench continues to be undertaken to increase further the efficacy of this therapy, while simultaneously minimizing the risk for treatment-related morbidities. In this review, we cover the history and evolution of CAR technology and its adaptation to targeting CD19. Furthermore, we discuss the future of CAR T cell therapy and the need to ask, as well as answer, critical questions as this treatment modality is being translated to the clinic. © Society for Leukocyte Biology.

  12. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

    PubMed

    Wang, Dashan

    2018-06-01

    The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.

  13. Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase.

    PubMed

    Yancopoulos, G D; Blackwell, T K; Suh, H; Hood, L; Alt, F W

    1986-01-31

    We have recently proposed that a common recombinase performs all of the many variable region gene assembly events in B and T cells, and that the specificity of these joining events is mediated by regulating the "accessibility" of the involved gene segments. To test this possibility, we have introduced "accessible" T cell receptor (TCR) variable region gene segments into a pre-B cell line capable of recombining endogenous and transfected immunoglobulin (Ig) variable region gene segments. Although the corresponding "inaccessible" endogenous TCR gene segments do not rearrange in this line or in B cells in general, the introduced TCR gene segments join very frequently and, in fact, closely resemble introduced Ig gene segments in their recombination characteristics. These observations suggest a new role for conventional Ig transcriptional enhancers--recombinational enhancement. Our studies provide insight into additional aspects of the joining mechanism such as N region insertion, aberrant joining, and recombination-recognition sequence requirements for joining.

  14. Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays.

    PubMed

    Zoutman, Willem H; Nell, Rogier J; Versluis, Mieke; van Steenderen, Debby; Lalai, Rajshri N; Out-Luiting, Jacoba J; de Lange, Mark J; Vermeer, Maarten H; Langerak, Anton W; van der Velden, Pieter A

    2017-03-01

    Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. A chimeric switch-receptor targeting PD1 augments the efficacy of second generation CAR T-Cells in advanced solid tumors

    PubMed Central

    Liu, Xiaojun; Ranganathan, Raghuveer; Jiang, Shuguang; Fang, Chongyun; Sun, Jing; Kim, Soyeon; Newick, Kheng; Lo, Albert; June, Carl H.; Zhao, Yangbing; Moon, Edmund K.

    2015-01-01

    Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy (ATC) has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally-occurring and genetically-modified tumor infiltrating lymphocytes (TILs) by inhibitory receptors (IRs), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T cell activity against solid tumors. To address this possibility, we introduced a genetically-engineered switch receptor construct, comprising the truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T-cells. We tested the effect of this supplement, “PD1CD28”, on human CAR T-cells targeting aggressive models of human solid tumors expressing relevant tumor antigens. Treatment of mice bearing large, established solid tumors with PD1CD28 CAR T-cells led to significant regression in tumor volume due to enhanced CAR TIL infiltrate, decreased susceptibility to tumor-induced hypofunction, and attenuation of IR expression compared to treatments with CAR T-cells alone or PD1 antibodies. Taken together, our findings suggest that the application of PD1CD28 to boost CAR T-cell activity is efficacious against solid tumors via a variety of mechanisms, prompting clinical investigation of this potentially promising treatment modality. PMID:26979791

  16. CD4+ T cells defined by their Vβ T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis

    PubMed Central

    Keesen, T S L; Antonelli, L R V; Faria, D R; Guimarães, L H; Bacellar, O; Carvalho, E M; Dutra, W O; Gollob, K J

    2011-01-01

    Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vβ region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vβ region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4+ T cells expressing Vβ 5·2 and Vβ 24 in CL compared to controls; (2) a Leishmania antigen-induced increase in CD4+ T cells expressing Vβ 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4+ Vβ 5·2-, 11- and 24-positive T cells, with higher expression of CD45RO, HLA-DR, interferon-γ, tumour necrosis factor-α and interleukin-10 compared to other Vβ-expressing subpopulations; (4) a positive correlation between higher frequencies of CD4+Vβ5·2+ T cells and larger lesions; and (5) biased homing of CD4+ T cells expressing Vβ 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long-lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology. PMID:21726211

  17. T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice.

    PubMed

    Szomolanyi-Tsuda, Eva; Seedhom, Mina O; Carroll, Michael C; Garcea, Robert L

    2006-08-15

    Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

  18. Chemokine Receptor Signatures in Allogeneic Stem Cell Transplantation

    DTIC Science & Technology

    2014-08-01

    versus-host disease (GHVD). We use T-cell receptor deep sequencing to characterize the repertoire of effector T-cells in allogeneic hematopoietic stem ... cell transplant (HSCT) recipients and identify the role of chemokine receptors in effector cell infiltration of target organs. In the recent funding

  19. Chimeric antigen receptor-modified T cells for the treatment of solid tumors: Defining the challenges and next steps☆

    PubMed Central

    Beatty, Gregory L.; O’Hara, Mark

    2016-01-01

    Chimeric antigen receptor (CAR) T cell therapy has shown promise in CD19 expressing hematologic malignancies, but how to translate this success to solid malignancies remains elusive. Effective translation of CAR T cells to solid tumors will require an understanding of potential therapeutic barriers, including factors that regulate CAR T cells expansion, persistence, trafficking, and fate within tumors. Herein, we describe the current state of CAR T cells in solid tumors; define key barriers to CAR T cell efficacy and mechanisms underlying these barriers, outline potential avenues for overcoming these therapeutic obstacles, and discuss the future of translating CAR T cells for the treatment of patients with solid malignancies. PMID:27373504

  20. The role of MAPK in CD4{sup +} T cells toll-like receptor 9-mediated signaling following HHV-6 infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chi, Jing; Wang, Fang; Li, Lingyun

    2012-01-05

    Human herpesvirus-6 (HHV-6) is an important immunosuppressive and immunomodulatory virus that primarily infects immune cells (mainly CD4{sup +} T cells) and strongly suppresses the proliferation of infected cells. Toll-like receptors are pattern-recognition receptors essential for the development of an appropriate innate immune defense against infection. To understand the role of CD4{sup +} T cells in the innate response to HHV-6 infection and the involvement of TLRs, we used an in vitro infection model and observed that the infection of CD4{sup +} T cells resulted in the activation of JNK/SAPK via up-regulation of toll-like receptor 9 (TLR9). Associated with JNK activation,more » annexin V-PI staining indicated that HHV-6A was a strong inducer of apoptosis. Apoptotic response associated cytokines, IL-6 and TNF-{alpha} also induced by HHV-6A infection.« less

  1. Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

    PubMed Central

    Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

    2017-01-01

    The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously

  2. Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets.

    PubMed

    Chiang, David; Chen, Xintong; Jones, Stacie M; Wood, Robert A; Sicherer, Scott H; Burks, A Wesley; Leung, Donald Y M; Agashe, Charuta; Grishin, Alexander; Dawson, Peter; Davidson, Wendy F; Newman, Leah; Sebra, Robert; Merad, Miriam; Sampson, Hugh A; Losic, Bojan; Berin, M Cecilia

    2018-06-01

    The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA). We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing. Patients with PA had tissue- and follicle-homing peanut-responsive CD4 + T cells with a heterogeneous pattern of T H 2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2-dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro, but cytokines associated with highly differentiated T H 2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4, IL5, IL9, IL13, and the IL-25 receptor IL17RB. These results demonstrate the presence of highly differentiated T H 2 cells producing T H 2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional T H 2 response was more evident than a Treg cell deficit among peanut-responsive T cells. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. All rights reserved.

  3. Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function.

    PubMed

    Robinson, George A; Waddington, Kirsty E; Pineda-Torra, Ines; Jury, Elizabeth C

    2017-01-01

    It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed.

  4. Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

    PubMed Central

    Robinson, George A.; Waddington, Kirsty E.; Pineda-Torra, Ines; Jury, Elizabeth C.

    2017-01-01

    It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signaling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here, we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β, peroxisome proliferator-activated receptor γ, estrogen receptors α/β (ERα/β), and sterol regulatory element-binding proteins in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid, and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed. PMID:29225604

  5. Sezary syndrome cells unlike normal circulating T lymphocytes fail to migrate following engagement of NT1 receptor.

    PubMed

    Magazin, Marilyn; Poszepczynska-Guigné, Ewa; Bagot, Martine; Boumsell, Laurence; Pruvost, Christelle; Chalon, Pascale; Culouscou, Jean-Michel; Ferrara, Pascual; Bensussan, Armand

    2004-01-01

    Circulating malignant Sezary cells are a clonal proliferation of CD4+CD45RO+ T lymphocytes primarily involving the skin. To study the biology of these malignant T lymphocytes, we tested their ability to migrate in chemotaxis assays. Previously, we had shown that the neuropeptide neurotensin (NT) binds to freshly isolated Sezary malignant cells and induces through NT1 receptors the cell migration of the cutaneous T cell lymphoma cell line Cou-L. Here, we report that peripheral blood Sezary cells as well as the Sezary cell line Pno fail to migrate in response to neurotensin although they are capable of migrating to the chemokine stromal-cell-derived factor 1 alpha. This is in contrast with normal circulating CD4+ or CD8+ lymphocytes, which respond to both types of chemoattractants except after ex vivo short-time anti-CD3 monoclonal antibody activation, which abrogates the neurotensin-induced lymphocyte migration. Furthermore, we demonstrate that neurotensin-responsive T lymphocytes express the functional NT1 receptor responsible for chemotaxis. In these cells, but not in Sezary cells, neurotensin induces recruitment of phosphatidylinositol-3 kinase, and redistribution of phosphorylated cytoplasmic tyrosine kinase focal adhesion kinase and filamentous actin. Taken together, these results, which show functional distinctions between normal circulating lymphocytes and Sezary syndrome cells, contribute to further understanding of the physiopathology of these atypical cells.

  6. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling.

    PubMed

    Daniels, Mark A; Teixeiro, Emma; Gill, Jason; Hausmann, Barbara; Roubaty, Dominique; Holmberg, Kaisa; Werlen, Guy; Holländer, Georg A; Gascoigne, Nicholas R J; Palmer, Ed

    2006-12-07

    A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.

  7. Human Epidermal Growth Factor Receptor 2 (HER2) -Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma.

    PubMed

    Ahmed, Nabil; Brawley, Vita S; Hegde, Meenakshi; Robertson, Catherine; Ghazi, Alexia; Gerken, Claudia; Liu, Enli; Dakhova, Olga; Ashoori, Aidin; Corder, Amanda; Gray, Tara; Wu, Meng-Fen; Liu, Hao; Hicks, John; Rainusso, Nino; Dotti, Gianpietro; Mei, Zhuyong; Grilley, Bambi; Gee, Adrian; Rooney, Cliona M; Brenner, Malcolm K; Heslop, Helen E; Wels, Winfried S; Wang, Lisa L; Anderson, Peter; Gottschalk, Stephen

    2015-05-20

    The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence. © 2015 by American Society of Clinical Oncology.

  8. Preclinical Models in Chimeric Antigen Receptor-Engineered T-Cell Therapy.

    PubMed

    Siegler, Elizabeth Louise; Wang, Pin

    2018-05-01

    Cancer immunotherapy has enormous potential in inducing long-term remission in cancer patients, and chimeric antigen receptor (CAR)-engineered T cells have been largely successful in treating hematological malignancies in the clinic. CAR-T therapy has not been as effective in treating solid tumors, in part due to the immunosuppressive tumor microenvironment. Additionally, CAR-T therapy can cause dangerous side effects, including off-tumor toxicity, cytokine release syndrome, and neurotoxicity. Animal models of CAR-T therapy often fail to predict such adverse events and frequently overestimate the efficacy of the treatment. Nearly all preclinical CAR-T studies have been performed in mice, including syngeneic, xenograft, transgenic, and humanized mouse models. Recently, a few studies have used primate models to mimic clinical side effects better. To date, no single model perfectly recapitulates the human immune system and tumor microenvironment, and some models have revealed CAR-T limitations that were contradicted or missed entirely in other models. Careful model selection based on the primary goals of the study is a crucial step in evaluating CAR-T treatment. Advancements are being made in preclinical models, with the ultimate objective of providing safer, more effective CAR-T therapy to patients.

  9. Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

    PubMed Central

    Stevenson, M; Zhang, X H; Volsky, D J

    1987-01-01

    Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327

  10. Thermodynamics of T cell receptor – peptide/MHC interactions: progress and opportunities

    PubMed Central

    Armstrong, Kathryn M.; Insaidoo, Francis K.; Baker, Brian M.

    2013-01-01

    αβ T cell receptors (TCR) recognize peptide antigens presented by class I or class II major histocompatibility complex molecules (pMHC). Here we review the use of thermodynamic measurements in the study of TCR-pMHC interactions, with attention to the diversity in binding thermodynamics and how this is related to the variation in TCR-pMHC interfaces. We show that there is no enthalpic or entropic signature for TCR binding; rather, enthalpy and entropy changes vary in a compensatory manner that reflects a narrow free energy window for the interactions that have been characterized. Binding enthalpy and entropy changes do not correlate with structural features such as buried surface area or the number of hydrogen bonds within TCR-pMHC interfaces, possibly reflecting the myriad of contributors to binding thermodynamics, but likely also reflecting a reliance on van’t Hoff over calorimetric measurements and the unaccounted influence of equilibria linked to binding. TCR-pMHC binding heat capacity changes likewise vary considerably. In some cases the heat capacity changes are consistent with conformational differences between bound and free receptors, but there is little data indicating these conformational differences represent the need to organize commonly disordered CDR loops. In this regard, we discuss how thermodynamics may provide additional insight into conformational changes occurring upon TCR binding. Finally, we highlight opportunities for the further use of thermodynamic measurements in the study of TCR-pMHC interactions, not only for understanding TCR binding in general, but for understanding specifics of individual interactions and the engineering of T cell receptors with desired molecular recognition properties. PMID:18496839

  11. Normal T lymphocytes can express two different T cell receptor beta chains: implications for the mechanism of allelic exclusion

    PubMed Central

    1995-01-01

    We have examined the extent of allelic exclusion at the T cell receptor (TCR) beta locus using monoclonal antibodies specific for V beta products. A small proportion (approximately 1%) of human peripheral blood T cells express two V beta as determined by flow cytometric analysis, isolation of representative clones, and sequencing of the corresponding V beta chains. Dual beta T cells are present in both the CD45R0+ and CD45R0- subset. These results indicate that dual beta expression is compatible with both central and peripheral selection. They also suggest that the substantial degree of TCR beta allelic exclusion is dependent only on asynchronous rearrangements at the beta locus, whereas the role of the pre-TCR is limited to signaling the presence of at least one functional beta protein. PMID:7699339

  12. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

    PubMed

    Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

    2015-02-03

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

  13. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

    PubMed Central

    Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

    2015-01-01

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

  14. TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia

    PubMed Central

    Catakovic, Kemal; Gassner, Franz Josef; Ratswohl, Christoph; Zaborsky, Nadja; Rebhandl, Stefan; Schubert, Maria; Steiner, Markus; Gutjahr, Julia Christine; Pleyer, Lisa; Egle, Alexander; Hartmann, Tanja Nicole; Greil, Richard; Geisberger, Roland

    2018-01-01

    ABSTRACT While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment. PMID:29296521

  15. Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever.

    PubMed

    de-Oliveira-Pinto, Luzia Maria; Marinho, Cíntia Ferreira; Povoa, Tiago Fajardo; de Azeredo, Elzinandes Leal; de Souza, Luiza Assed; Barbosa, Luiza Damian Ribeiro; Motta-Castro, Ana Rita C; Alves, Ada M B; Ávila, Carlos André Lins; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Paes, Marciano Viana; Kubelka, Claire Fernandes

    2012-01-01

    Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by

  16. Regulation of Inflammatory Chemokine Receptors on Blood T Cells Associated to the Circulating Versus Liver Chemokines in Dengue Fever

    PubMed Central

    Povoa, Tiago Fajardo; de Azeredo, Elzinandes Leal; de Souza, Luiza Assed; Barbosa, Luiza Damian Ribeiro; Motta-Castro, Ana Rita C.; Alves, Ada M. B.; Ávila, Carlos André Lins; de Souza, Luiz José; da Cunha, Rivaldo Venâncio; Damasco, Paulo Vieira; Paes, Marciano Viana; Kubelka, Claire Fernandes

    2012-01-01

    Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES+ cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44HIGH and CD127LOW markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines

  17. How nonuniform contact profiles of T cell receptors modulate thymic selection outcomes

    NASA Astrophysics Data System (ADS)

    Chen, Hanrong; Chakraborty, Arup K.; Kardar, Mehran

    2018-03-01

    T cell receptors (TCRs) bind foreign or self-peptides attached to major histocompatibility complex (MHC) molecules, and the strength of this interaction determines T cell activation. Optimizing the ability of T cells to recognize a diversity of foreign peptides yet be tolerant of self-peptides is crucial for the adaptive immune system to properly function. This is achieved by selection of T cells in the thymus, where immature T cells expressing unique, stochastically generated TCRs interact with a large number of self-peptide-MHC; if a TCR does not bind strongly enough to any self-peptide-MHC, or too strongly with at least one self-peptide-MHC, the T cell dies. Past theoretical work cast thymic selection as an extreme value problem and characterized the statistical enrichment or depletion of amino acids in the postselection TCR repertoire, showing how T cells are selected to be able to specifically recognize peptides derived from diverse pathogens yet have limited self-reactivity. Here, we investigate how the diversity of the postselection TCR repertoire is modified when TCRs make nonuniform contacts with peptide-MHC. Specifically, we were motivated by recent experiments showing that amino acids at certain positions of a TCR sequence have large effects on thymic selection outcomes, and crystal structure data that reveal a nonuniform contact profile between a TCR and its peptide-MHC ligand. Using a representative TCR contact profile as an illustration, we show via simulations that the statistical enrichment or depletion of amino acids now varies by position according to the contact profile, and, importantly, it depends on the implementation of nonuniform contacts during thymic selection. We explain these nontrivial results analytically. Our study has implications for understanding the selection forces that shape the functionality of the postselection TCR repertoire.

  18. T cell receptor cross-reactivity between similar foreign and self peptides influences naïve cell population size and autoimmunity

    PubMed Central

    Nelson, Ryan W.; Beisang, Daniel; Tubo, Noah J.; Dileepan, Thamotharampillai; Wiesner, Darin L.; Nielsen, Kirsten; Wüthrich, Marcel; Klein, Bruce S.; Kotov, Dmitri I.; Spanier, Justin A.; Fife, Brian T.; Moon, James J.; Jenkins, Marc K.

    2014-01-01

    SUMMARY T cell receptor (TCR) cross-reactivity between major histocompatibility complex II (MHCII)-binding self and foreign peptides could influence the naïve CD4+ T cell repertoire and autoimmunity. We found that nonamer peptides that bind to the same MHCII molecule only need to share five amino acids to cross-react on the same TCR. This property was biologically relevant since systemic expression of a self peptide reduced the size of a naïve cell population specific for a related foreign peptide by deletion of cells with cross-reactive TCRs. Reciprocally, an incompletely deleted naïve T cell population specific for a tissue-restricted self peptide could be triggered by related microbial peptides to cause autoimmunity. Thus, TCR cross-reactivity between similar self and foreign peptides can reduce the size of certain foreign peptide-specific T cell populations, and may allow T cell populations specific for tissue-restricted self peptides to cause autoimmunity after infection. PMID:25601203

  19. Core Fucosylation on T Cells, Required for Activation of T-Cell Receptor Signaling and Induction of Colitis in Mice, Is Increased in Patients With Inflammatory Bowel Disease.

    PubMed

    Fujii, Hironobu; Shinzaki, Shinichiro; Iijima, Hideki; Wakamatsu, Kana; Iwamoto, Chizuru; Sobajima, Tomoaki; Kuwahara, Ryusuke; Hiyama, Satoshi; Hayashi, Yoshito; Takamatsu, Shinji; Uozumi, Naofumi; Kamada, Yoshihiro; Tsujii, Masahiko; Taniguchi, Naoyuki; Takehara, Tetsuo; Miyoshi, Eiji

    2016-06-01

    Attachment of a fucose molecule to the innermost N-glycan in a glycoprotein (core fucosylation) regulates the activity of many growth factor receptors and adhesion molecules. The process is catalyzed by α1-6 fucosyltransferase (FUT8) and required for immune regulation, but it is not clear whether this process is dysregulated during disease pathogenesis. We investigated whether core fucosylation regulates T-cell activation and induction of colitis in mice, and is altered in patients with inflammatory bowel disease (IBD). Biopsy samples were collected from inflamed and noninflamed regions of intestine from patients (8 with Crohn's disease, 4 with ulcerative colitis, and 4 without IBD [controls]) at Osaka University Hospital. Colitis was induced in FUT8-deficient (Fut8(-/-)) mice and Fut8(+/+) littermates by administration of trinitrobenzene sulfonic acid. Intestinal tissues were collected and analyzed histologically. Immune cells were collected and analyzed by lectin flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction, as well as for production of cytokines and levels of T-cell receptor (TCR) in lipid raft fractions. T-cell function was analyzed by intraperitoneal injection of CD4(+)CD62L(+) naïve T cells into RAG2-deficient mice. Levels of core fucosylation were increased on T cells from mice with colitis, compared with mice without colitis, as well as on inflamed mucosa from patients with IBD, compared with their noninflamed tissues or tissues from control patients. Fut8(-/-) mice developed less-severe colitis than Fut8(+/+) mice, and T cells from Fut8(-/-) mice produced lower levels of T-helper 1 and 2 cytokines. Adoptive transfer of Fut8(-/-) T cells to RAG2-deficient mice reduced the severity of colitis. Compared with CD4(+) T cells from Fut8(+/+) mice, those from Fut8(-/-) mice expressed similar levels of TCR and CD28, but these proteins did not contain core fucosylation. TCR complexes formed on CD4(+) T cells from Fut8

  20. Adoptive immunotherapy utilizing anti-CD19 chimeric antigen receptor T-cells for B-cell malignancies.

    PubMed

    Oh, Iekuni; Oh, Yukiko; Ohmine, Ken

    2016-01-01

    Genetically modified T-cells with forced expression of anti-CD19 chimeric antigen receptor (CD19 CAR) have demonstrated promising clinical results for relapsed and refractory B cell malignancies in early clinical trial settings. The first beneficial tumor regressions were identified among approximately half of CLL patients in 2011. Similarly, CD19 CAR T-cells achieved remissions in about 80% of aggressive B-cell lymphomas in 2012. Furthermore, in 2013 this cellular therapy showed an extremely high rate of efficacy against refractory CD19 positive acute lymphoid leukemia, which had been regarded as the most difficult to treat hematologic disease. Recently, despite the absence of CD19 expression by neoplastic plasma cells, patients with refractory multiple myeloma achieved stringent complete remission after this therapy coupled with high dose chemotherapy and autologous stem cell transplantation. However, there are significant toxicities. Cytokine releasing syndrome and neurotoxicity are recognized as life-threatening adverse events. Although phase I/II clinical trials have just started in Japan, given the exciting results obtained to date, this cellular therapy is expected to be a novel breakthrough immunotherapy for treating refractory B-cell malignancies.

  1. Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells.

    PubMed

    Badache, A; Hynes, N E

    2001-01-01

    Interleukin (IL)-6, a multifunctional regulator of immune response, hematopoiesis, and acute phase reactions, has also been shown to regulate cancer cell proliferation. We have investigated IL-6 signaling pathways and cellular responses in the T47D breast carcinoma cell line. The IL-6-type cytokines, IL-6 and oncostatin M, simultaneously inhibited cell proliferation and increased cell migration. In T47D cells, IL-6 stimulated the activation of Janus-activated kinase 1 tyrosine kinase and signal transducers and activators of transcription (STAT) 1 and STAT3 transcription factors. Expression of dominant negative STAT3 in the cells strongly reduced IL-6-mediated growth inhibition but did not prevent IL-6-induced cell migration. IL-6 treatment led to activation of the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3'-kinase (PI3K) pathways. Inhibition of MAPK or PI3K activity reversed IL-6- and oncostatin M-stimulated migration. Because cross-talk between cytokine receptors and members of the ErbB family of receptor tyrosine kinases has been described previously, we have examined their interaction in T47D cells. Down-regulation of ErbB receptor activity, through the use of specific pharmacological inhibitors or dominant negative receptor constructs, revealed that IL-6-induced MAPK activation was largely dependent on epidermal growth factor (EGF) receptor activity, but not on ErbB-2 activity. Using a monoclonal antibody that interferes with EGF receptor-ligand interaction, we have shown that in T47D cells, IL-6 cooperates with an EGF receptor autocrine activity loop for signaling through the MAPK and PI3K pathways and for cell migration. Both the tyrosine phosphatase SHP-2 and the multisubstrate docking molecule Gab1, which are potential links between IL-6 and the MAPK/PI3K pathways, were constitutively associated with the active EGF receptor. On IL-6 stimulation, SHP-2 and Gab1 were recruited to the gp130 subunit of the IL-6 receptor and tyrosine

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

    PubMed Central

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

    2016-01-01

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

  3. Immune Tolerance Maintained by Cooperative Interactions between T Cells and Antigen Presenting Cells Shapes a Diverse TCR Repertoire

    PubMed Central

    Best, Katharine; Chain, Benny; Watkins, Chris

    2015-01-01

    The T cell population in an individual needs to avoid harmful activation by self peptides while maintaining the ability to respond to an unknown set of foreign peptides. This property is acquired by a combination of thymic and extra-thymic mechanisms. We extend current models for the development of self/non-self discrimination to consider the acquisition of self-tolerance as an emergent system level property of the overall T cell receptor repertoire. We propose that tolerance is established at the level of the antigen presenting cell/T cell cluster, which facilitates and integrates cooperative interactions between T cells of different specificities. The threshold for self-reactivity is therefore imposed at a population level, and not at the level of the individual T cell/antigen encounter. Mathematically, the model can be formulated as a linear programing optimization problem that can be implemented as a multiplicative update algorithm, which shows a rapid convergence to a stable state. The model constrains self-reactivity within a predefined threshold, but maintains repertoire diversity and cross reactivity which are key characteristics of human T cell immunity. We show further that the size of individual clones in the model repertoire becomes heterogeneous, and that new clones can establish themselves even when the repertoire has stabilized. Our study combines the salient features of the “danger” model of self/non-self discrimination with the concepts of quorum sensing, and extends repertoire generation models to encompass the establishment of tolerance. Furthermore, the dynamic and continuous repertoire reshaping, which underlies tolerance in this model, suggests opportunities for therapeutic intervention to achieve long-term tolerance following transplantation. PMID:26300880

  4. Improve T Cell Therapy in Neuroblastoma

    DTIC Science & Technology

    2014-07-01

    or non- lymphoid tissue (132). Their potential value as CAR-expressing effec- tor cells is considered below. Provision of costimulation to enhance CAR-T... lymphoid leukemia. N Engl J Med 2011;365:725–733. 42. Kalos M, et al. T cells with chimeric antigen receptors have potent antitumor effects and can...antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:1509–1518. 45. Till BG, et al. Adoptive immunotherapy for indolent non

  5. Working in "NK Mode": Natural Killer Group 2 Member D and Natural Cytotoxicity Receptors in Stress-Surveillance by γδ T Cells.

    PubMed

    Silva-Santos, Bruno; Strid, Jessica

    2018-01-01

    Natural killer cell receptors (NKRs) are germline-encoded transmembrane proteins that regulate the activation and homeostasis of NK cells as well as other lymphocytes. For γδ T cells, NKRs play critical roles in discriminating stressed (transformed or infected) cells from their healthy counterparts, as proposed in the "lymphoid stress-surveillance" theory. Whereas the main physiologic role is seemingly fulfilled by natural killer group 2 member D, constitutively expressed by γδ T cells, enhancement of their therapeutic potential may rely on natural cytotoxicity receptors (NCRs), like NKp30 or NKp44, that can be induced selectively on human Vδ1 + T cells. Here, we review the contributions of NCRs, NKG2D, and their multiple ligands, to γδ T cell biology in mouse and human.

  6. Receptor-mediated cell mechanosensing

    PubMed Central

    Chen, Yunfeng; Ju, Lining; Rushdi, Muaz; Ge, Chenghao; Zhu, Cheng

    2017-01-01

    Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components of force, stress, and strain but also substrate rigidity, topology, and adhesiveness. This ability is crucial for the cell to respond to the surrounding mechanical cues and adapt to the changing environment. Examples of responses and adaptation include (de)activation, proliferation/apoptosis, and (de)differentiation. Receptor-mediated cell mechanosensing is a multistep process that is initiated by binding of cell surface receptors to their ligands on the extracellular matrix or the surface of adjacent cells. Mechanical cues are presented by the ligand and received by the receptor at the binding interface; but their transmission over space and time and their conversion into biochemical signals may involve other domains and additional molecules. In this review, a four-step model is described for the receptor-mediated cell mechanosensing process. Platelet glycoprotein Ib, T-cell receptor, and integrins are used as examples to illustrate the key concepts and players in this process. PMID:28954860

  7. Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy.

    PubMed

    Yeku, Oladapo O; Brentjens, Renier J

    2016-04-15

    Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-1BB in addition to CD3ζ provided T-cell activation signal 2 and further improved the efficacy and persistence of these second generation CAR T-cells. Third generation CAR T-cells which utilize two tandem costimulatory domains have also been reported. In this review, we discuss a different approach to optimization of CAR T-cells. Through additional genetic modifications, these resultant armored CAR T-cells are typically modified second generation CAR T-cells that have been further optimized to inducibly or constitutively secrete active cytokines or express ligands that further armor CAR T-cells to improve efficacy and persistence. The choice of the 'armor' agent is based on knowledge of the tumour microenvironment and the roles of other elements of the innate and adaptive immune system. Although there are several variants of armored CAR T-cells under investigation, here we focus on three unique approaches using interleukin-12 (IL-12), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T-cell efficacy and persistence in the face of a hostile tumour microenvironment via different mechanisms. © 2016 Authors; published by Portland Press Limited.

  8. Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy

    PubMed Central

    Yeku, Oladapo O.; Brentjens, Renier J.

    2017-01-01

    Chimaeric antigen receptor (CAR) T-cells are T-cells that have been genetically modified to express an artificial construct consisting of a synthetic T-cell receptor (TCR) targeted to a predetermined antigen expressed on a tumour. Coupling the T-cell receptor to a CD3ζ signalling domain paved the way for first generation CAR T-cells that were efficacious against cluster of differentiation (CD)19-expressing B-cell malignancies. Optimization with additional signalling domains such as CD28 or 4-1BB in addition to CD3ζ provided T-cell activation signal 2 and further improved the efficacy and persistence of these second generation CAR T-cells. Third generation CAR T-cells which utilize two tandem costimulatory domains have also been reported. In this review, we discuss a different approach to optimization of CAR T-cells. Through additional genetic modifications, these resultant armored CAR T-cells are typically modified second generation CAR T-cells that have been further optimized to inducibly or constitutively secrete active cytokines or express ligands that further armor CAR T-cells to improve efficacy and persistence. The choice of the ‘armor’ agent is based on knowledge of the tumour microenvironment and the roles of other elements of the innate and adaptive immune system. Although there are several variants of armored CAR T-cells under investigation, here we focus on three unique approaches using interleukin-12 (IL-12), CD40L and 4-1BBL. These agents have been shown to further enhance CAR T-cell efficacy and persistence in the face of a hostile tumour microenvironment via different mechanisms. PMID:27068948

  9. Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model.

    PubMed

    Chapelin, Fanny; Gao, Shang; Okada, Hideho; Weber, Thomas G; Messer, Karen; Ahrens, Eric T

    2017-12-18

    Discovery of effective cell therapies against cancer can be accelerated by the adaptation of tools to rapidly quantitate cell biodistribution and survival after delivery. Here, we describe the use of nuclear magnetic resonance (NMR) 'cytometry' to quantify the biodistribution of immunotherapeutic T cells in intact tissue samples. In this study, chimeric antigen receptor (CAR) T cells expressing EGFRvIII targeting transgene were labeled with a perfluorocarbon (PFC) emulsion ex vivo and infused into immunocompromised mice bearing subcutaneous human U87 glioblastomas expressing EGFRvIII and luciferase. Intact organs were harvested at day 2, 7 and 14 for whole-sample fluorine-19 ( 19 F) NMR to quantitatively measure the presence of PFC-labeled CAR T cells, followed by histological validation. NMR measurements showed greater CAR T cell homing and persistence in the tumors and spleen compared to untransduced T cells. Tumor growth was monitored with bioluminescence imaging, showing that CAR T cell treatment resulted in significant tumor regression compared to untransduced T cells. Overall, 19 F NMR cytometry is a rapid and quantitative method to evaluate cell biodistribution, tumor homing, and fate in preclinical studies.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-09-08

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

  12. Gab-family adapter proteins act downstream of cytokine and growth factor receptors and T- and B-cell antigen receptors.

    PubMed

    Nishida, K; Yoshida, Y; Itoh, M; Fukada, T; Ohtani, T; Shirogane, T; Atsumi, T; Takahashi-Tezuka, M; Ishihara, K; Hibi, M; Hirano, T

    1999-03-15

    We previously found that the adapter protein Gab1 (110 kD) is tyrosine-phosphorylated and forms a complex with SHP-2 and PI-3 kinase upon stimulation through either the interleukin-3 receptor (IL-3R) or gp130, the common receptor subunit of IL-6-family cytokines. In this report, we identified another adapter molecule (100 kD) interacting with SHP-2 and PI-3 kinase in response to various stimuli. The molecule displays striking homology to Gab1 at the amino acid level; thus, we named it Gab2. It contains a PH domain, proline-rich sequences, and tyrosine residues that bind to SH2 domains when they are phosphorylated. Gab1 is phosphorylated on tyrosine upon stimulation through the thrombopoietin receptor (TPOR), stem cell factor receptor (SCFR), and T-cell and B-cell antigen receptors (TCR and BCR, respectively), in addition to IL-3R and gp130. Tyrosine phosphorylation of Gab2 was induced by stimulation through gp130, IL-2R, IL-3R, TPOR, SCFR, and TCR. Gab1 and Gab2 were shown to be substrates for SHP-2 in vitro. Overexpression of Gab2 enhanced the gp130 or Src-related kinases-mediated ERK2 activation as that of Gab1 did. These data indicate that Gab-family molecules act as adapters for transmitting various signals.

  13. Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

    PubMed

    Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

    2017-01-01

    Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  14. Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

    PubMed Central

    Mamalaki, Clio; Elliott, James; Norton, Trisha; Yannoutsos, Nicholas; Townsend, Alain R.; Chandler, Phillip; Simpson, Elizabeth

    1993-01-01

    A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired. PMID:8281031

  15. 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.

  16. 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

  17. Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

    PubMed

    Giudice, Valentina; Feng, Xingmin; Lin, Zenghua; Hu, Wei; Zhang, Fanmao; Qiao, Wangmin; Ibanez, Maria Del Pilar Fernandez; Rios, Olga; Young, Neal S

    2018-05-01

    Oligoclonal expansion of CD8 + CD28 - lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 + CD28 - cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8 + CD28 - CD57 + cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8 + CD57 + cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 + cells from aplastic anemia patients with CD8 + CD57 + cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8 + CD57 + cells, which also showed decreased diversity compared to total CD4 + and CD8 + cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 + cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 + T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at clinicaltrials.gov identifiers: 00001620, 01623167, 00001397, 00071045, 00081523, 00961064 ). Copyright © 2018 Ferrata Storti Foundation.

  18. Decoy receptor 3 attenuates collagen-induced arthritis by modulating T cell activation and B cell expansion.

    PubMed

    Cheng, Chia-Pi; Sytwu, Huey-Kang; Chang, Deh-Ming

    2011-12-01

    To investigate the immune-modulated effects of decoy receptor 3 (DCR3) in an experimental model of rheumatoid arthritis (RA). We delivered DCR3 plasmid into collagen-induced arthritis (CIA) mice using the hydrodynamic method and evaluated the serum level of DCR3 protein by ELISA. After immunization, we assessed disease severity of arthritis incidence, arthritis scores, paw thickness, and means of arthritic limbs, and used hematoxylin and eosin staining to observe synovial hyperplasia. We analyzed numbers of murine splenocytes and inguinal lymphocyte cells, cell populations, and serum proinflammatory cytokines by flow cytometry. We investigated B cell proliferation by carboxyfluorescein succinimidyl ester assay. We evaluated serum levels of total IgG2a and type II collagen-specific IgG and IgG2a using ELISA. DCR3 expression in sera significantly attenuated disease severity in CIA mice. We found that DCR3 inhibited the volume of inguinal lymph nodes, numbers of CD19+ B cells, and populations of interferon-γ, interleukin 4 (IL-4), IL-17A, and Foxp3-producing CD4+ T cell in vivo. We found that DCR3 inhibited Pam3CSK4 (Toll-like receptor 1/2 ligand)-induced B220+ B cell proliferation in vitro. DCR3 treatment reduced the serum level of IL-6, total IgG2a, and CII-specific IgG2a antibody. We postulated that the protective effects of DCR3 in CIA resulted from modulation of the immune system by maintaining the B/T cell balance and decreasing lymphocyte expansion. We suggest DCR3 as a prophylactic and potential therapeutic agent in the treatment of RA.

  19. 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

  20. Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses.

    PubMed

    Choi, Dong Hoon; Kim, Kwang Soon; Yang, Se Hwan; Chung, Doo Hyun; Song, Boyeong; Sprent, Jonathan; Cho, Jae Ho; Sung, Young Chul

    2011-12-15

    Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

  1. Chimeric antigen receptor-engineered natural killer and natural killer T cells for cancer immunotherapy.

    PubMed

    Bollino, Dominique; Webb, Tonya J

    2017-09-01

    Natural killer (NK) cells of the innate immune system and natural killer T (NKT) cells, which have roles in both the innate and adaptive responses, are unique lymphocyte subsets that have similarities in their functions and phenotypes. Both cell types can rapidly respond to the presence of tumor cells and participate in immune surveillance and antitumor immune responses. This has incited interest in the development of novel cancer therapeutics based on NK and NKT cell manipulation. Chimeric antigen receptors (CARs), generated through the fusion of an antigen-binding region of a monoclonal antibody or other ligand to intracellular signaling domains, can enhance lymphocyte targeting and activation toward diverse malignancies. Most of the CAR studies have focused on their expression in T cells; however, the functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. CAR-modified NK and NKT cells are becoming more prevalent because they provide a method to direct these cells more specifically to target cancer cells, with less risk of adverse effects. This review will outline current NK and NKT cell CAR constructs and how they compare to conventional CAR T cells, and discuss future modifications that can be explored to advance adoptive cell transfer of NK and NKT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Expansions of NK-like αβT cells with chronologic aging: Novel lymphocyte effectors that compensate for functional deficits of conventional NK cells and T cells

    PubMed Central

    Vallejo, Abbe N.; Mueller, Robert G.; Hamel, David L.; Way, Amanda; Dvergsten, Jeffrey A.; Griffin, Patricia; Newman, Anne B.

    2010-01-01

    As the repertoire of αβT cell receptors (TCR) contracts with advancing age, there is an associated age-dependent accumulation of oligoclonal T cells expressing of a variety of receptors (NKR), normally expressed on natural killer (NK) cells. Evidences for differential regulation of expression of particular NKRs between T cells and NK cells suggest that NKR expression on T cells is physiologically programmed rather than a random event of the aging process. Experimental studies show NKRs on aged αβT cells may function either as independent receptors, and/or as costimulatory receptors to the TCR. Considering the reported deficits of conventional αβTCR-driven activation and also functional deficits of classical NK cells, NKR+ αβT cells likely represent novel immune effectors that are capable of combining innate and adaptive functions. Inasmuch as immunity is a determinant of individual fitness, the type and density of NKRs could be important contributing factors to the wide heterogeneity of health characteristics of older adults, ranging from institutionalized frail elders who are unable to mount immune responses to functionally independent community-dwelling elders who exhibit protective immunity. Understanding the biology of NKR+ αβT cells could lead to new avenues for age-specific intervention to improve protective immunity. PMID:20932941

  3. Peroxisome Proliferator-Activated Receptor γ Deficiency in T Cells Accelerates Chronic Rejection by Influencing the Differentiation of CD4+ T Cells and Alternatively Activated Macrophages

    PubMed Central

    Ye, Ping; Cheng, Chao; Wu, Jie; Wang, Sihua; Sun, Yuan; Liu, Zheng; Xie, Aini; Xia, Jiahong

    2014-01-01

    Background In a previous study, activation of the peroxisome proliferator–activated receptor γ (PPARγ) inhibited chronic cardiac rejection. However, because of the complexity of chronic rejection and the fact that PPARγ is widely expressed in immune cells, the mechanism of the PPARγ - induced protective effect was unclear. Materials and Methods A chronic rejection model was established using B6.C-H-2bm12KhEg (H-2bm12) mice as donors, and MHC II-mismatched T-cell-specific PPARγ knockout mice or wild type (WT) littermates as recipients. The allograft lesion was assessed by histology and immunohistochemistry. T cells infiltrates in the allograft were isolated, and cytokines and subpopulations were detected using cytokine arrays and flow cytometry. Transcription levels in the allograft were measured by RT-PCR. In vitro, the T cell subset differentiation was investigated after culture in various polarizing conditions. PPARγ-deficient regularory T cells (Treg) were cocultured with monocytes to test their ability to induce alternatively activated macrophages (AAM). Results T cell-specific PPARγ knockout recipients displayed reduced cardiac allograft survival and an increased degree of pathology compared with WT littermates. T cell-specific PPARγ knockout resulted in more CD4+ T cells infiltrating into the allograft and altered the Th1/Th2 and Th17/Treg ratios. The polarization of AAM was also reduced by PPARγ deficiency in T cells through the action of Th2 and Treg. PPARγ-deficient T cells eliminated the pioglitazone-induced polarization of AAM and reduced allograft survival. Conclusions PPARγ-deficient T cells influenced the T cell subset and AAM polarization in chronic allograft rejection. The mechanism of PPARγ activation in transplantation tolerance could yield a novel treatment without side effects. PMID:25383620

  4. Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNAmore » staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.« less

  5. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cellmore » migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.« less

  6. Death receptor 3 signaling enhances proliferation of human regulatory T cells.

    PubMed

    Bittner, Sebastian; Knoll, Gertrud; Ehrenschwender, Martin

    2017-04-01

    Exploiting regulatory T cells (Tregs) to control aberrant immune reactions is a promising therapeutic approach, but is hampered by their relative paucity. In mice, activation of death receptor 3 (DR3), a member of the TNF-receptor superfamily (TNFRSF), increases Treg frequency and efficiently controls exuberant immune activation. For human Tregs, neither DR3 expression nor potential functions have been described. Here, we show that human Tregs express DR3 and demonstrate DR3-mediated activation of p38, ERK, and NFκB. DR3 stimulation enhances Treg expansion ex vivo while retaining their suppressive capacity. In summary, our results establish a functional role for DR3 signaling in human Tregs and could potentially help to tailor Treg-based therapies. © 2017 Federation of European Biochemical Societies.

  7. Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.

    PubMed

    Chinnasamy, Dhanalakshmi; Yu, Zhiya; Kerkar, Sid P; Zhang, Ling; Morgan, Richard A; Restifo, Nicholas P; Rosenberg, Steven A

    2012-03-15

    We investigated the feasibility of delivering the proinflammatory cytokine interleukin (IL)-12 into tumor using T cells genetically engineered to express a chimeric antigen receptor (CAR) against the VEGF receptor-2 (VEGFR-2). Two different strains of mice bearing five different established subcutaneous tumors were treated with syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressed single-chain murine IL-12 or an inducible IL-12 gene after host lymphodepletion. Tumor regression, survival of mice, and persistence of the transferred cells were evaluated. Adoptive transfer of syngeneic T cells cotransduced with an anti-VEGFR-2 CAR and a constitutively expressing single-chain IL-12 resulted in the regression of five different established tumors of different histologies without the need for IL-2 administration. T cells transduced with either anti-VEGFR-2 CAR or single-chain IL-12 alone did not alter the tumor growth indicating that both of them had to be expressed in the same cell to mediate tumor regression. Anti-VEGFR-2 CAR and IL-12-cotransduced T cells infiltrated the tumors, expanded, and persisted for prolonged periods. The antitumor effect did not require the presence of host T and B cells but was dependent on host IL-12R-expressing cells. The anti-VEGFR-2 CAR changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b(+)Gr1(+) myeloid suppressor cell subsets that expressed VEGFR-2. These results suggest that targeted delivery of IL-12 into the tumor environment with T cells redirected against VEGFR-2 is a promising approach for treating patients with a variety of solid tumor types.

  8. Carcinoma autoantigens T and Tn and their cleavage products interact with Gal/GalNAc-specific receptors on rat Kupffer cells and hepatocytes.

    PubMed

    Schlepper-Schäfer, J; Springer, G F

    1989-10-09

    We studied interactions of isolated Thomsen-Friedenreich (T)- and Tn-specific glycoproteins with the Gal/GalNAc-specific receptors on rat Kupffer cells and compared them to those with rat hepatocytes. Immunoreactive T and Tn are specific pancarcinoma epitopes. Electron microscopy of gold-labelled T and Tn antigens revealed their specific binding to Kupffer cells, followed by their uptake via the coated pit/vesicle pathway of receptor-mediated endocytosis. Preincubation of Kupffer cells with GalNAc and GalNAc-BSA, but not GlcNAc or GlcNAc-BSA specifically inhibited binding of the T and Tn glycoproteins. Desialylated, isologous erythrocytes (T RBC) are known to bind to the Gal/GalNAc receptors of rat Kupffer cells and hepatocytes. This attachment was specifically inhibited by T and Tn in a concentration-dependent manner: 50% T RBC-Kupffer cell contacts were inhibited at 8.5.10(-6) mM T and 8.5.10(-5) mM Tn antigen concentrations, respectively. The corresponding figures for hepatocytes were 6.10(-6) mM T and 1.2.10(-6) mM Tn antigen. Amino-terminal cleavage products of the T glycoprotein, possessing clusters terminating in non-reducing Gal/GalNAc, inhibited T RBC binding to Kupffer cells and hepatocytes usually at 10(-2) to 10(-5) mM concentrations, whereas GalNAc, galactose and galactose glycosides inhibited at millimolar concentrations. Galactose-unrelated carbohydrates were inactive at concentrations greater than or equal to 50 mM.

  9. Acetylcholine released from T cells regulates intracellular Ca2+, IL-2 secretion and T cell proliferation through nicotinic acetylcholine receptor.

    PubMed

    Mashimo, Masato; Iwasaki, Yukari; Inoue, Shoko; Saito, Shoko; Kawashima, Koichiro; Fujii, Takeshi

    2017-03-01

    T lymphocytes synthesize acetylcholine (ACh) and express muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively) responsible for increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ). Our aim in the present study was to assess whether autocrine ACh released from T lymphocytes regulates their physiological functions. MOLT-3 human leukemic cell line and murine splenocytes were loaded with fura-2 to monitor [Ca 2+ ] i changes in the absence or presence of several AChR antagonists, including mecamylamine, methyllycaconitine and scopolamine. Real-time PCR and ELISA were performed to measure interleukin-2 (IL-2) mRNA and protein levels. T lymphocytes constitutively produce sufficient amounts of ACh to elicit autocrine changes in [Ca 2+ ] i . These autocrine ACh-evoked [Ca 2+ ] i transients were mediated by nAChRs and then influx of extracellular Ca 2+ . Mecamylamine, a nAChR inhibitor, suppressed not only these [Ca 2+ ] i transients, but also IL-2 release and T cell proliferation. Here, we confirmed that T lymphocytes utilize ACh as a tool to interact with each other and that autocrine ACh-activated nAChRs are involved in cytokine release and cell proliferation. These findings suggest the possibility that nAChR agonists and antagonists and smoking are able to modulate immune function, which in turn suggests the therapeutic potential of immune activation or suppression using nAChR agonists or antagonists. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V subgroup synteny and chain-specific evolution

    USGS Publications Warehouse

    Breaux, Breanna; Hunter, Margaret; Cruz-Schneider, Maria Paula; Sena, Leonardo; Bonde, Robert K.; Criscitiello, Michael F.

    2018-01-01

    The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostrisand human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies.

  11. The Florida manatee (Trichechus manatus latirostris) T cell receptor loci exhibit V subgroup synteny and chain-specific evolution.

    PubMed

    Breaux, Breanna; Hunter, Margaret E; Cruz-Schneider, Maria Paula; Sena, Leonardo; Bonde, Robert K; Criscitiello, Michael F

    2018-08-01

    The Florida manatee (Trichechus manatus latirostris) has limited diversity in the immunoglobulin heavy chain. We therefore investigated the antigen receptor loci of the other arm of the adaptive immune system: the T cell receptor. Manatees are the first species from Afrotheria, a basal eutherian superorder, to have an in-depth characterization of all T cell receptor loci. By annotating the genome and expressed transcripts, we found that each chain has distinct features that correlates to their individual functions. The genomic organization also plays a role in modulating sequence conservation between species. There were extensive V subgroup synteny blocks in the TRA and TRB loci between T. m. latirostris and human. Increased genomic locus complexity correlated to increased locus synteny. We also identified evidence for a VHD pseudogene for the first time in a eutherian mammal. These findings emphasize the value of including species within this basal eutherian radiation in comparative studies. Copyright © 2018. Published by Elsevier Ltd.

  12. A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

    PubMed

    Belmont, Judson; Gu, Tao; Mudd, Ashley; Salomon, Arthur R

    2017-08-04

    Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca 2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr 192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr 192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

  13. A Cleavable N-Terminal Signal Peptide Promotes Widespread Olfactory Receptor Surface Expression in HEK293T Cells

    PubMed Central

    Shepard, Blythe D.; Natarajan, Niranjana; Protzko, Ryan J.; Acres, Omar W.; Pluznick, Jennifer L.

    2013-01-01

    Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. PMID:23840901

  14. 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.

  15. Broad cross-reactive T cell receptor repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

    PubMed Central

    Clute, Shalyn C.; Naumov, Yuri N.; Watkin, Levi B.; Aslan, Nuray; Sullivan, John L.; Thorley-Lawson, David A.; Luzuriaga, Katherine; Welsh, Raymond M.; Puzone, Roberto; Celada, Franco; Selin, Liisa K.

    2013-01-01

    Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show here, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M158-66 epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1280-288 epitope (GLCTLVAML), that under some conditions heterologous immunity can lead to a significant broadening rather than a narrowing of the T cell receptor repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad rather than narrow T cell repertoire if there is a lack of dominant high affinity clones, and this hypothesis is supported by computer simulation. PMID:21048112

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

    DTIC Science & Technology

    2014-05-01

    in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

  17. Changing the threshold-Signals and mechanisms of mast cell priming.

    PubMed

    Halova, Ivana; Rönnberg, Elin; Draberova, Lubica; Vliagoftis, Harissios; Nilsson, Gunnar P; Draber, Petr

    2018-03-01

    Mast cells play a key role in allergy and other inflammatory diseases involving engagement of multivalent antigen with IgE bound to high-affinity IgE receptors (FcεRIs). Aggregation of FcεRIs on mast cells initiates a cascade of signaling events that eventually lead to degranulation, secretion of leukotrienes and prostaglandins, and cytokine and chemokine production contributing to the inflammatory response. Exposure to pro-inflammatory cytokines, chemokines, bacterial and viral products, as well as some other biological products and drugs, induces mast cell transition from the basal state into a primed one, which leads to enhanced response to IgE-antigen complexes. Mast cell priming changes the threshold for antigen-mediated activation by various mechanisms, depending on the priming agent used, which alone usually do not induce mast cell degranulation. In this review, we describe the priming processes induced in mast cells by various cytokines (stem cell factor, interleukins-4, -6 and -33), chemokines, other agents acting through G protein-coupled receptors (adenosine, prostaglandin E 2 , sphingosine-1-phosphate, and β-2-adrenergic receptor agonists), toll-like receptors, and various drugs affecting the cytoskeleton. We will review the current knowledge about the molecular mechanisms behind priming of mast cells leading to degranulation and cytokine production and discuss the biological effects of mast cell priming induced by several cytokines. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation.

    PubMed

    Bajgain, Pradip; Tawinwung, Supannikar; D'Elia, Lindsey; Sukumaran, Sujita; Watanabe, Norihiro; Hoyos, Valentina; Lulla, Premal; Brenner, Malcolm K; Leen, Ann M; Vera, Juan F

    2018-05-10

    The adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site. First (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells. In the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo. Our findings demonstrate the feasibility of targeting breast

  19. Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T Cells specifically inhibit Ewing sarcoma growth in vitro and in vivo

    PubMed Central

    Kirschner, Andreas; Thiede, Melanie; Rubio, Rebeca Alba; Schirmer, David; Kirchner, Thomas; Richter, Gunther H.S.; Mall, Sabine; Klar, Richard; Riddell, Stanley; Busch, Dirk H.; Krackhardt, Angela; Grunewald, Thomas G.P.; Burdach, Stefan

    2016-01-01

    The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction. After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells. CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic. PMID:27281613

  20. P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells.

    PubMed

    Figliuolo, Vanessa R; Savio, Luiz Eduardo Baggio; Safya, Hanaa; Nanini, Hayandra; Bernardazzi, Cláudio; Abalo, Alessandra; de Souza, Heitor S P; Kanellopoulos, Jean; Bobé, Pierre; Coutinho, Cláudia M L M; Coutinho-Silva, Robson

    2017-06-01

    P2X7 receptor activation contributes to inflammation development in different pathologies. We previously reported that the P2X7 receptor is over-expressed in the gut mucosa of patients with inflammatory bowel disease, and that P2X7 inhibition protects against chemically induced colitis. Here, we investigated in detail the role of the P2X7 receptor in inflammatory bowel disease development, by treating P2X7 knockout (KO) and WT mice with two different (and established) colitis inductors. P2X7 KO mice were protected against gut inflammation induced by 2,4,6-trinitrobenzenesulfonic acid or oxazolone, with no weight loss or gut histological alterations after treatment. P2X7 receptor knockout induced regulatory T cell accumulation in the colon, as evaluated by qRT-PCR for FoxP3 expression and immunostaining for CD90/CD45RB low . Flow cytometry analysis of mesenteric lymph node cells showed that P2X7 activation (by ATP) triggered regulatory T cell death. In addition, such cells from P2X7 KO mice expressed more CD103, suggesting increased migration of regulatory T cells to the colon (relative to the WT). Our results show that the P2X7 has a key role during inflammation development in inflammatory bowel disease, by triggering the death and retention in the mesenteric lymph nodes of regulatory T cells that would otherwise promote immune system tolerance in the gut. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. T-helper cell receptors from long-term survivors after telomerase cancer vaccination for use in adoptive cell therapy.

    PubMed

    Kyte, Jon Amund; Gaudernack, Gustav; Faane, Anne; Lislerud, Kari; Inderberg, Else Marit; Brunsvig, Paal; Aamdal, Steinar; Kvalheim, Gunnar; Wälchli, Sébastien; Pule, Martin

    2016-01-01

    We herein report retargeting of T-helper (Th) cells against the universal cancer antigen telomerase for use in adoptive cell therapy. The redirected Th cells may counter tumor tolerance, transform the inflammatory milieu, and induce epitope spreading and cancer senescence. We have previously conducted a series of trials evaluating vaccination with telomerase peptides. From long-term survivors, we isolated >100 CD4 + Th-cell clones recognizing telomerase epitopes. The clones were characterized with regard to HLA restriction, functional avidity, fine specificity, proliferative capacity, cytokine profile, and recognition of naturally processed epitopes. DP4 is the most prevalent HLA molecule worldwide. Two DP4-restricted T-cell clones with different functional avidity, C13 and D71, were selected for molecular T-cell receptor (TCR) cloning. Both clones showed a high proliferative capacity, recognition of naturally processed telomerase epitopes, and a polyfunctional and Th1-weighted cytokine profile. TCR C13 and D71 were cloned into the retroviral vector MP71 together with the compact and GMP-applicable marker/suicide gene RQR8. Both TCRs were expressed well in recipient T cells after PBMC transduction. The transduced T cells co-expressed RQR8 and acquired the desired telomerase specificity, with a polyfunctional response including production of TNFa, IFNγ, and CD107a. Interestingly, the DP4-restricted TCRs were expressed and functional both in CD4 + and CD8 + T cells. The findings demonstrate that the cloned TCRs confer recipient T cells with the desired hTERT-specificity and functionality. We hypothesize that adoptive therapy with Th cells may offer a powerful novel approach for overcoming tumor tolerance and synergize with other forms of immunotherapy.

  2. Impact of lipid rafts on the T -cell-receptor and peptide-major-histocompatibility-complex interactions under different measurement conditions

    NASA Astrophysics Data System (ADS)

    Li, Long; Xu, Guang-Kui; Song, Fan

    2017-01-01

    The interactions between T-cell receptor (TCR) and peptide-major-histocompatibility complex (pMHC), which enable T-cell development and initiate adaptive immune responses, have been intensively studied. However, a central issue of how lipid rafts affect the TCR-pMHC interactions remains unclear. Here, by using a statistical-mechanical membrane model, we show that the binding affinity of TCR and pMHC anchored on two apposing cell membranes is significantly enhanced because of the lipid raft-induced signaling protein aggregation. This finding may provide an alternative insight into the mechanism of T-cell activation triggered by very low densities of pMHC. In the case of cell-substrate adhesion, our results indicate that the loss of lateral mobility of the proteins on the solid substrate leads to the inhibitory effect of lipid rafts on TCR-pMHC interactions. Our findings help to understand why different experimental methods for measuring the impact of lipid rafts on the receptor-ligand interactions have led to contradictory conclusions.

  3. 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. © 2016 British Society for Immunology.

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

    PubMed

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

    2016-01-01

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

  5. Leukocyte-specific protein 1 regulates T-cell migration in rheumatoid arthritis

    PubMed Central

    Hwang, Seong-Hye; Jung, Seung-Hyun; Lee, Saseong; Choi, Susanna; Yoo, Seung-Ah; Park, Ji-Hwan; Hwang, Daehee; Shim, Seung Cheol; Sabbagh, Laurent; Kim, Ki-Jo; Park, Sung Hwan; Cho, Chul-Soo; Kim, Bong-Sung; Leng, Lin; Montgomery, Ruth R.; Bucala, Richard; Chung, Yeun-Jun; Kim, Wan-Uk

    2015-01-01

    Copy number variations (CNVs) have been implicated in human diseases. However, it remains unclear how they affect immune dysfunction and autoimmune diseases, including rheumatoid arthritis (RA). Here, we identified a novel leukocyte-specific protein 1 (LSP1) deletion variant for RA susceptibility located in 11p15.5. We replicated that the copy number of LSP1 gene is significantly lower in patients with RA, which correlates positively with LSP1 protein expression levels. Differentially expressed genes in Lsp1-deficient primary T cells represent cell motility and immune and cytokine responses. Functional assays demonstrated that LSP1, induced by T-cell receptor activation, negatively regulates T-cell migration by reducing ERK activation in vitro. In mice with T-cell–dependent chronic inflammation, loss of Lsp1 promotes migration of T cells into the target tissues as well as draining lymph nodes, exacerbating disease severity. Moreover, patients with RA show diminished expression of LSP1 in peripheral T cells with increased migratory capacity, suggesting that the defect in LSP1 signaling lowers the threshold for T-cell activation. To our knowledge, our work is the first to demonstrate how CNVs result in immune dysfunction and a disease phenotype. Particularly, our data highlight the importance of LSP1 CNVs and LSP1 insufficiency in the pathogenesis of RA and provide previously unidentified insights into the mechanisms underlying T-cell migration toward the inflamed synovium in RA. PMID:26554018

  6. Stop and go: hematopoietic cell transplantation in the era of chimeric antigen receptor T cells and checkpoint inhibitors.

    PubMed

    Ghosh, Arnab; Politikos, Ioannis; Perales, Miguel-Angel

    2017-11-01

    For several decades, hematopoietic cell transplantation (HCT) has been considered the standard curative therapy for many patients with hematological malignancies. In addition to the cytotoxic effects of the chemotherapy and radiation used in the conditioning regimen, the benefits of HCT are derived from a reset of the immune system and harnessing the ability of donor T cells to eliminate malignant cells. With the dawn of the era of immunotherapies in the form of checkpoint inhibitors and chimeric antigen receptor (CAR) T cells, the role of HCT has evolved. Immunotherapy with checkpoint inhibitors is increasingly being used for relapsed Hodgkin and non-Hodgkin lymphoma after autologous HCT. Checkpoint inhibitors are also being tested after allogeneic HCT with observable benefits in treating hematological malignancies, but with a potential risk of increased graft versus host disease and transplant-related mortality. Immunotherapy with Cluster of differentiation 19 CAR T cells are powerful options with aggressive B-cell malignancies both for therapy and as induction leading to allogeneic HCT. Although immunotherapies with checkpoint inhibition and CAR T cells are increasingly being used to treat hematological malignancies, HCT remains a standard of care for most of the diseases with the best chance of cure. Combination of these therapies with HCT has the potential to more effectively treat hematological malignancies.

  7. Adoptive transfer of MART-1 T cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma

    PubMed Central

    Chodon, Thinle; Comin-Anduix, Begonya; Chmielowski, Bartosz; Koya, Richard C; Wu, Zhongqi; Auerbach, Martin; Ng, Charles; Avramis, Earl; Seja, Elizabeth; Villanueva, Arturo; McCannel, Tara A.; Ishiyama, Akira; Czernin, Johannes; Radu, Caius G.; Wang, Xiaoyan; Gjertson, David W.; Cochran, Alistair J.; Cornetta, Kenneth; Wong, Deborah J.L.; Kaplan-lefko, Paula; Hamid, Omid; Samlowski, Wolfram; Cohen, Peter A.; Daniels, Gregory A.; Mukherji, Bijay; Yang, Lili; Zack, Jerome A.; Kohn, Donald B.; Heath, James R.; Glaspy, John A.; Witte, Owen N.; Baltimore, David; Economou, James S.; Ribas, Antoni

    2014-01-01

    Purpose It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short one-week manufacture protocol to determine the feasibility, safety and antitumor efficacy of this double cell therapy. Experimnetal Design A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and re-infused with (n = 10) or without (n = 3) prior cryopreservation. Results 14 patients with metastatic melanoma were enrolled and nine out of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within two weeks of ACT indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared to cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using non-cryopreserved T cells. Conclusion Double cell therapy with ACT of TCR engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. PMID:24634374

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

    PubMed Central

    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.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and 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, 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-kB, 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 non-constitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or non-constitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  9. 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

  10. Tuning of activation thresholds explains flexibility in the selection and development of T cells in the thymus

    PubMed Central

    Grossman, Zvi; Singer, Alfred

    1996-01-01

    Immature CD4+CD8+ thymocytes expressing T-cell antigen receptors (TCR) are selected by TCR-mediated recognition of peptides associated with major histocompatibility complex molecules on thymic stromal cells. Selection ensures reactivity of the mature cells to foreign antigens and tolerance to self. Although much has been learned about the factors that determine whether a thymocyte with a given specificity will be positively or negatively selected, selection as an aspect of the developmental process as a whole is less well-understood. Here we invoke a model in which thymocytes tune their response characteristics individually and dynamically in the course of development. Cellular development and selection are driven by receptor-mediated metabolic perturbations. Perturbation is a measure of the net intracellular change induced by external stimulation. It results from the integration of several signals and countersignals over time and therefore depends on the environment and the maturation stage of the cell. Individual cell adaptation limits the range of perturbations. Such adaptation renders thymocytes less sensitive to the level of stimulation per se, but responsive to environmental changes in that level. This formulation begins to explain the mechanisms that link developmental and selection events to each other. PMID:8962126

  11. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

    PubMed Central

    Gross, Catharina C.; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G.; Wiendl, Heinz

    2016-01-01

    Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4+ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4+ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4+ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4+ T cells and the cytolytic activity of NK cells. PMID:27162345

  12. 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. Copyright © 2016. Published by Elsevier Inc.

  13. Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

    DTIC Science & Technology

    2008-05-01

    adoptive therapy using CD19- specific chimeric antigen receptor re-directed T cells for recurrent/refractory follicular lymphoma. Mol Ther...T- cell therapies for B- cell malignancies we have developed a chimeric antigen receptor (CAR) which when expressed on the cell surface redirects T...that both CD4+ and CD8+ T cells expressing CD19-specific chimeric antigen receptor (CAR) can be generated usmg a novel non-viral gene

  14. Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

    PubMed

    Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

    1997-01-15

    Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

  15. The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

    PubMed Central

    Proust, Richard; Bertoglio, Jacques; Gesbert, Franck

    2012-01-01

    Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex. PMID:22912825

  16. Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms.

    PubMed

    Waldmann, T A; Davis, M M; Bongiovanni, K F; Korsmeyer, S J

    1985-09-26

    The T alpha and T beta chains of the heterodimeric T-lymphocyte antigen receptor are encoded by separated DNA segments that recombine during T-cell development. We have used rearrangements of the T beta gene as a widely applicable marker of clonality in the T-cell lineage. We show that the T beta genes are used in both the T8 and T4 subpopulations of normal T cells and that Sézary leukemia, adult T-cell leukemia, and the non-B-lineage acute lymphoblastic leukemias are clonal expansions of T cells. Furthermore, circulating T cells from a patient with the T8-cell-predominantly lymphocytosis associated with granulocytopenia are shown to be monoclonal. Finally, the sensitivity and specificity of this tumor-associated marker have been exploited to monitor the therapy of a patient with adult T-cell leukemia. These unique DNA rearrangements provide insights into the cellular origin, clonality, and natural history of T-cell neoplasia.

  17. CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

    PubMed

    Ayala, Victor I; Deleage, Claire; Trivett, Matthew T; Jain, Sumiti; Coren, Lori V; Breed, Matthew W; Kramer, Joshua A; Thomas, James A; Estes, Jacob D; Lifson, Jeffrey D; Ott, David E

    2017-06-01

    Follicular helper CD4 T cells, T FH , residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8 hCXCR5 ) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8 hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8 hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8 hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8 hCXCR5 T cells were present throughout the follicles with some observed near infected T FH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication. IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, T FH , present inside B-cell follicles represent a

  18. Modulation of T-cell receptor functional sensitivity via the opposing actions of protein tyrosine kinases and phosphatases: a mathematical model.

    PubMed

    Szomolay, Barbara; van den Berg, Hugo A

    2014-12-01

    Combining receptor kinetics and stochastic modelling of receptor activation, we show that a T-cell can specifically augment its functional sensitivity to one particular peptide ligand while simultaneously decreasing its sensitivity to other ligands, by coordinating the expression levels of the co-receptor CD8 and the relative activities of kinases and phosphatases in the vicinity of the T-cell receptor (TCR). We propose that this focusable degeneracy of epitope recognition allows a TCR to have a wide range of potential ligands but be specifically sensitive to only one or a few of these at any one time, which resolves the paradox of how a relatively small number of clones (∼10(6)) can maintain the potential to respond to a vast space of ligands (∼20(9)) whilst avoiding auto-immunity. We validate the model against experimental data and predict shifts in functional sensitivity following a shift in the kinase/phosphatase balance (which could in principle be induced by experimental means). Moreover, we propose that in vivo, the T-cell gauges ligand quality by monitoring changes in TCR triggering rate concomitant with shifts in this balance, for instance as the immunological synapse matures.

  19. Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

    PubMed

    Bueno, Clara; Lemke, Caitlin D; Criado, Gabriel; Baroja, Miren L; Ferguson, Stephen S G; Rahman, A K M Nur-Ur; Tsoukas, Constantine D; McCormick, John K; Madrenas, Joaquin

    2006-07-01

    The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

  20. T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis.

    PubMed

    Held, Kathrin; Beltrán, Eduardo; Moser, Markus; Hohlfeld, Reinhard; Dornmair, Klaus

    2015-09-01

    Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161(hi)TRAV1-2(+) T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161(hi)TRAV1-2(+) TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  1. T Cell Receptor Engineering and Analysis Using the Yeast Display Platform

    PubMed Central

    Smith, Sheena N.; Harris, Daniel T.; Kranz, David M.

    2017-01-01

    The αβ heterodimeric T cell receptor (TCR) recognizes peptide antigens that are transported to the cell surface as a complex with a protein encoded by the major histocompatibility complex (MHC). T cells thus evolved a strategy to sense these intracellular antigens, and to respond either by eliminating the antigen-presenting cell (e.g. a virus-infected cell) or by secreting factors that recruit the immune system to the site of the antigen. The central role of the TCR in the binding of antigens as peptide-MHC (pepMHC) ligands has now been studied thoroughly. Interestingly, despite their exquisite sensitivity (e.g. T cell activation by as few as 1 to 3 pepMHC complexes on a single target cell), TCRs are known to have relatively low affinities for pepMHC, with KD values in the micromolar range. There has been interest in engineering the affinity of TCRs in order to use this class of molecules in ways similar to now done with antibodies. By doing so, it would be possible to harness the potential of TCRs as therapeutics against a much wider array of antigens that include essentially all intracellular targets. To engineer TCRs, and to analyze their binding features more rapidly, we have used a yeast display system as a platform. Expression and engineering of a single-chain form of the TCR, analogous to scFv fragments from antibodies, allow the TCR to be affinity matured with a variety of possible pepMHC ligands. In addition, the yeast display platform allows one to rapidly generate TCR variants with diverse binding affinities and to analyze specificity and affinity without the need for purification of soluble forms of the TCRs. The present chapter describes the methods for engineering and analyzing single-chain TCRs using yeast display. PMID:26060072

  2. The phosphatase JKAP/DUSP22 inhibits t-cell receptor signalling and autoimmunity by inactivating Lck

    USDA-ARS?s Scientific Manuscript database

    JNK pathway-associated phosphatase (JKAP, also known as DUSP22 or JSP-1) is a JNK activator. The in vivo role of JKAP in immune regulation remains unclear. Here we report that JKAP directly inactivates Lck by dephosphorylating tyrosine-394 residue during T-cell receptor (TCR) signalling. JKAP-knocko...

  3. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

    PubMed

    Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

    2015-02-18

    Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). Copyright © 2015, American Association for the Advancement of Science.

  4. Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

    PubMed

    Wolf, Insa M A; Diercks, Björn-Philipp; Gattkowski, Ellen; Czarniak, Frederik; Kempski, Jan; Werner, René; Schetelig, Daniel; Mittrücker, Hans-Willi; Schumacher, Valéa; von Osten, Manuel; Lodygin, Dimitri; Flügel, Alexander; Fliegert, Ralf; Guse, Andreas H

    2015-10-13

    The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals. Copyright © 2015, American Association for the Advancement of Science.

  5. Conjugated Bilirubin Differentially Regulates CD4+ T Effector Cells and T Regulatory Cell Function through Outside-In and Inside-Out Mechanisms: The Effects of HAV Cell Surface Receptor and Intracellular Signaling

    PubMed Central

    Corral-Jara, Karla F.; Gómez-Leyva, Juan F.; Rosenstein, Yvonne; Jose-Abrego, Alexis; Roman, Sonia

    2016-01-01

    We recently reported an immune-modulatory role of conjugated bilirubin (CB) in hepatitis A virus (HAV) infection. During this infection the immune response relies on CD4+ T lymphocytes (TLs) and it may be affected by the interaction of HAV with its cellular receptor (HAVCR1/TIM-1) on T cell surface. How CB might affect T cell function during HAV infection remains to be elucidated. Herein, in vitro stimulation of CD4+ TLs from healthy donors with CB resulted in a decrease in the degree of intracellular tyrosine phosphorylation and an increase in the activity of T regulatory cells (Tregs) expressing HAVCR1/TIM-1. A comparison between CD4+ TLs from healthy donors and HAV-infected patients revealed changes in the TCR signaling pathway relative to changes in CB levels. The proportion of CD4+CD25+ TLs increased in patients with low CB serum levels and an increase in the percentage of Tregs expressing HAVCR1/TIM-1 was found in HAV-infected patients relative to controls. A low frequency of 157insMTTTVP insertion in the viral receptor gene HAVCR1/TIM-1 was found in patients and controls. Our data revealed that, during HAV infection, CB differentially regulates CD4+ TLs and Tregs functions by modulating intracellular pathways and by inducing changes in the proportion of Tregs expressing HAVCR1/TIM-1. PMID:27578921

  6. The Promise of Chimeric Antigen Receptor Engineered T cells in the Treatment of Hematologic Malignancies

    PubMed Central

    Nagle, Sarah J.; Garfall, Alfred L.; Stadtmauer, Edward A.

    2015-01-01

    Relapsed and refractory hematologic malignancies have a very poor prognosis. Chimeric antigen receptor (CAR) T cells are emerging as a powerful therapy in this setting. Early clinical trials of genetically modified T cells for the treatment of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) have shown high complete response rates in patients with few therapeutic options. Exploration is ongoing for other hematologic malignancies including multiple myeloma (MM), acute myeloid leukemia (AML) and Hodgkin lymphoma (HL). At the same time, the design and production of CAR T cells is being advanced so that this therapy can be more widely utilized. Cytokine release syndrome (CRS) and neurotoxicity are common, but they are treatable and fully reversible. This review will review currently available data as well as future developments and challenges in the field. PMID:26841014

  7. T lymphocyte recruitment into renal cell carcinoma tissue: a role for chemokine receptors CXCR3, CXCR6, CCR5, and CCR6.

    PubMed

    Oldham, Kimberley A; Parsonage, Greg; Bhatt, Rupesh I; Wallace, D Michael A; Deshmukh, Nayneeta; Chaudhri, Shalini; Adams, David H; Lee, Steven P

    2012-02-01

    Evidence suggests that some patients with renal cell carcinoma (RCC) respond to immunomodulatory therapies that activate T lymphocytes. A prerequisite for effective T cell therapy is efficient targeting of effector T cells to the tumour site, yet the molecular basis of T cell recruitment to RCC is unknown. Furthermore, some T cells that naturally infiltrate this cancer are regulatory T cells (Tregs) that may suppress antitumour immune responses. Determine the mechanisms of effector and regulatory T cell recruitment to RCC to allow targeted therapy that promotes local anti-tumour immunity. Tumour-infiltrating and peripheral blood T cells were collected from 70 patients undergoing nephrectomy for RCC. T cells were analysed by multicolour flow cytometry for expression of 19 chemokine receptors and 7 adhesion molecules. Receptors that were expressed at higher levels on tumour-infiltrating lymphocytes (TILs) compared with matched peripheral blood lymphocytes (PBLs) were analysed further for their ability to mediate migration responses in TILs and for expression of corresponding ligands in tumour tissue. Three chemokine receptors-CCR5, CXCR3, and CXCR6-were significantly overexpressed on TILs compared with matched PBLs (n=16 cases) and were capable of promoting migration in vitro. Their corresponding ligands CCL4-5, CXCL9-11, and CXCL16 were all detected in RCC tissue. However, since they were present in all cases studied, it was not possible to correlate ligand expression with levels of T cell infiltration. Foxp3(+) Tregs were enriched within TILs compared with matched PBLs and expressed high levels of CCR5, CXCR3, and CXCR6, as well as CCR6, the ligand for which (CCL20) was detectable in RCC tissue. Our data support a role for CCR5, CXCR3, and CXCR6 in the selective recruitment of T cells into RCC tissue and, together with CCR6, in the recruitment of Tregs. Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  8. Sulfamethoxazole Induces a Switch Mechanism in T Cell Receptors Containing TCRVβ20-1, Altering pHLA Recognition

    PubMed Central

    Watkins, Stephan; Pichler, Werner J.

    2013-01-01

    T cell receptors (TCR) containing Vβ20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vβ20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2β domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vβ20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable β or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2β loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2β loop amino acid positions. These residues, TYRβ57, ASPβ64, and LYSβ65 initially hold hydrogen bonds from the CDR2β to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2β of Vβ20-1 acts as a ligand controlled switch affecting overall TCR

  9. Determinants of public T cell responses.

    PubMed

    Li, Hanjie; Ye, Congting; Ji, Guoli; Han, Jiahuai

    2012-01-01

    Historically, sharing T cell receptors (TCRs) between individuals has been speculated to be impossible, considering the dramatic discrepancy between the potential enormity of the TCR repertoire and the limited number of T cells generated in each individual. However, public T cell response, in which multiple individuals share identical TCRs in responding to a same antigenic epitope, has been extensively observed in a variety of immune responses across many species. Public T cell responses enable individuals within a population to generate similar antigen-specific TCRs against certain ubiquitous pathogens, leading to favorable biological outcomes. However, the relatively concentrated feature of TCR repertoire may limit T cell response in a population to some other pathogens. It could be a great benefit for human health if public T cell responses can be manipulated. Therefore, the mechanistic insight of public TCR generation is important to know. Recently, high-throughput DNA sequencing has revolutionized the study of immune receptor repertoires, which allows a much better understanding of the factors that determine the overlap of TCR repertoire among individuals. Here, we summarize the current knowledge on public T-cell response and discuss future challenges in this field.

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

    PubMed

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

    2016-12-21

    Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC 50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases. Copyright © 2016, American Association for the Advancement of Science.

  11. Mechanical regulation of T-cell functions

    PubMed Central

    Chen, Wei; Zhu, Cheng

    2013-01-01

    Summary T cells are key players of the mammalian adaptive immune system. They experience different mechanical microenvironments during their life cycles, from the thymus, secondary lymph organs, and peripheral tissues that are free of externally applied force but display variable substrate rigidities, to the blood and lymphatic circulation systems where complicated hydrodynamic forces are present. Regardless of whether T cells are subject to external forces or generate their own internal forces, they response and adapt to different biomechanical cues to modulate their adhesion, migration, trafficking, and triggering of immune functions through mechanical regulation of various molecules that bear force. These include adhesive receptors, immunoreceptors, motor proteins, cytoskeletal proteins, and their associated molecules. Here we discuss the forces acting on various surface and cytoplasmic proteins of a T cell in different mechanical milieus. We review existing data on how force regulates protein conformational changes and interactions with counter molecules, including integrins, actin, and the T-cell receptor, and how each relates to T-cell functions. PMID:24117820

  12. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yin, Yiyuan; Wang, Xin Xiang; Mariuzza, Roy A

    2012-07-11

    Adaptive immunity depends on specific recognition by a T-cell receptor (TCR) of an antigenic peptide bound to a major histocompatibility complex (pMHC) molecule on an antigen-presenting cell (APC). In addition, T-cell activation generally requires binding of this same pMHC to a CD4 or CD8 coreceptor. Here, we report the structure of a complete TCR-pMHC-CD4 ternary complex involving a human autoimmune TCR, a myelin-derived self-peptide bound to HLA-DR4, and CD4. The complex resembles a pointed arch in which TCR and CD4 are each tilted ~65° relative to the T-cell membrane. By precluding direct contacts between TCR and CD4, the structure explainsmore » how TCR and CD4 on the T cell can simultaneously, yet independently, engage the same pMHC on the APC. The structure, in conjunction with previous mutagenesis data, places TCR-associated CD3εγ and CD3εδ subunits, which transmit activation signals to the T cell, inside the TCR-pMHC-CD4 arch, facing CD4. By establishing anchor points for TCR and CD4 on the T-cell membrane, the complex provides a basis for understanding how the CD4 coreceptor focuses TCR on MHC to guide TCR docking on pMHC during thymic T-cell selection.« less

  13. 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

  14. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia

    PubMed Central

    Porter, David L.; Hwang, Wei-Ting; Frey, Noelle V.; Lacey, Simon F.; Shaw, Pamela A.; Loren, Alison W.; Bagg, Adam; Marcucci, Katherine T.; Shen, Angela; Gonzalez, Vanessa; Ambrose, David; Grupp, Stephan A.; Chew, Anne; Zheng, Zhaohui; Milone, Michael C.; Levine, Bruce L.; Melenhorst, Jan J.; June, Carl H.

    2018-01-01

    Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)–modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 108 to 11 × 108 CTL019 cells (median, 1.6 × 108 cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL. PMID:26333935

  15. Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

    PubMed

    Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

    2009-08-15

    In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

  16. Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, So Yong; Lim, Ju Hyun; Choi, Sung Won

    2010-04-09

    Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundlymore » induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.« less

  17. Inhibitory masking controls the threshold sensitivity of retinal ganglion cells

    PubMed Central

    Pan, Feng; Toychiev, Abduqodir; Zhang, Yi; Atlasz, Tamas; Ramakrishnan, Hariharasubramanian; Roy, Kaushambi; Völgyi, Béla; Akopian, Abram

    2016-01-01

    Key points Retinal ganglion cells (RGCs) in dark‐adapted retinas show a range of threshold sensitivities spanning ∼3 log units of illuminance.Here, we show that the different threshold sensitivities of RGCs reflect an inhibitory mechanism that masks inputs from certain rod pathways.The masking inhibition is subserved by GABAC receptors, probably on bipolar cell axon terminals.The GABAergic masking inhibition appears independent of dopaminergic circuitry that has been shown also to affect RGC sensitivity.The results indicate a novel mechanism whereby inhibition controls the sensitivity of different cohorts of RGCs. This can limit and thereby ensure that appropriate signals are carried centrally in scotopic conditions when sensitivity rather than acuity is crucial. Abstract The responses of rod photoreceptors, which subserve dim light vision, are carried through the retina by three independent pathways. These pathways carry signals with largely different sensitivities. Retinal ganglion cells (RGCs), the output neurons of the retina, show a wide range of sensitivities in the same dark‐adapted conditions, suggesting a divergence of the rod pathways. However, this organization is not supported by the known synaptic morphology of the retina. Here, we tested an alternative idea that the rod pathways converge onto single RGCs, but inhibitory circuits selectively mask signals so that one pathway predominates. Indeed, we found that application of GABA receptor blockers increased the sensitivity of most RGCs by unmasking rod signals, which were suppressed. Our results indicate that inhibition controls the threshold responses of RGCs under dim ambient light. This mechanism can ensure that appropriate signals cross the bottleneck of the optic nerve in changing stimulus conditions. PMID:27350405

  18. Gamma Interferon-Induced T-Cell Loss in Virulent Mycobacterium avium Infection

    PubMed Central

    Flórido, Manuela; Pearl, John E.; Solache, Alejandra; Borges, Margarida; Haynes, Laura; Cooper, Andrea M.; Appelberg, Rui

    2005-01-01

    Infection by virulent Mycobacterium avium caused progressive severe lymphopenia in C57BL/6 mice due to increased apoptosis rates. T-cell depletion did not occur in gamma interferon (IFN-γ)-deficient mice which showed increased T-cell numbers and proliferation; in contrast, deficiency in nitric oxide synthase 2 did not prevent T-cell loss. Although T-cell loss was IFN-γ dependent, expression of the IFN-γ receptor on T cells was not required for depletion. Similarly, while T-cell loss was optimal if the T cells expressed IFN-γ, CD8+ T-cell depletion could occur in the absence of T-cell-derived IFN-γ. Depletion did not require that the T cells be specific for mycobacterial antigen and was not affected by deficiencies in the tumor necrosis factor receptors p55 or p75, the Fas receptor (CD95), or the respiratory burst enzymes or by forced expression of bcl-2 in hematopoietic cells. PMID:15908387

  19. Primer sets for cloning the human repertoire of T cell Receptor Variable regions.

    PubMed

    Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

    2008-08-29

    Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

  20. The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

    PubMed Central

    Raab, M; Yamamoto, M; Rudd, C E

    1994-01-01

    CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration. Images PMID:7513045

  1. The Short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction

    PubMed Central

    Chen, Lanfen; Chen, Zhangguo; Baker, Kristi; Halvorsen, E lizabeth M.; da Cunha, Andre Pires; Flak, Magdalena B.; Gerber, Georg; Huang, Yu-Hwa; Hosomi, Shuhei; Arthur, J anelle C.; Dery, Ken J.; Nagaishi, Takashi; Beauchemin, Nicole; Holmes, Kathryn V.; Ho, Joshua W. K.; Shively, John E.; Jobin, Christian; Onderdonk, Andrew B.; Bry, Lynn; Weiner, Howard L.; Higgins, Darren E.; Blumberg, Richard S.

    2012-01-01

    Summary Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens. PMID:23123061

  2. Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

    PubMed

    Coursey, Terry G; Gandhi, Niral B; Volpe, Eugene A; Pflugfelder, Stephen C; de Paiva, Cintia S

    2013-01-01

    CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.

  3. Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

    PubMed Central

    Coursey, Terry G.; Gandhi, Niral B.; Volpe, Eugene A.; Pflugfelder, Stephen C.; de Paiva, Cintia S.

    2013-01-01

    CD4+ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4+ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4+ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4+ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease. PMID:24223818

  4. Chemokine Receptor Signatures in Allogeneic Stem Cell Transplantation

    DTIC Science & Technology

    2015-08-01

    T - cells in allogeneic hematopoietic stem - cell transplant (HSCT) recipients and identify the role of chemokine receptors in...immune responses after allogeneic hematopoietic stem - cell transplantation (HSCT) in humans. Control of donor T - cells recruitment into target organs...effector T - cells after allogeneic stem - cell transplantation (Aim 1). To characterize the clonal diversity that correlates with

  5. Role of IL-4 receptor α-positive CD4(+) T cells in chronic airway hyperresponsiveness.

    PubMed

    Kirstein, Frank; Nieuwenhuizen, Natalie E; Jayakumar, Jaisubash; Horsnell, William G C; Brombacher, Frank

    2016-06-01

    TH2 cells and their cytokines are associated with allergic asthma in human subjects and with mouse models of allergic airway disease. IL-4 signaling through the IL-4 receptor α (IL-4Rα) chain on CD4(+) T cells leads to TH2 cell differentiation in vitro, implying that IL-4Rα-responsive CD4(+) T cells are critical for the induction of allergic asthma. However, mechanisms regulating acute and chronic allergen-specific TH2 responses in vivo remain incompletely understood. This study defines the requirements for IL-4Rα-responsive CD4(+) T cells and the IL-4Rα ligands IL-4 and IL-13 in the development of allergen-specific TH2 responses during the onset and chronic phase of experimental allergic airway disease. Development of acute and chronic ovalbumin (OVA)-induced allergic asthma was assessed weekly in CD4(+) T cell-specific IL-4Rα-deficient BALB/c mice (Lck(cre)IL-4Rα(-/lox)) and respective control mice in the presence or absence of IL-4 or IL-13. During acute allergic airway disease, IL-4 deficiency did not prevent the onset of TH2 immune responses and OVA-induced airway hyperresponsiveness or goblet cell hyperplasia, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. In contrast, deficiency of IL-13 prevented allergic asthma, irrespective of the presence or absence of IL-4Rα-responsive CD4(+) T cells. Importantly, chronic allergic inflammation and airway hyperresponsiveness were dependent on IL-4Rα-responsive CD4(+) T cells. Deficiency in IL-4Rα-responsive CD4(+) T cells resulted in increased numbers of IL-17-producing T cells and, consequently, increased airway neutrophilia. IL-4-responsive T helper cells are dispensable for acute OVA-induced airway disease but crucial in maintaining chronic asthmatic pathology. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia

    PubMed Central

    Kenderian, Saad S.; Shen, Feng; Ruella, Marco; Shestova, Olga; Kozlowski, Miroslaw; Li, Yong; Schrank-Hacker, April; Morrissette, Jennifer J. D.; Carroll, Martin; June, Carl H.; Grupp, Stephan A.; Gill, Saar

    2017-01-01

    We and others previously reported potent antileukemia efficacy of CD123-redirected chimeric antigen receptor (CAR) T cells in preclinical human acute myeloid leukemia (AML) models at the cost of severe hematologic toxicity. This observation raises concern for potential myeloablation in patients with AML treated with CD123-redirected CAR T cells and mandates novel approaches for toxicity mitigation. We hypothesized that CAR T-cell depletion with optimal timing after AML eradication would preserve leukemia remission and allow subsequent hematopoietic stem cell transplantation. To test this hypothesis, we compared 3 CAR T-cell termination strategies: (1) transiently active anti-CD123 messenger RNA–electroporated CART (RNA-CART123); (2) T-cell ablation with alemtuzumab after treatment with lentivirally transduced anti–CD123-4-1BB-CD3ζ T cells (CART123); and (3) T-cell ablation with rituximab after treatment with CD20-coexpressing CART123 (CART123-CD20). All approaches led to rapid leukemia elimination in murine xenograft models of human AML. Subsequent antibody-mediated depletion of CART123 or CART123-CD20 did not impair leukemia remission. Time-course studies demonstrated that durable leukemia remission required CAR T-cell persistence for 4 weeks prior to ablation. Upon CAR T-cell termination, we further demonstrated successful hematopoietic engraftment with a normal human donor to model allogeneic stem cell rescue. Results from these studies will facilitate development of T-cell depletion strategies to augment the feasibility of CAR T-cell therapy for patients with AML. PMID:28246194

  7. PiggyBac-mediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor

    PubMed Central

    Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

    2011-01-01

    Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy. PMID:21772253

  8. PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor.

    PubMed

    Nakazawa, Yozo; Huye, Leslie E; Salsman, Vita S; Leen, Ann M; Ahmed, Nabil; Rollins, Lisa; Dotti, Gianpietro; Gottschalk, Stephen M; Wilson, Matthew H; Rooney, Cliona M

    2011-12-01

    Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can be modified to function as heterologous tumor directed effector cells that survive longer in vivo than tumor directed T cells without virus specificity, due to chronic stimulation by viral antigens expressed during persistent infection in seropositive individuals. We evaluated the nonviral piggyBac (PB) transposon system as a platform for modifying EBV-CTLs to express a functional human epidermal growth factor receptor 2-specific chimeric antigen receptor (HER2-CAR) thereby directing virus-specific, gene modified CTLs towards HER2-positive cancer cells. Peripheral blood mononuclear cells (PBMCs) were nucleofected with transposons encoding a HER2-CAR and a truncated CD19 molecule for selection followed by specific activation and expansion of EBV-CTLs. HER2-CAR was expressed in ~40% of T cells after CD19 selection with retention of immunophenotype, polyclonality, and function. HER2-CAR-modified EBV-CTLs (HER2-CTLs) killed HER2-positive brain tumor cell lines in vitro, exhibited transient and reversible increases in HER2-CAR expression following antigen-specific stimulation, and stably expressed HER2-CAR beyond 120 days. Adoptive transfer of PB-modified HER2-CTLs resulted in tumor regression in a murine xenograft model. Our results demonstrate that PB can be used to redirect virus-specific CTLs to tumor targets, which should prolong tumor-specific T cell survival in vivo producing more efficacious immunotherapy.

  9. Glutamine supplementation attenuates expressions of adhesion molecules and chemokine receptors on T cells in a murine model of acute colitis.

    PubMed

    Hou, Yu-Chen; Wu, Jin-Ming; Wang, Ming-Yang; Wu, Ming-Hsun; Chen, Kuen-Yuan; Yeh, Sung-Ling; Lin, Ming-Tsan

    2014-01-01

    Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

  10. Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

    PubMed Central

    Bruzzone, Santina; Kunerth, Svenja; Zocchi, Elena; De Flora, Antonio; Guse, Andreas H.

    2003-01-01

    The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38− cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 ± 5.2 and 50.5 ± 8.0 pmol/mg protein). P2Y receptor stimulation of CD38− cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave. PMID:14623867

  11. Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical Hematopoietic Progenitor-Cell Transplant

    DTIC Science & Technology

    2009-05-01

    adoptive therapy using CD19-specific chimeric antigen receptor re-directed T cells for recurrent/refrctory follicular lymphoma...Beauty (SB) transposon/transposase system to express a CD19-specific chimeric antigen receptor (CAR). T cells that have undergone transposition...accomplished using genetic engineering to express a chimeric antigen receptor (CAR) to redirect the specificity of T cells for CD19 on malignant B cells

  12. Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10{sup −/−} mice by attenuating the activation of T cells and promoting their apoptosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Singh, Udai P.; Singh, Narendra P.; Singh, Balwan

    2012-01-15

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptor induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10{sup −/−} mice. JWH-133 effectively attenuated the overall clinical score, and reversed colitis-associated pathogenesis and decrease in body weight in IL-10{sup −/−} mice. After JWH-133 treatment, the percentage of CD4{sup +} Tmore » cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells declined in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) of mice with chronic colitis. JWH-133 was also effective in ameliorating dextran sodium sulfate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodo-pravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, and suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD. -- Highlights: ► JWH-133, a cannnabinoid receptor-2 agonist ameliorates experimental colitis. ► JWH-133 suppressed inflammation

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

    PubMed

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

    2010-11-11

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)-expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19-CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19-CAR-transduced T cells completely eliminated normal B cells from mice for at least 143 days. Anti-CD19-CAR-transduced T cells eradicated intraperitoneally injected lymphoma cells and large subcutaneous lymphoma masses. The antilymphoma efficacy of anti-CD19-CAR-transduced T cells was critically dependent on irradiation of mice before anti-CD19-CAR-transduced T-cell infusion. Anti-CD19-CAR-transduced T cells had superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived. Our results demonstrated impressive antilymphoma activity and profound destruction of normal B cells caused by anti-CD19-CAR-transduced T cells in a clinically relevant murine model.

  14. Anergic self-reactive B cells present self antigen and respond normally to CD40-dependent T-cell signals but are defective in antigen-receptor-mediated functions.

    PubMed Central

    Eris, J M; Basten, A; Brink, R; Doherty, K; Kehry, M R; Hodgkin, P D

    1994-01-01

    B-cell tolerance to soluble protein self antigens such as hen egg lysozyme (HEL) is mediated by clonal anergy. Anergic B cells fail to mount antibody responses even in the presence of carrier-primed T cells, suggesting an inability to activate or respond to T helper cells. To investigate the nature of this defect, B cells from tolerant HEL/anti-HEL double-transgenic mice were incubated with a membrane preparation from activated T-cell clones expressing the CD40 ligand. These membranes, together with interleukin 4 and 5 deliver the downstream antigen-independent CD40-dependent B-cell-activating signals required for productive T-B collaboration. Anergic B cells responded to this stimulus by proliferating and secreting antibody at levels comparable to or better than control B cells. Furthermore, anergic B cells presented HEL acquired in vivo and could present the unrelated antigen, conalbumin, targeted for processing via surface IgD. In contrast, the low immunoglobulin receptor levels on anergic B cells were associated with reduced de novo presentation of HEL and a failure to upregulate costimulatory ligands for CD28. These defects in immunoglobulin-receptor-mediated functions could be overcome in vivo, suggesting a number of mechanisms for induction of autoantibody responses. Images PMID:7514304

  15. 64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET

    PubMed Central

    Griessinger, Christoph M.; Maurer, Andreas; Kesenheimer, Christian; Kehlbach, Rainer; Reischl, Gerald; Ehrlichmann, Walter; Bukala, Daniel; Harant, Maren; Cay, Funda; Brück, Jürgen; Nordin, Renate; Kohlhofer, Ursula; Rammensee, Hans-Georg; Quintanilla-Martinez, Leticia; Schaller, Martin; Röcken, Martin; Pichler, Bernd J.; Kneilling, Manfred

    2015-01-01

    T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific 64Cu-monoclonal antibody (mAb)–TCR complex enables a stable labeling of T cells. The TCR–mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied 64Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover. PMID:25587131

  16. Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8+ T Cells following Antiviral Treatment of Chronic Hepatitis B▿

    PubMed Central

    Ma, Shi-Wu; Li, Yong-Yin; Zhang, Guang-Wen; Huang, Xuan; Sun, Jian; Li, Chris; Abbott, William G. H.; Hou, Jin-Lin

    2011-01-01

    An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B. PMID:21098256

  17. Divide, Conquer, and Sense: CD8+CD28- T Cells in Perspective.

    PubMed

    Arosa, Fernando A; Esgalhado, André J; Padrão, Carolina A; Cardoso, Elsa M

    2016-01-01

    Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8 + T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the "signal 2" CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8 + T cells, also known as CD8 + CD28 - , CD8 + KIR + , NK-like CD8 + T cells, or innate CD8 + T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8 + T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis.

  18. Lenalidomide enhances the function of chimeric antigen receptor T cells against the epidermal growth factor receptor variant III by enhancing immune synapses.

    PubMed

    Kuramitsu, S; Ohno, M; Ohka, F; Shiina, S; Yamamichi, A; Kato, A; Tanahashi, K; Motomura, K; Kondo, G; Kurimoto, M; Senga, T; Wakabayashi, T; Natsume, A

    2015-10-01

    The epidermal growth factor receptor variant III (EGFRvIII) is exclusively expressed on the cell surface in ~50% of glioblastoma multiforme (GBM). This variant strongly and persistently activates the phosphatidylinositol 3-kinase-Akt signaling pathway in a ligand-independent manner resulting in enhanced tumorigenicity, cellular motility and resistance to chemoradiotherapy. Our group generated a recombinant single-chain variable fragment (scFv) antibody specific to the EGFRvIII, referred to as 3C10-scFv. In the current study, we constructed a lentiviral vector transducing the chimeric antigen receptor (CAR) that consisted of 3C10-scFv, CD3ζ, CD28 and 4-1BB (3C10-CAR). The 3C10-CAR-transduced peripheral blood mononuclear cells (PBMCs) and CD3(+) T cells specifically lysed the glioma cells that express EGFRvIII. Moreover, we demonstrated that CAR CD3(+) T cells migrated to the intracranial xenograft of GBM in the mice treated with 3C10-CAR PBMCs. An important and novel finding of our study was that a thalidomide derivative lenalidomide induced 3C10-CAR PBMC proliferation and enhanced the persistent antitumor effect of the cells in vivo. Lenalidomide also exhibited enhanced immunological synapses between the effector cells and the target cells as determined by CD11a and F-actin polymerization. Collectively, lentiviral-mediated transduction of CAR effectors targeting the EGFRvIII showed specific efficacy, and lenalidomide even intensified CAR cell therapy by enhanced formation of immunological synapses.

  19. Adoptive T-cell therapy for cancer: The era of engineered T cells.

    PubMed

    Bonini, Chiara; Mondino, Anna

    2015-09-01

    Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T-cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR-modified T cells and chimeric antigen receptor gene-modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. TCRmodel: high resolution modeling of T cell receptors from sequence.

    PubMed

    Gowthaman, Ragul; Pierce, Brian G

    2018-05-22

    T cell receptors (TCRs), along with antibodies, are responsible for specific antigen recognition in the adaptive immune response, and millions of unique TCRs are estimated to be present in each individual. Understanding the structural basis of TCR targeting has implications in vaccine design, autoimmunity, as well as T cell therapies for cancer. Given advances in deep sequencing leading to immune repertoire-level TCR sequence data, fast and accurate modeling methods are needed to elucidate shared and unique 3D structural features of these molecules which lead to their antigen targeting and cross-reactivity. We developed a new algorithm in the program Rosetta to model TCRs from sequence, and implemented this functionality in a web server, TCRmodel. This web server provides an easy to use interface, and models are generated quickly that users can investigate in the browser and download. Benchmarking of this method using a set of nonredundant recently released TCR crystal structures shows that models are accurate and compare favorably to models from another available modeling method. This server enables the community to obtain insights into TCRs of interest, and can be combined with methods to model and design TCR recognition of antigens. The TCRmodel server is available at: http://tcrmodel.ibbr.umd.edu/.

  1. Retinoic acid receptor alpha drives cell cycle progression and is associated with increased sensitivity to retinoids in T-cell lymphoma.

    PubMed

    Wang, Xueju; Dasari, Surendra; Nowakowski, Grzegorz S; Lazaridis, Konstantinos N; Wieben, Eric D; Kadin, Marshall E; Feldman, Andrew L; Boddicker, Rebecca L

    2017-04-18

    Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with generally poor outcomes following standard therapy. Few candidate therapeutic targets have been identified to date. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to retinoids. While retinoids have been used to treat some cutaneous T-cell lymphomas (CTCLs), their mechanism of action and the role of RARA in CTCL and other mature T-cell lymphomas remain poorly understood. After identifying a PTCL with a RARAR394Q mutation, we sought to characterize the role of RARA in T-cell lymphoma cells. Overexpressing wild-type RARA or RARAR394Q significantly increased cell growth in RARAlow cell lines, while RARA knockdown induced G1 arrest and decreased expression of cyclin-dependent kinases CDK2/4/6 in RARAhigh cells. The retinoids, AM80 (tamibarotene) and all-trans retinoic acid, caused dose-dependent growth inhibition, G1 arrest, and CDK2/4/6 down-regulation. Genes down-regulated in transcriptome data were enriched for cell cycle and G1-S transition. Finally, RARA overexpression augmented chemosensitivity to retinoids. In conclusion, RARA drives cyclin-dependent kinase expression, G1-S transition, and cell growth in T-cell lymphoma. Synthetic retinoids inhibit these functions in a dose-dependent fashion and are most effective in cells with high RARA expression, indicating RARA may represent a therapeutic target in some PTCLs.

  2. T-Cell Artificial Focal Triggering Tools: Linking Surface Interactions with Cell Response

    PubMed Central

    Carpentier, Benoît; Pierobon, Paolo; Hivroz, Claire; Henry, Nelly

    2009-01-01

    T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy. PMID:19274104

  3. Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

    PubMed

    Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša

    2013-03-01

    Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways

  4. Pancreatic cancer therapy with combined mesothelin-redirected chimeric antigen receptor T cells and cytokine-armed oncolytic adenoviruses

    PubMed Central

    Watanabe, Keisuke; Luo, Yanping; Da, Tong; Scholler, John; Keith, Brian; Young, Regina M.; Sorsa, Suvi; Siurala, Mikko; Havunen, Riikka; Tähtinen, Siri; Hemminki, Akseli

    2018-01-01

    Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA. PMID:29618658

  5. Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements

    PubMed Central

    Cushman-Vokoun, Allison M.; Connealy, Solomon; Greiner, Timothy C.

    2010-01-01

    Interpretation of capillary electrophoresis results derived from multiplexed fluorochrome-labeled primer sets can be complicated by small peaks, which may be incorrectly interpreted as clonal T-cell receptor-γ gene rearrangements. In this report, different assay designs were used to illustrate how design may adversely affect specificity. Ten clinical cases, with subclonal peaks containing one of the two infrequently used joining genes, were identified with a tri-color, one-tube assay. The DNA was amplified with the same NED fluorochrome on all three joining primers, first combined (one-color assay) and then amplified separately using a single NED-labeled joining primer. The single primer assay design shows how insignificant peaks could easily be wrongly interpreted as clonal T-cell receptor-γ gene rearrangements. Next, the performance of the one-tube assay was compared with the two-tube BIOMED-2-based TCRG Gene Clonality Assay in a series of 44 cases. Whereas sensitivity was similar between the two methods (92.9% vs. 96.4%; P = 0.55), specificity was significantly less in the BIOMED-2 assay (87.5% vs. 56.3%; P = 0.049) when a 2× ratio was used to define clonality. Specificity was improved to 81.3% by the use of a 5× peak height ratio (P = 0.626). These findings illustrate how extra caution is needed in interpreting a design with multiple, separate distributions, which is more difficult to interpret than a single distribution assay. PMID:20959612

  6. Nasal-type NK/T-cell lymphomas are more frequently T rather than NK lineage based on T-cell receptor gene, RNA, and protein studies: lineage does not predict clinical behavior.

    PubMed

    Hong, Mineui; Lee, Taehee; Young Kang, So; Kim, Suk-Jin; Kim, Wonseog; Ko, Young-Hyeh

    2016-05-01

    Extranodal natural killer (NK)/T-cell lymphoma (ENKTL), nasal type, comprises NK or cytotoxic T cells. We evaluated the clinical impact of cell type and the usefulness of T-cell receptor (TCR) gene transcripts in distinguishing cell lineage. One hundred and eight cases of ENKTL were analyzed for TCR gene rearrangements using the BIOMED-2 protocol and for TCR gene expression using immunohistochemistry for TCR-βF1 and TCR-cγM1, and RNA in situ hybridization for TCR gene transcripts. Prognostic factors were analyzed. Among the 108 cases, 44 were monoclonal for a TCR rearrangement (40%) while 64 (60%) were undefinable. The monoclonal cases expressed TCR-βF1 in 14 out of 40 cases (35%) and TCR-cγM1 in 1 out of 44 cases (2%). The 64 undetermined cases expressed TCR-βF1 in 15 cases (23%) and TCR-cγM1 in 1 (2%). Thirteen of 40 TCR-β constant gene transcript-positive cases (33%) expressed TCR-βF1 and one of nine TCR-γ constant gene transcript-positive cases (11%) expressed TCR-cγM1. TCR gene transcripts were not useful in the distinction of cell lineages. TCR gene transcripts were positive in ENKTLs as well as in normal B cells and aggressive NK-cell leukemia. Based on gene rearrangements and immunohistochemistry for TCR, there were 60 T-cell type cases (56%), 32 NK-cell type cases (30%), and 16 cases with an undetermined cell type (14%). TCR protein was expressed in 30/60 T-ENKTLs (50%) in a variable fraction of tumor cells. There were no significant differences in clinical findings or overall patient survival between T- or NK-cell types of ENKTL, although those with a T-cell type tended to show a better prognosis for those with localized nasal lymphomas. Univariate and multivariate analysis showed that a non-nasal ENKTL, age >60 years, high level of lactate dehydrogenase, bone marrow involvement, and the absence of radiotherapy were independent prognostic factors.

  7. Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma.

    PubMed

    Panjwani, M Kazim; Smith, Jenessa B; Schutsky, Keith; Gnanandarajah, Josephine; O'Connor, Colleen M; Powell, Daniel J; Mason, Nicola J

    2016-09-01

    Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans.

  8. Feasibility and Safety of RNA-transfected CD20-specific Chimeric Antigen Receptor T Cells in Dogs with Spontaneous B Cell Lymphoma

    PubMed Central

    Panjwani, M Kazim; Smith, Jenessa B; Schutsky, Keith; Gnanandarajah, Josephine; O'Connor, Colleen M; Powell, Daniel J; Mason, Nicola J

    2016-01-01

    Preclinical murine models of chimeric antigen receptor (CAR) T cell therapy are widely applied, but are greatly limited by their inability to model the complex human tumor microenvironment and adequately predict safety and efficacy in patients. We therefore sought to develop a system that would enable us to evaluate CAR T cell therapies in dogs with spontaneous cancers. We developed an expansion methodology that yields large numbers of canine T cells from normal or lymphoma-diseased dogs. mRNA electroporation was utilized to express a first-generation canine CD20-specific CAR in expanded T cells. The canine CD20 (cCD20) CAR expression was efficient and transient, and electroporated T cells exhibited antigen-specific interferon-gamma (IFN-γ) secretion and lysed cCD20+ targets. In a first-in-canine study, autologous cCD20-ζ CAR T cells were administered to a dog with relapsed B cell lymphoma. Treatment was well tolerated and led to a modest, but transient, antitumor activity, suggesting that stable CAR expression will be necessary for durable clinical remissions. Our study establishes the methodologies necessary to evaluate CAR T cell therapy in dogs with spontaneous malignancies and lays the foundation for use of outbred canine cancer patients to evaluate the safety and efficacy of next-generation CAR therapies and their optimization prior to translation into humans. PMID:27401141

  9. Cannabinoids Receptor-2 (CB2) agonist ameliorates colitis in IL-10−/− mice by attenuating the activation of T cells and promoting their apoptosis

    PubMed Central

    Singh, Udai P.; Singh, Narendra P.; Singh, Balwan; Price, Robert L.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2014-01-01

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptors induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10−/− mice. JWH-133 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis and decrease in body weight in IL-10−/− mice. After JWH-133 treatment, the percentage of CD4+ T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells in the LP of colitis mice declined after JWH-133 treatment in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN). JWH-133 was also effective in ameliorating dextran sodium sulphate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodopravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD. PMID:22119709

  10. Critical role of γ4 chain in the expression of functional Vγ4Vδ1 T cell receptor of gastric tumour-infiltrating γδT lymphocytes.

    PubMed

    Jiang, Y; Tang, F; Li, Z; Cui, L; He, W

    2012-01-01

    Vγ4Vδ1 T cell receptor (TCRγ4δ1)-expressing γδT cells were the most dominant subset in gastric tumour-infiltrating γδT cells (γδTIL) we recently analyzed. To study the essential roles of γ and δ chains in assembly and function of TCRγ4δ1, we sequenced and constructed them into lentiviral vectors for the reconstitution of TCRγ4δ1 using different modalities of transduction. We were able to efficiently reconstitute TCRγ4δ1 with functional activities when both γ4 and δ1 chains are coexpressed in TCR-negative J.RT3-T3.5 cells. However, the expression of δ1 chain is greatly diminished when γ4 expression is absent, suggesting that the coexpressing γ4 is critical in maintaining the folding and stability of δ1 product. To functionally study the reconstituted TCRγ4δ1, we examined the cytolytic activity of TCRγ4δ1-reconstituted J.RT3-T3.5 cells and cytokine secretion and found the receptors are fully functional, but their functionality also requires the presence of γ4. Our results demonstrated that γ4 is critical for the stability of δ1 and the function of TCRγ4δ1. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

  11. Taste responses in mice lacking taste receptor subunit T1R1

    PubMed Central

    Kusuhara, Yoko; Yoshida, Ryusuke; Ohkuri, Tadahiro; Yasumatsu, Keiko; Voigt, Anja; Hübner, Sandra; Maeda, Katsumasa; Boehm, Ulrich; Meyerhof, Wolfgang; Ninomiya, Yuzo

    2013-01-01

    The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1−/−) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1−/− mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1−/− mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/− mice responded to sweet and umami compounds, whereas those in T1R1−/− mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1−/− than in T1R1+/− mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1−/− and T1R1+/− mice. Conditioned taste aversion tests demonstrated that both T1R1−/− and T1R1+/− mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds. PMID:23339178

  12. Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia

    PubMed Central

    Porter, David L.; Levine, Bruce L.; Kalos, Michael; Bagg, Adam; June, Carl H.

    2012-01-01

    SUMMARY We designed a lentiviral vector expressing a chimeric antigen receptor with specificity for the B-cell antigen CD19, coupled with CD137 (a costimulatory receptor in T cells [4-1BB]) and CD3-zeta (a signal-transduction component of the T-cell antigen receptor) signaling domains. A low dose (approximately 1.5×105 cells per kilogram of body weight) of autologous chimeric antigen receptor–modified T cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo, with delayed development of the tumor lysis syndrome and with complete remission. Apart from the tumor lysis syndrome, the only other grade 3/4 toxic effect related to chimeric antigen receptor T cells was lymphopenia. Engineered cells persisted at high levels for 6 months in the blood and bone marrow and continued to express the chimeric antigen receptor. A specific immune response was detected in the bone marrow, accompanied by loss of normal B cells and leukemia cells that express CD19. Remission was ongoing 10 months after treatment. Hypogammaglobulinemia was an expected chronic toxic effect. PMID:21830940

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

    PubMed Central

    Guo, Yelei; Wang, Yao; Han, Weidong

    2016-01-01

    Recent studies have highlighted the successes of chimeric antigen receptor-modified T- (CART-) cell-based therapy for B-cell malignancies, and early phase clinical trials have been launched in recent years. The few published clinical studies of CART cells in solid tumors have addressed safety and feasibility, but the clinical outcome data are limited. Although antitumor effects were confirmed in vitro and in animal models, CART-cell-based therapy still faces several challenges when directed towards solid tumors, and it has been difficult to achieve the desired outcomes in clinical practice. Many studies have struggled to improve the clinical responses to and benefits of CART-cell treatment of solid tumors. In this review, the status quo of CART cells and their clinical applications for solid tumors will be summarized first. Importantly, we will suggest improvements that could increase the therapeutic effectiveness of CART cells for solid tumors and their future clinical applications. These interventions will make treatment with CART cells an effective and routine therapy for solid tumors. PMID:26998495

  14. The function of the chemokine receptor CXCR6 in the T cell response of mice against Listeria monocytogenes.

    PubMed

    Heesch, Kira; Raczkowski, Friederike; Schumacher, Valéa; Hünemörder, Stefanie; Panzer, Ulf; Mittrücker, Hans-Willi

    2014-01-01

    The chemokine receptor CXCR6 is expressed on different T cell subsets and up-regulated following T cell activation. CXCR6 has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16 on liver sinusoidal endothelial cells. Here, we analyzed the role of CXCR6 in CD8+ T cell responses to infection of mice with Listeria monocytogenes. CD8+ T cells responding to listerial antigens acquired high expression levels of CXCR6. However, deficiency of mice in CXCR6 did not impair control of the L. monocytogenes infection. CXCR6-deficient mice were able to generate listeria-specific CD4+ and CD8+ T cell responses and showed accumulation of T cells in the infected liver. In transfer assays, we detected reduced accumulation of listeria-specific CXCR6-deficient CD8+ T cells in the liver at early time points post infection. Though, CXCR6 was dispensable at later time points of the CD8+ T cell response. When transferred CD8+ T cells were followed for extended time periods, we observed a decline in CXCR6-deficient CD8+ T cells. The manifestation of this cell loss depended on the tissue analyzed. In conclusion, our results demonstrate that CXCR6 is not required for the formation of a T cell response to L. monocytogenes and for the accumulation of T cells in the infected liver but CXCR6 appears to influence long-term survival and tissue distribution of activated cells.

  15. The Function of the Chemokine Receptor CXCR6 in the T Cell Response of Mice against Listeria monocytogenes

    PubMed Central

    Heesch, Kira; Raczkowski, Friederike; Schumacher, Valéa; Hünemörder, Stefanie; Panzer, Ulf; Mittrücker, Hans-Willi

    2014-01-01

    The chemokine receptor CXCR6 is expressed on different T cell subsets and up-regulated following T cell activation. CXCR6 has been implicated in the localization of cells to the liver due to the constitutive expression of its ligand CXCL16 on liver sinusoidal endothelial cells. Here, we analyzed the role of CXCR6 in CD8+ T cell responses to infection of mice with Listeria monocytogenes. CD8+ T cells responding to listerial antigens acquired high expression levels of CXCR6. However, deficiency of mice in CXCR6 did not impair control of the L. monocytogenes infection. CXCR6-deficient mice were able to generate listeria-specific CD4+ and CD8+ T cell responses and showed accumulation of T cells in the infected liver. In transfer assays, we detected reduced accumulation of listeria-specific CXCR6-deficient CD8+ T cells in the liver at early time points post infection. Though, CXCR6 was dispensable at later time points of the CD8+ T cell response. When transferred CD8+ T cells were followed for extended time periods, we observed a decline in CXCR6-deficient CD8+ T cells. The manifestation of this cell loss depended on the tissue analyzed. In conclusion, our results demonstrate that CXCR6 is not required for the formation of a T cell response to L. monocytogenes and for the accumulation of T cells in the infected liver but CXCR6 appears to influence long-term survival and tissue distribution of activated cells. PMID:24832098

  16. Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

    PubMed

    Pasetto, Anna; Frelin, Lars; Brass, Anette; Yasmeen, Anila; Koh, Sarene; Lohmann, Volker; Bartenschlager, Ralf; Magalhaes, Isabelle; Maeurer, Markus; Sällberg, Matti; Chen, Margaret

    2012-02-01

    Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

  17. Vav1-phospholipase C-γ1 (Vav1-PLC-γ1) pathway initiated by T cell antigen receptor (TCRγδ) activation is required to overcome inhibition by ubiquitin ligase Cbl-b during γδT cell cytotoxicity.

    PubMed

    Yin, Shanshan; Zhang, Jianmin; Mao, Yujia; Hu, Yu; Cui, Lianxian; Kang, Ning; He, Wei

    2013-09-13

    T cell antigen receptor γδ (TCRγδ) and natural killer group 2, member D (NKG2D) are two crucial receptors for γδT cell cytotoxicity. Compelling evidences suggest that γδT cell cytotoxicity is TCRγδ-dependent and can be co-stimulated by NKG2D. However, the molecular mechanism of underlying TCRγδ-dependent activation of γδT cells remains unclear. In this study we demonstrated that TCRγδ but not NKG2D engagement induced lytic granule polarization and promoted γδT cell cytotoxicity. TCRγδ activation alone was sufficient to trigger Vav1-dependent phospholipase C-γ1 signaling, resulting in lytic granule polarization and effective killing, whereas NKG2D engagement alone failed to trigger cytotoxicity-related signaling to overcome the inhibitory effect of Cbl-b; therefore, NKG2D engagement alone could not induce effective killing. However, NKG2D ligation augmented the activation of γδT cell cytotoxicity through the Vav1-phospholipase C-γ1 pathway. Vav1 overexpression or Cbl-b knockdown not only enhanced TCRγδ activation-initiated killing but also enabled NKG2D activation alone to induce γδT cell cytotoxicity. Taken together, our results suggest that the activation of γδT cell cytotoxicity requires a strong activation signal to overcome the inhibitory effect of Cbl-b. Our finding provides new insights into the molecular mechanisms underlying the initiation of γδT cell cytotoxicity and likely implications for optimizing γδT cell-based cancer immunotherapy.

  18. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System

    PubMed Central

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence JN; Rosenberg, Steven A

    2016-01-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAKS2580F or ERBB2H473Y or the HLA-DQB*0601-restricted neoantigen ERBB2IPE805G were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ+ T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27−CD45RA−) and less-differentiated (CD27+CD45RA+) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006

  19. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-24

    ... contemplating the grant of an exclusive patent license, subject to existing non-exclusive licenses and current non-exclusive license applications under consideration, to practice the inventions embodied in U.S... equivalents thereof entitled ``Anti-MAGE-A3 T cell receptors and related materials and methods of use'' (HHS...

  20. Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.

    PubMed

    Ilchmann, Anne; Burgdorf, Sven; Scheurer, Stephan; Waibler, Zoe; Nagai, Ryoji; Wellner, Anne; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Henle, Thomas; Kurts, Christian; Kalinke, Ulrich; Vieths, Stefan; Toda, Masako

    2010-01-01

    The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy. This study aimed at defining the T-cell immunogenicity of food AGEs by using ovalbumin (OVA) as a model allergen. AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGEs, as well as mDCs deficient for these receptors. Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs. SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  1. The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.

    PubMed

    Bertin, Samuel; Aoki-Nonaka, Yukari; Lee, Jihyung; de Jong, Petrus R; Kim, Peter; Han, Tiffany; Yu, Timothy; To, Keith; Takahashi, Naoki; Boland, Brigid S; Chang, John T; Ho, Samuel B; Herdman, Scott; Corr, Maripat; Franco, Alessandra; Sharma, Sonia; Dong, Hui; Akopian, Armen N; Raz, Eyal

    2017-09-01

    Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca 2+ )-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca 2+ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1 -/- CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1 + TRPV1 + T cell infiltration in colonic biopsies from patients with IBD. We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1 -/- CD4+ T cells have increased T-cell receptor-induced Ca 2+ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1 + TRPV1 + T cells in the colon of patients with IBD. Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  2. Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions.

    PubMed

    Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Frickel, Eva-Maria; Ploegh, Hidde L

    2016-11-01

    T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. © 2016 The Authors.

  3. Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells.

    PubMed

    Yee, Karen K; Sukumaran, Sunil K; Kotha, Ramana; Gilbertson, Timothy A; Margolskee, Robert F

    2011-03-29

    Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium-glucose cotransporter (SGLT1), and two components of the ATP-gated K(+) (K(ATP)) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K(ATP) channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K(ATP) channels constitute a major portion of K(+) channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K(ATP) may serve as mediators of the T1r-independent sweet taste of sugars.

  4. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

    PubMed Central

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

    2016-01-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  5. Severity of Acute Infectious Mononucleosis Correlates with Cross-Reactive Influenza CD8 T-Cell Receptor Repertoires.

    PubMed

    Aslan, Nuray; Watkin, Levi B; Gil, Anna; Mishra, Rabinarayan; Clark, Fransenio G; Welsh, Raymond M; Ghersi, Dario; Luzuriaga, Katherine; Selin, Liisa K

    2017-12-05

    Fifty years after the discovery of Epstein-Barr virus (EBV), it remains unclear how primary infection with this virus leads to massive CD8 T-cell expansion and acute infectious mononucleosis (AIM) in young adults. AIM can vary greatly in severity, from a mild transient influenza-like illness to a prolonged severe syndrome. We questioned whether expansion of a unique HLA-A2.01-restricted, cross-reactive CD8 T-cell response between influenza virus A-M1 58 (IAV-M1) and EBV BMLF1 280 (EBV-BM) could modulate the immune response to EBV and play a role in determining the severity of AIM in 32 college students. Only ex vivo total IAV-M1 and IAV-M1+EBV-BM cross-reactive tetramer + frequencies directly correlated with AIM severity and were predictive of severe disease. Expansion of specific cross-reactive memory IAV-M1 T-cell receptor (TCR) Vβ repertoires correlated with levels of disease severity. There were unique profiles of qualitatively different functional responses in the cross-reactive and EBV-specific CD8 T-cell responses in each of the three groups studied, severe-AIM patients, mild-AIM patients, and seropositive persistently EBV-infected healthy donors, that may result from differences in TCR repertoire use. IAV-M1 tetramer + cells were functionally cross-reactive in short-term cultures, were associated with the highest disease severity in AIM, and displayed enhanced production of gamma interferon, a cytokine that greatly amplifies immune responses, thus frequently contributing to induction of immunopathology. Altogether, these data link heterologous immunity via CD8 T-cell cross-reactivity to CD8 T-cell repertoire selection, function, and resultant disease severity in a common and important human infection. In particular, it highlights for the first time a direct link between the TCR repertoire with pathogenesis and the diversity of outcomes upon pathogen encounter. IMPORTANCE The pathogenic impact of immune responses that by chance cross-react to unrelated

  6. T-cell receptor excision circle levels and safety of paediatric immunization: A population-based self-controlled case series analysis.

    PubMed

    Wilson, Kumanan; Duque, Daniel Rodriguez; Murphy, Malia Sq; Hawken, Steven; Pham-Huy, Anne; Kwong, Jeffrey; Deeks, Shelley L; Potter, Beth K; Crowcroft, Natasha S; Bulman, Dennis E; Chakraborty, Pranesh; Little, Julian

    2018-02-08

    T-cell receptor excision circle levels are a surrogate marker of T-cell production and immune system function. We sought to determine whether non-pathological levels of infant T-cell receptor excision circles were associated with adverse events following immunization. A self-controlled case series design was applied on a sample of 231,693 children who completed newborn screening for severe combined immunodeficiency in Ontario, Canada between August 2013 and December 2015. Exposures included routinely administered pediatric vaccines up to 15 months of age. Main outcomes were combined health services utilization for recognized adverse events following immunization. 1,406,981 vaccination events were included in the final dataset. 103,007 children received the Pneu-C-13 or Men-C-C vaccine and 97,998 received the MMR vaccine at 12 months of age. 67,725 children received the varicella immunization at 15 months. Our analysis identified no association between newborn T-cell receptor excision circle levels and subsequent health services utilization events following DTa-IPV-Hib, Pneu-C-13, and Men-C-C vaccinations at 2-month (RI 0.94[95%CI 0.87-1.02]), 4-month (RI 0.82[95%CI 0.75-0.9]), 6-month (RI 0.63[95%CI 0.57-0.7]) and 12-month (RI 0.49[95%CI 0.44-0.55]). We also found no trends in health services utilization following MMR (RI 1.43[95%1.34-1.52]) or varicella (RI 1.14[95%CI 1.05-1.23]) vaccination. Our findings provide further support for the safety of pediatric vaccinations.

  7. TCR tuning of T cell subsets.

    PubMed

    Cho, Jae-Ho; Sprent, Jonathan

    2018-05-01

    After selection in the thymus, the post-thymic T cell compartments comprise heterogenous subsets of naive and memory T cells that make continuous T cell receptor (TCR) contact with self-ligands bound to major histocompatibility complex (MHC) molecules. T cell recognition of self-MHC ligands elicits covert TCR signaling and is particularly important for controlling survival of naive T cells. Such tonic TCR signaling is tightly controlled and maintains the cells in a quiescent state to avoid autoimmunity. Here, we review how naive and memory T cells are differentially tuned and wired for TCR sensitivity to self and foreign ligands. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. A critical role for transcription factor Smad4 in T cell function independent of transforming growth factor beta receptor signaling

    PubMed Central

    Gu, Ai-Di; Zhang, Song; Wang, Yunqi; Xiong, Hui; Curtis, Thomas A.; Wan, Yisong Y.

    2014-01-01

    Summary Transforming growth factor-beta (TGF-β) suppresses T cell function to maintain self-tolerance and to promote tumor immune evasion. Yet how Smad4, a transcription factor component of TGF-β signaling, regulates T cell function remains unclear. Here we have demonstrated an essential role for Smad4 in promoting T cell function during autoimmunity and anti-tumor immunity. Smad4 deletion rescued the lethal autoimmunity resulting from transforming growth factor-beta receptor (TGF-βR) deletion and compromised T-cell-mediated tumor rejection. While Smad4 was dispensable for T cell generation, homeostasis and effector function, it was essential for T cell proliferation following activation in vitro and in vivo. The transcription factor Myc was identified to mediate Smad4-controlled T cell proliferation. This study thus reveals a requirement of Smad4 for T-cell-mediated autoimmunity and tumor rejection, which is beyond the current paradigm. It highlights a TGF-βR-independent role for Smad4 in promoting T cell function, autoimmunity and anti-tumor immunity. PMID:25577439

  9. IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules.

    PubMed

    Knolle, P A; Uhrig, A; Hegenbarth, S; Löser, E; Schmitt, E; Gerken, G; Lohse, A W

    1998-12-01

    Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-gamma) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2 (PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver.

  10. T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection.

    PubMed

    Wang, George C; Dash, Pradyot; McCullers, Jonathan A; Doherty, Peter C; Thomas, Paul G

    2012-04-04

    A diverse T cell receptor (TCR) repertoire capable of recognizing a broad range of antigenic peptides is thought to be central to effective pathogen-specific immunity by counteracting escape mutations, selecting high-avidity T cells, and providing T cell specificities with comprehensive functional characteristics. However, evidence that TCR diversity is important for the successful control of human infections is limited. A single-cell strategy for the clonotypic analysis of human CD8⁺ TCRαβ repertoires was used to probe the diversity and magnitude of individual human cytomegalovirus (CMV)-specific CD8⁺ T cells recovered directly ex vivo. We found that CD8⁺ TCRαβ repertoire diversity, but not the size of the CD8⁺ T cell response, was inversely related to circulating CMV-specific antibody levels, a measure that has been correlated epidemiologically with differential mortality risks and found here to be higher in persons with detectable (versus undetectable) CMV viral loads. Overall, our findings indicate that CD8⁺ T cell diversity may be more important than T cell abundance in limiting the negative consequences of CMV persistence, demonstrate high prevalence of both TCRα and TCRβ public motif usage, and suggest that a highly diverse TCRαβ repertoire may be an important benchmark and target in the success of immunotherapeutic strategies.

  11. Superior Therapeutic Index in Lymphoma Therapy: CD30+ CD34+ Hematopoietic Stem Cells Resist a Chimeric Antigen Receptor T-cell Attack

    PubMed Central

    Hombach, Andreas A; Görgens, André; Chmielewski, Markus; Murke, Florian; Kimpel, Janine; Giebel, Bernd; Abken, Hinrich

    2016-01-01

    Recent clinical trials with chimeric antigen receptor (CAR) redirected T cells targeting CD19 revealed particular efficacy in the treatment of leukemia/lymphoma, however, were accompanied by a lasting depletion of healthy B cells. We here explored CD30 as an alternative target, which is validated in lymphoma therapy and expressed by a broad variety of Hodgkin's and non-Hodgkin's lymphomas. As a safty concern, however, CD30 is also expressed by lymphocytes and hematopoietic stem and progenitor cells (HSPCs) during activation. We revealed that HRS3scFv-derived CAR T cells are superior since they were not blocked by soluble CD30 and did not attack CD30+ HSPCs while eliminating CD30+ lymphoma cells. Consequently, normal hemato- and lymphopoiesis was not affected in the long-term in the humanized mouse; the number of blood B and T cells remained unchanged. We provide evidence that the CD30+ HSPCs are protected against a CAR T-cell attack by substantially lower CD30 levels than lymphoma cells and higher levels of the granzyme B inactivating SP6/PI9 serine protease, which furthermore increased upon activation. Taken together, adoptive cell therapy with anti-CD30 CAR T cells displays a superior therapeutic index in the treatment of CD30+ malignancies leaving healthy activated lymphocytes and HSPCs unaffected. PMID:27112062

  12. Divide, Conquer, and Sense: CD8+CD28− T Cells in Perspective

    PubMed Central

    Arosa, Fernando A.; Esgalhado, André J.; Padrão, Carolina A.; Cardoso, Elsa M.

    2017-01-01

    Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8+ T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the “signal 2” CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8+ T cells, also known as CD8+CD28−, CD8+KIR+, NK-like CD8+ T cells, or innate CD8+ T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8+ T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis. PMID:28096804

  13. QUANTITATION OF ABERRANT INTERLOCUS T-CELL RECEPTOR REARRANGEMENTS IN MOUSE THYMOCYTES AND THE EFFECT OF THE HERBICIDE 2,4- DICHLOROPHENOXYACETIC ACID

    EPA Science Inventory

    Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4- Dichlorophenoxyacetic acid

    Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene a...

  14. Primer sets for cloning the human repertoire of T cell Receptor Variable regions

    PubMed Central

    Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

    2008-01-01

    Background Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Results Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. Conclusion This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets. PMID:18759974

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

    PubMed

    Hombach, Andreas A; Abken, Hinrich

    2013-01-01

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

  16. Site-Selective Regulation of Platelet-Derived Growth Factor β Receptor Tyrosine Phosphorylation by T-Cell Protein Tyrosine Phosphatase

    PubMed Central

    Persson, Camilla; Sävenhed, Catrine; Bourdeau, Annie; Tremblay, Michel L.; Markova, Boyka; Böhmer, Frank D.; Haj, Fawaz G.; Neel, Benjamin G.; Elson, Ari; Heldin, Carl-Henrik; Rönnstrand, Lars; Östman, Arne; Hellberg, Carina

    2004-01-01

    The platelet-derived growth factor (PDGF) β receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF β receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF β receptor, we compared PDGF β receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF β receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cγ1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cγ1 activity and migratory hyperresponsiveness to PDGF. PDGF β receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPɛ ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF β receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors. PMID:14966296

  17. Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.

    PubMed

    Xiao, Lin; Chen, Can; Li, Zhendong; Zhu, Sumin; Tay, Johan Ck; Zhang, Xi; Zha, Shijun; Zeng, Jieming; Tan, Wee Kiat; Liu, Xin; Chng, Wee Joo; Wang, Shu

    2018-03-01

    Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  18. Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

    PubMed Central

    Joshi, Rubin N.; Binai, Nadine A.; Marabita, Francesco; Sui, Zhenhua; Altman, Amnon; Heck, Albert J. R.; Tegnér, Jesper; Schmidt, Angelika

    2017-01-01

    Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer. PMID:28993769

  19. Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

    PubMed

    Utz, U; Banks, D; Jacobson, S; Biddison, W E

    1996-02-01

    Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.

  20. An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis

    PubMed Central

    Quintana, Francisco J.; Murugaiyan, Gopal; Farez, Mauricio F.; Mitsdoerffer, Meike; Tukpah, Ann-Marcia; Burns, Evan J.; Weiner, Howard L.

    2010-01-01

    The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3+ Treg, Tr1 cells, and IL-17–producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3+ Treg compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3+ Treg in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3+ Treg in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3+ Treg that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3+ Treg differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders. PMID:21068375

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

    PubMed

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

    2015-02-01

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

  2. Driving CAR T-cells forward.

    PubMed

    Jackson, Hollie J; Rafiq, Sarwish; Brentjens, Renier J

    2016-06-01

    The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in haematological and solid malignancies. In this Review, we discuss these trials and present strategies that can increase the antitumour efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting.

  3. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

    PubMed Central

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

    2014-01-01

    Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

  4. Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/β-Catenin Signaling Thresholds in NIH3T3 Fibroblasts*

    PubMed Central

    Willis, Catherine M.; Klüppel, Michael

    2012-01-01

    Aberrant activation of the Wnt/β-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/β-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/β-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/β-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds. PMID:22915582

  5. The Effects of TLR Activation on T-Cell Development and Differentiation

    PubMed Central

    Jin, Bo; Sun, Tao; Yu, Xiao-Hong; Yang, Ying-Xiang; Yeo, Anthony E. T.

    2012-01-01

    Invading pathogens have unique molecular signatures that are recognized by Toll-like receptors (TLRs) resulting in either activation of antigen-presenting cells (APCs) and/or costimulation of T cells inducing both innate and adaptive immunity. TLRs are also involved in T-cell development and can reprogram Treg cells to become helper cells. T cells consist of various subsets, that is, Th1, Th2, Th17, T follicular helper (Tfh), cytotoxic T lymphocytes (CTLs), regulatory T cells (Treg) and these originate from thymic progenitor thymocytes. T-cell receptor (TCR) activation in distinct T-cell subsets with different TLRs results in differing outcomes, for example, activation of TLR4 expressed in T cells promotes suppressive function of regulatory T cells (Treg), while activation of TLR6 expressed in T cells abrogates Treg function. The current state of knowledge of regarding TLR-mediated T-cell development and differentiation is reviewed. PMID:22737174

  6. Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit.

    PubMed

    Tanaka, Junji; Sugita, Junichi; Kato, Naoko; Toubai, Tomomi; Ibata, Makoto; Shono, Yusuke; Ota, Shuichi; Kondo, Takeshi; Kobayashi, Takahiko; Kobayashi, Masanobu; Asaka, Masahiro; Imamura, Masahiro

    2007-10-01

    Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.

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

    PubMed

    Alrifai, Doraid; Sarker, Debashis; Maher, John

    2016-01-01

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

  8. Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease

    PubMed Central

    Han, Arnold; Newell, Evan W.; Glanville, Jacob; Fernandez-Becker, Nielsen; Khosla, Chaitan; Chien, Yueh-hsiu; Davis, Mark M.

    2013-01-01

    Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4+ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4+ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8+ αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8+ αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases. PMID:23878218

  9. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains onmore » the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.« less

  10. γδ T cells affect IL-4 production and B-cell tolerance

    PubMed Central

    Huang, Yafei; Heiser, Ryan A.; Detanico, Thiago O.; Getahun, Andrew; Kirchenbaum, Greg A.; Casper, Tamara L.; Aydintug, M. Kemal; Carding, Simon R.; Ikuta, Koichi; Huang, Hua; Cambier, John C.; Wysocki, Lawrence J.; O’Brien, Rebecca L.; Born, Willi K.

    2015-01-01

    γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4–producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4–regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4–inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance. PMID:25535377

  11. γδ T cells affect IL-4 production and B-cell tolerance.

    PubMed

    Huang, Yafei; Heiser, Ryan A; Detanico, Thiago O; Getahun, Andrew; Kirchenbaum, Greg A; Casper, Tamara L; Aydintug, M Kemal; Carding, Simon R; Ikuta, Koichi; Huang, Hua; Cambier, John C; Wysocki, Lawrence J; O'Brien, Rebecca L; Born, Willi K

    2015-01-06

    γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

  12. BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

    PubMed

    Dörrie, Jan; Babalija, Lek; Hoyer, Stefanie; Gerer, Kerstin F; Schuler, Gerold; Heinzerling, Lucie; Schaft, Niels

    2018-01-18

    BRAF and MEK inhibitors (BRAFi/MEKi), the standard treatment for patients with BRAF V600 mutated melanoma, are currently explored in combination with various immunotherapies, notably checkpoint inhibitors and adoptive transfer of receptor-transfected T cells. Since two BRAFi/MEKi combinations with similar efficacy are approved, potential differences in their effects on immune cells would enable a rational choice for triple therapies. Therefore, we characterized the influence of the clinically approved BRAFi/MEKi combinations dabrafenib (Dabra) and trametinib (Tram) vs. vemurafenib (Vem) and cobimetinib (Cobi) on the activation and functionality of chimeric antigen receptor (CAR)-transfected T cells. We co-cultured CAR-transfected CD8⁺ T cells and target cells with clinically relevant concentrations of the inhibitors and determined the antigen-induced cytokine secretion. All BRAFi/MEKi reduced this release as single agents, with Dabra having the mildest inhibitory effect, and Dabra + Tram having a clearly milder inhibitory effect than Vem + Cobi. A similar picture was observed for the upregulation of the activation markers CD25 and CD69 on CAR-transfected T cells after antigen-specific stimulation. Most importantly, the cytolytic capacity of the CAR-T cells was significantly inhibited by Cobi and Vem + Cobi, whereas the other kinase inhibitors showed no effect. Therefore, the combination Dabra + Tram would be more suitable for combining with T-cell-based immunotherapy than Vem + Cobi.

  13. Role of T cell receptor delta gene in susceptibility to celiac disease.

    PubMed

    Roschmann, E; Wienker, T F; Volk, B A

    1996-02-01

    There is a strong genetic influence on the susceptibility to celiac disease. Although in the vast majority of patients with celiac disease, the HLA-DQ(alpha1*0501, beta1*0201) heterodimer encoded by the alleles HLA-DQA1*0501 and HLA-DQB1*0201 seems to confer the primary disease susceptibility, it cannot be excluded that other genes contribute to disease susceptibility, as indicated by the difference in concordance rates between monozygotic twins and HLA identical siblings (70% vs. 30%). Obviously other genes involved in the genetic control of T cell mediated immune response could potentially influence susceptibility to celiac disease. The density of T cells using the gammadelta T cell receptor (TCR) is considerably increased in the jejunal epithelium of patients with celiac disease, an abnormality considered to be specific for celiac disease. This suggests an involvement of gammadelta T cells in the pathogenesis of the disease. To ascertain whether the TCR delta (TCRD) gene contributes to celiac disease susceptibility we carried out an association study and genetic linkage analysis using a highly polymorphic microsatellite marker at the TCRD locus on chromosome 14q11.2. The association study demonstrated no significant difference in allele frequencies of the TCRD gene marker between celiac disease patients and controls; accordingly, the relative risk estimates did not reach the level of statistical significance. In the linkage analysis, performed in 23 families, the logarithm of the odds (LOD) scores calculated for celiac disease versus the TCRD gene marker excluded linkage, suggesting that there is no determinant contributing to celiac disease status at or 5 cM distant to the analyzed TCRD gene marker. In conclusion, the results of the present study provide no evidence that the analyzed TCRD gene contributes substantially to celiac disease susceptibility.

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

    PubMed

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

    2016-06-07

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

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

    PubMed Central

    Hombach, Andreas A.; Abken, Hinrich

    2013-01-01

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

  16. Membrane Microdomains and Cytoskeleton Organization Shape and Regulate the IL-7 Receptor Signalosome in Human CD4 T-cells*

    PubMed Central

    Tamarit, Blanche; Bugault, Florence; Pillet, Anne-Hélène; Lavergne, Vincent; Bochet, Pascal; Garin, Nathalie; Schwarz, Ulf; Thèze, Jacques; Rose, Thierry

    2013-01-01

    Interleukin (IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signaling pathways, mainly JAK/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation, and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few min induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton, supporting the formation of the signaling complex, the signalosome, on the IL-7 receptor cytoplasmic domains. Here we describe by stimulated emission depletion microscopy the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated JAK/STAT signaling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7 activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced JAK1 and JAK3 phosphorylation. Drug-induced disassembly of the cytoskeleton inhibits phospho-STAT5 formation, transport, and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism. Activated IL-7 receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus 2 min after IL-7 stimulation. Radial microtubules disappear 15 min later, while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center. PMID:23329834

  17. Impact of karyotype organization on interlocus recombination between T cell receptor genes in Equidae.

    PubMed

    Drbalova, Jitka; Musilova, Petra; Kubickova, Svatava; Sebestova, Hana; Vahala, Jiri; Rubes, Jiri

    2014-01-01

    The T cell receptor (TCR) genes (TRA, TRB, TRD and TRG) reside in 3 different chromosomal regions. During the maturation of T lymphocytes, the TCR genes are rearranged by site-specific recombination, a process that also predisposes T cells to aberrant rearrangements. Illegitimate recombination between the TCR genes occurs at a low level in healthy individuals, but this frequency may correlate with the risk of lymphoma. The aim of this work was to investigate interlocus recombination in equids. Illegitimate rearrangements were studied in peripheral blood lymphocytes by FISH with painting and BAC probes and by sequencing of PCR products, and the frequencies of recombination were assessed in horses and 4 other equids. The presence of several trans-rearrangement products between the TRA and TRG genes was verified by PCR in all investigated equids. Frequencies of trans-rearrangements in horses are higher than in humans, and colocalization of the TCR genes on the same chromosome increases the incidence of trans-rearrangements between them. The orientation of the TCR genes does not impact interlocus recombination itself but does affect the viability of cells carrying its products and consequently the number of trans-rearrangements observed in lymphocytes.

  18. Efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy in patients with haematological and solid malignancies: protocol for a systematic review and meta-analysis.

    PubMed

    Grigor, Emma J M; Fergusson, Dean A; Haggar, Fatima; Kekre, Natasha; Atkins, Harold; Shorr, Risa; Holt, Robert A; Hutton, Brian; Ramsay, Tim; Seftel, Matthew; Jonker, Derek; Daugaard, Mads; Thavorn, Kednapa; Presseau, Justin; Lalu, Manoj M

    2017-12-29

    Patients with relapsed or refractory malignancies have a poor prognosis. Immunotherapy with chimeric antigen receptor T (CAR-T) cells redirects a patient's immune cells against the tumour antigen. CAR-T cell therapy has demonstrated promise in treating patients with several haematological malignancies, including acute B-cell lymphoblastic leukaemia and B-cell lymphomas. CAR-T cell therapy for patients with other solid tumours is also being tested. Safety is an important consideration in CAR-T cell therapy given the potential for serious adverse events, including death. Previous reviews on CAR-T cell therapy have been limited in scope and methodology. Herein, we present a protocol for a systematic review to identify CAR-T cell interventional studies and examine the safety and efficacy of this therapy in patients with haematology malignancies and solid tumours. We will search MEDLINE, including In-Process and Epub Ahead of Print, EMBASE and the Cochrane Central Register of Controlled Trials from 1946 to 22 February 2017. Studies will be screened by title, abstract and full text independently and in duplicate. Studies that report administering CAR-T cells of any chimeric antigen receptor construct targeting antigens in patients with haematological malignancies and solid tumours will be eligible for inclusion. Outcomes to be extracted will include complete response rate (primary outcome), overall response rate, overall survival, relapse and adverse events. A meta-analysis will be performed to synthesise the prevalence of outcomes reported as proportions with 95% CIs. The potential for bias within included studies will be assessed using a modified Institute of Health Economics tool. Heterogeneity of effect sizes will be determined using the Cochrane I 2 statistic. The review findings will be submitted for peer-reviewed journal publication and presented at relevant conferences and scientific meetings to promote knowledge transfer. CRD42017075331. © Article author(s) (or

  19. Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

    PubMed

    Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

    2009-01-01

    MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

  20. Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T Cell Receptors in High-risk MYCN-not-amplified Human Neuroblastoma.

    PubMed

    Wei, Jun S; Kuznetsov, Igor B; Zhang, Shile; Song, Young K; Asgharzadeh, Shahab; Sindiri, Sivasish; Wen, Xinyu; Patidar, Rajesh; Nagaraj, Sushma; Walton, Ashley; Guidry Auvil, Jaime M; Gerhard, Daniela S; Yuksel, Aysen; Catchpoole, Daniel R; Hewitt, Stephen M; Sondel, Paul M; Seeger, Robert C; Maris, John M; Khan, Javed

    2018-05-21

    High-risk neuroblastoma is an aggressive disease. DNA sequencing studies have revealed a paucity of actionable genomic alterations and a low mutation burden, posing challenges to develop effective novel therapies. We used RNA sequencing (RNA-seq) to investigate the biology of this disease including a focus on tumor-infiltrating lymphocytes (TILs). We performed deep RNA-seq on pre-treatment diagnostic tumors from 129 high-risk and 21 low- or intermediate-risk patients with neuroblastomas. We used single-sample gene set enrichment analysis to detect gene expression signatures of TILs in tumors and examined their association with clinical and molecular parameters including patient outcome. The expression profiles of 190 additional pre-treatment diagnostic neuroblastomas, a neuroblastoma tissue microarray, and T-cell receptor (TCR) sequencing were used to validate our findings. We found that MYCN -not-amplified ( MYCN -NA) tumors had significant higher cytotoxic TIL signatures compared to MYCN -amplified ( MYCN -A) tumors. A reported MYCN-activation-signature was significantly associated with poor outcome for high-risk patients with MYCN -NA tumors; however, a subgroup of these patients who had elevated activated NK cells, CD8+ T-cells, and cytolytic signatures showed improved outcome and expansion of infiltrating T-cell receptor (TCR) clones. Furthermore, we observed up-regulation of immune exhaustion marker genes, indicating an immune suppressive microenvironment in these neuroblastomas. Conclusions: This study provides evidence that RNA signatures of cytotoxic TIL are associated with the presence of activated NK-/T-cells and improved outcomes in high-risk neuroblastoma patients harboring MYCN -NA tumors. Our findings suggest that these high-risk patients with MYCN -NA neuroblastoma may benefit from additional immunotherapies incorporated into the current therapeutic strategies. Copyright ©2018, American Association for Cancer Research.

  1. Limited Model Antigen Expression by Transgenic Fungi Induces Disparate Fates during Differentiation of Adoptively Transferred T Cell Receptor Transgenic CD4+ T Cells: Robust Activation and Proliferation with Weak Effector Function during Recall

    PubMed Central

    Ersland, Karen; Pick-Jacobs, John C.; Gern, Benjamin H.; Frye, Christopher A.; Sullivan, Thomas D.; Brennan, Meghan B.; Filutowicz, Hanna I.; O'Brien, Kevin; Korthauer, Keegan D.; Schultz-Cherry, Stacey; Klein, Bruce S.

    2012-01-01

    CD4+ T cells are the key players of vaccine resistance to fungi. The generation of effective T cell-based vaccines requires an understanding of how to induce and maintain CD4+ T cells and memory. The kinetics of fungal antigen (Ag)-specific CD4+ T cell memory development has not been studied due to the lack of any known protective epitopes and clonally restricted T cell subsets with complementary T cell receptors (TCRs). Here, we investigated the expansion and function of CD4+ T cell memory after vaccination with transgenic (Tg) Blastomyces dermatitidis yeasts that display a model Ag, Eα-mCherry (Eα-mCh). We report that Tg yeast led to Eα display on Ag-presenting cells and induced robust activation, proliferation, and expansion of adoptively transferred TEa cells in an Ag-specific manner. Despite robust priming by Eα-mCh yeast, antifungal TEa cells recruited and produced cytokines weakly during a recall response to the lung. The addition of exogenous Eα-red fluorescent protein (RFP) to the Eα-mCh yeast boosted the number of cytokine-producing TEa cells that migrated to the lung. Thus, model epitope expression on yeast enables the interrogation of Ag presentation to CD4+ T cells and primes Ag-specific T cell activation, proliferation, and expansion. However, the limited availability of model Ag expressed by Tg fungi during T cell priming blunts the downstream generation of effector and memory T cells. PMID:22124658

  2. Chimeric Antigen Receptor T Cells and Hematopoietic Cell Transplantation: How Not to Put the CART Before the Horse

    PubMed Central

    Kenderian, Saad S.; Porter, David L.; Gill, Saar

    2016-01-01

    Hematopoietic cell transplantation (HCT) remains an important and potentially curative option in most hematological malignancies. As a form of immunotherapy, allogeneic HCT offers the potential for durable remissions but is limited by transplant related morbidity and mortality due to organ toxicity, infection and graft versus host disease. The recent positive outcomes of chimeric antigen receptor T (CART) cell therapy in B cell malignancies may herald a paradigm shift in the management of these disorders and perhaps other hematological malignancies. Clinical trials will now need to address the relative roles of CART cells and HCT in the context of transplant-eligible patients. In this review we summarize the state of the art of the development of CART cell therapy for leukemia, lymphoma and myeloma and discuss our perspective of how CART cell therapy can be applied in the context of HCT. PMID:27638367

  3. Leptin Directly Promotes T Cell Glycolytic Metabolism to Drive Effector T cell Differentiation in Autoimmunity

    PubMed Central

    Gerriets, Valerie A.; Danzaki, Keiko; Kishton, Rigel J.; Eisner, William; Nichols, Amanda G.; Saucillo, Donte C.; Shinohara, Mari L.; MacIver, Nancie J.

    2016-01-01

    Upon activation, T cells require energy for growth, proliferation and function. Effector T cells (Teff), such as Th1 and Th17, utilize high levels of glucose uptake and glycolysis to fuel proliferation and function. In contrast, Treg instead require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg metabolism is altered in settings of malnutrition, when nutrients are limited and circulating leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff number, function, and glucose metabolism, but did not alter Treg metabolism or suppressive function. Using the autoimmune model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff, but not Treg, glucose metabolism and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg. PMID:27222115

  4. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

    PubMed

    Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

    2010-11-15

    Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

  5. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor

    PubMed Central

    Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M.; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G.; Scholler, John; Levine, Bruce L.; Albelda, Steven M.; June, Carl H.

    2010-01-01

    Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CARs). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week post electroporation. Multiple injections of RNA CAR electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(−/−) mice. Dramatic tumor reduction also occurred when the pre-existing intraperitoneal human-derived tumors, that had been growing in vivo for over 50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes demonstrating that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. PMID:20926399

  6. Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma.

    PubMed

    Skuljec, Jelena; Chmielewski, Markus; Happle, Christine; Habener, Anika; Busse, Mandy; Abken, Hinrich; Hansen, Gesine

    2017-01-01

    Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

  7. Driving CAR T-cells forward

    PubMed Central

    Jackson, Hollie J.; Rafiq, Sarwish; Brentjens, Renier J.

    2017-01-01

    The engineered expression of chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T-cell specificity. Early clinical trials using CAR T cells for the treatment of patients with cancer showed modest results, but the impressive outcomes of several trials of CD19-targeted CAR T cells in the treatment of patients with B-cell malignancies have generated an increased enthusiasm for this approach. Important lessons have been derived from clinical trials of CD19-specific CAR T cells, and ongoing clinical trials are testing CAR designs directed at novel targets involved in haematological and solid malignancies. In this Review, we discuss these trials and present strategies that can increase the antitumour efficacy and safety of CAR T-cell therapy. Given the fast-moving nature of this field, we only discuss studies with direct translational application currently or soon-to-be tested in the clinical setting. PMID:27000958

  8. Type I Interferon Elevates Co-Regulatory Receptor Expression on CMV- and EBV-Specific CD8 T Cells in Chronic Hepatitis C

    PubMed Central

    Owusu Sekyere, Solomon; Suneetha, Pothakamuri Venkata; Hardtke, Svenja; Falk, Christine Susanne; Hengst, Julia; Manns, Michael Peter; Cornberg, Markus; Wedemeyer, Heiner; Schlaphoff, Verena

    2015-01-01

    Hepatitis C virus (HCV) readily sets up persistence in a large fraction of infected hosts. Mounting epidemiological and immunological evidence suggest that HCV’s persistence could influence immune responses toward unrelated pathogens and vaccines. Nonetheless, the fundamental contribution of the inflammatory milieu during persistent HCV infection in impacting immune cells specific for common pathogens such as CMV and EBV has not been fully studied. As the co-regulatory receptors PD-1, Tim-3, and 2B4 have all been shown to be vital in regulating CD8+ T cell function, we assessed their expression on CMV/EBV-specific CD8+ T cells from patients with chronic hepatitis C (CHC) and healthy controls ex vivo and upon stimulation with virus-specific peptides in vitro. Total and CMV/EBV-specific CD8+ T cells expressing PD-1, Tim-3, and 2B4 were highly enriched in patients with CHC compared to healthy individuals ex vivo. In vitro peptide stimulation further potentiated the differential co-regulatory receptor expression of PD-1, Tim-3, and 2B4, which then culminated in an enhanced functionality of CMV/EBV-specific CD8+ T cells in CHC patients. Comprehensively analyzing plasma cytokines between the two cohorts, we observed that not only was IFNα-2a dominant among 21 other inflammatory mediators elevated in CHC patients but it also correlated with PD-1 and Tim-3 expressions ex vivo. Importantly, IFNα-2a further caused upregulation of these markers upon in vitro peptide stimulation. Finally, we could prospectively study patients receiving novel IFN-free antiviral therapy. Here, we observed that treatment-induced clearance of HCV resulted in a partial reversion of the phenotype of CMV/EBV-specific CD8+ T cells in patients with CHC. These data reveal an alteration of the plasma concentrations of IFNα-2a together with other inflammatory mediators during CHC, which appeared to pervasively influence co-regulatory receptor expression on CMV/EBV-specific CD8+ T cells. PMID:26113847

  9. Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

    NASA Astrophysics Data System (ADS)

    Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.

    2002-05-01

    The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.

  10. Toward precision manufacturing of immunogene T-cell therapies.

    PubMed

    Xu, Jun; Melenhorst, J Joseph; Fraietta, Joseph A

    2018-05-01

    Cancer can be effectively targeted using a patient's own T cells equipped with synthetic receptors, including chimeric antigen receptors (CARs) that redirect and reprogram these lymphocytes to mediate tumor rejection. Over the past two decades, several strategies to manufacture genetically engineered T cells have been proposed, with the goal of generating optimally functional cellular products for adoptive transfer. Based on this work, protocols for manufacturing clinical-grade CAR T cells have been established, but these complex methods have been used to treat only a few hundred individuals. As CAR T-cell therapy progresses into later-phase clinical trials and becomes an option for more patients, a major consideration for academic institutions and industry is developing robust manufacturing processes that will permit scaling-out production of immunogene T-cell therapies in a reproducible and efficient manner. In this review, we will discuss the steps involved in cell processing, the major obstacles surrounding T-cell manufacturing platforms and the approaches for improving cellular product potency. Finally, we will address the challenges of expanding CAR T-cell therapy to a global patient population. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Topical resiquimod can induce disease regression and enhance T-cell effector functions in cutaneous T-cell lymphoma.

    PubMed

    Rook, Alain H; Gelfand, Joel M; Gelfand, Joel C; Wysocka, Maria; Troxel, Andrea B; Benoit, Bernice; Surber, Christian; Elenitsas, Rosalie; Buchanan, Marie A; Leahy, Deborah S; Watanabe, Rei; Kirsch, Ilan R; Kim, Ellen J; Clark, Rachael A

    2015-09-17

    Early-stage cutaneous T-cell lymphoma (CTCL) is a skin-limited lymphoma with no cure aside from stem cell transplantation. Twelve patients with stage IA-IIA CTCL were treated in a phase 1 trial of 0.03% and 0.06% topical resiquimod gel, a Toll-like receptor 7/8 agonist. Treated lesions significantly improved in 75% of patients and 30% had clearing of all treated lesions. Resiquimod also induced regression of untreated lesions. Ninety-two percent of patients had more than a 50% improvement in body surface area involvement by the modified Severity-Weighted Assessment Tool analysis and 2 patients experienced complete clearing of disease. Four of 5 patients with folliculotropic disease also improved significantly. Adverse effects were minor and largely skin limited. T-cell receptor sequencing and flow cytometry studies of T cells from treated lesions demonstrated decreased clonal malignant T cells in 90% of patients and complete eradication of malignant T cells in 30%. High responses were associated with recruitment and expansion of benign T-cell clones in treated skin, increased skin T-cell effector functions, and a trend toward increased natural killer cell functions. In patients with complete or near eradication of malignant T cells, residual clinical inflammation was associated with cytokine production by benign T cells. Fifty percent of patients had increased activation of circulating dendritic cells, consistent with a systemic response to therapy. In summary, topical resiquimod is safe and effective in early-stage CTCL and the first topical therapy to our knowledge that can induce clearance of untreated lesions and complete remissions in some patients. This trial was registered at www.clinicaltrials.gov as #NCT813320. © 2015 by The American Society of Hematology.

  12. Engineered Interleukin-2 Antagonists for the Inhibition of Regulatory T cells

    PubMed Central

    Liu, David V.; Maier, Lisa M.; Hafler, David A.; Wittrup, K. Dane

    2014-01-01

    The immunosuppressive effects of CD4+ CD25high regulatory T cells interfere with anti-tumor immune responses in cancer patients. Here, we present a novel class of engineered human Interleukin (IL)-2 analogues that antagonize the IL-2 receptor, for inhibiting regulatory T cell suppression. These antagonists have been engineered for high affinity to the α subunit of the IL-2 receptor and very low affinity to either the β or γ subunit, resulting in a signaling-deficient IL-2 analogue that sequesters the IL-2 receptor α subunit from wild type IL-2. Two variants, “V91R” and “Q126T” with residue substitutions that disrupt the β and γ subunit binding interfaces, respectively, have been characterized in both a T cell line and in human primary regulatory T cells. These mutants retain their high affinity binding to IL-2 receptor α subunit, but do not activate STAT5 phosphorylation or stimulate T cell growth. The two mutants competitively antagonize wild-type IL-2 signaling through the IL-2 receptor with similar efficacy, with inhibition constants of 183 pM for V91R and 216 pM for Q126T. Here, we present a novel approach to CD25-mediated Treg inhibition, with the use of an engineered human IL-2 analogue that antagonizes the IL-2 receptor. PMID:19816193

  13. Oligoclonal T cell receptor gene rearrangements in blood lymphocytes of patients with acute Epstein-Barr virus-induced infectious mononucleosis.

    PubMed Central

    Strickler, J G; Movahed, L A; Gajl-Peczalska, K J; Horwitz, C A; Brunning, R D; Weiss, L M

    1990-01-01

    Gene rearrangement studies were performed on blood lymphocytes from eight patients with acute Epstein-Barr virus-induced infectious mononucleosis. The diagnosis in each case was based on characteristic clinical, hematologic, and serologic findings. The blood lymphocytes in each patient consisted predominantly of CD8+ T cells. EBV DNA was detected in seven patients by Southern blot analysis (EBV Bam HI W probe, Bam HI). A germline configuration was found for the immunoglobulin heavy and light chain genes (JH probe, Bam HI and Eco RI; C kappa probe, Bam HI; and C lambda probe, Eco RI). T cell receptor gene rearrangements were detected with J gamma and J beta 1 + 2 probes. Using a J gamma probe with two different restriction enzymes (Bgl II and Eco RI), the blood from each patient showed several bands corresponding to the polyclonal pattern previously described in the blood of normal individuals. Using J beta 1 + 2 probes with two different restriction enzymes (Bgl II and Bam HI), each case showed from 3 to about 12 extragermline bands of varying intensity and in different locations from case to case. In addition, each case showed relative deletion of the J beta 1 germline band. This oligoclonal pattern of T cell receptor gene rearrangements has not been previously reported in benign or malignant T cell populations. Images PMID:2170451

  14. Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists

    PubMed Central

    Zhong, RuiKun; Li, Hongying; Messer, Karen; Lane, Thomas A.; Zhou, Jiehua; Ball, Edward D.

    2016-01-01

    This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLADR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T + L + R) than that by T alone. CTL induced from T + L + R, T + R, T + L, L + R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T + L + R was significantly higher than T or L alone, and T + R was significantly higher than T alone. CTL generated from T + L + R, T + L, T + R, L + R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T + L + R and T + R was significantly higher than T or R alone. These results indicate that the combination of T + L + R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients. PMID:25795133

  15. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  16. Toll-like receptor agonist imiquimod facilitates antigen-specific CD8+ T-cell accumulation in the genital tract leading to tumor control through IFNγ.

    PubMed

    Soong, Ruey-Shyang; Song, Liwen; Trieu, Janson; Knoff, Jayne; He, Liangmei; Tsai, Ya-Chea; Huh, Warner; Chang, Yung-Nien; Cheng, Wen-Fang; Roden, Richard B S; Wu, T-C; Trimble, Cornelia L; Hung, Chien-Fu

    2014-11-01

    Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. Using an orthotopic HPV16 E6/E7(+) syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8(+) T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8(+) T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8(+) T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3(+) CD8(+) T cells to the genital tract. Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. ©2014 American Association for Cancer Research.

  17. Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

    PubMed Central

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

  18. Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

    PubMed

    Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Chacon, Jessica A; Figini, Mariangela; Powell, Daniel J

    2016-07-20

    The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.

  19. Toll-like Receptors and B-cell Receptors Synergize to Induce Immunoglobulin Class Switch DNA Recombination: Relevance to Microbial Antibody Responses

    PubMed Central

    Pone, Egest J.; Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Xu, Zhenming; Casali, Paolo

    2011-01-01

    Differentiation of naïve B cells, including immunoglobulin (Ig) class switch DNA recombination (CSR), is critical for the immune response and depends on the extensive integration of signals from the B cell receptor (BCR), tumor necrosis factor (TNF) receptor family members, Toll-like receptors (TLRs) and cytokine receptors. TLRs and BCR synergize to induce CSR in T cell-dependent and T cell-independent antibody responses to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced B cell differentiation and antibody responses. The requirement of both BCR and TLR engagement would ensure appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak. Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified by the activation of B cells by initial TLR engagement, leading to the upregulation of co-stimulatory CD80 and MHC-II receptors, which, in turn, result in more efficient interactions with T cells, thereby enhancing the germinal center (GC) reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from the pathogen or immune cells and their products, determine the ensuing B cell antibody response. PMID:20370617

  20. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liang Pengfei; Jiang Bimei; Yang Xinghua

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, anmore » EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.« less