Sample records for t-cell receptor-transduced cell

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

    PubMed Central

    Berdien, Belinda; Reinhard, Henrike; Meyer, Sabrina; Spöck, Stefanie; Kröger, Nicolaus; Atanackovic, Djordje; Fehse, Boris

    2013-01-01

    Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies. PMID:23428899

  7. Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts

    PubMed Central

    2013-01-01

    Background Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. Methods We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. Results CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. Conclusion These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM. PMID:24829757

  8. Expression of miR-17-92 enhances anti-tumor activity of T-cells transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts.

    PubMed

    Ohno, Masasuke; Ohkuri, Takayuki; Kosaka, Akemi; Tanahashi, Kuniaki; June, Carl H; Natsume, Atsushi; Okada, Hideho

    2013-01-01

    Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM.

  9. 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.

  10. 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

  11. 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

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

    PubMed Central

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

    2013-01-01

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

  13. 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.

  14. 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.

  15. 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.

  16. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor–transduced T cells

    PubMed Central

    Dudley, Mark E.; Feldman, Steven A.; Wilson, Wyndham H.; Spaner, David E.; Maric, Irina; Stetler-Stevenson, Maryalice; Phan, Giao Q.; Hughes, Marybeth S.; Sherry, Richard M.; Yang, James C.; Kammula, Udai S.; Devillier, Laura; Carpenter, Robert; Nathan, Debbie-Ann N.; Morgan, Richard A.; Laurencot, Carolyn; Rosenberg, Steven A.

    2012-01-01

    We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti–CD19-CAR–transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19+ B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti–CD19-CAR–transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti–CD19-CAR–transduced T-cell infusions. Anti–CD19-CAR–transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19+ cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR–transduced T-cell infusions. This trial was registered with ClinicalTrials.gov as NCT00924326. PMID:22160384

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. Functional and genomic analyses of FOXP3-transduced Jurkat-T cells as regulatory T (Treg)-like cells

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

    Kim, Joon-Young; Kim, Han-Jong; Hurt, Elaine M.

    2007-10-12

    FOXP3, a forkhead transcription factor is essential for the development and function of CD4{sup +}CD25{sup +} regulatory T cells (Tregs). In contrast to conversion of murine naive T cells to Tregs by transduction of Foxp3, it is controversial whether ectopic expression of FOXP3 in human CD4{sup +} T cells is sufficient for acquisition of suppressive activity. Here, we show that retroviral transduction of FOXP3 induces a Treg phenotype in human leukemic CD4{sup +} Jurkat-T cells, evidenced by increased expression of Treg-associated cell surface markers as well as inhibition of cytokine production. Furthermore, FOXP3-transduced Jurkat-T cells suppress the proliferation of humanmore » CD4{sup +}CD25{sup -} T cells. Additionally, DNA microarray analysis identifies Treg-related genes regulated by FOXP3. Our study demonstrates that enforced expression of FOXP3 confers Treg-like properties on Jurkat-T cells, which can be a convenient and efficient Treg-like cell model for further study to identify Treg cell surface markers and target genes regulated by FOXP3.« less

  7. 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.

  8. 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

  9. Construction of an anti-programmed death-ligand 1 chimeric antigen receptor and determination of its antitumor function with transduced cells

    PubMed Central

    Xie, Jiasen; Zhou, Zishan; Jiao, Shunchang; Li, Xiaokun

    2018-01-01

    A chimeric antigen receptor (CAR) is a type of fusion protein that comprises an antigen-recognition domain and signaling domains. In the present study, a programmed death-ligand 1 (PD-L1)-specific CAR, comprised of a single-chain variable fragment (scFv) derived from a monoclonal antibody, co-stimulatory domains of cluster of differentiation (CD) 28 and 4-1BB and a T-cell-activation domain derived from CD3ζ, was designed. The construction was cloned and packaged into the lentiviral vector pLVX. Flow cytometry confirmed that peripheral blood mononuclear cells were efficiently transduced and that the CAR was successfully expressed on T cells. The cytotoxicity of transduced T cells was detected using PD-L1-positive NCI-H358 bronchioalveolar carcinoma cells and A549 lung adenocarcinoma cells (with a low expression of PD-L1, only in the A549 cells). The results demonstrated mild cytotoxicity at an effector-to-target ratio of 10:1. An ELISA revealed a significant increase in the level of interferon-γ released from T cells transduced with scFv-28Bz when the cells were co-cultured with PD-L1-positive NCI-H358 cells, while interkeukin-2 and tumor necrosis factor-α levels remained unchanged. These data indicated a potential method for the treatment of solid tumors. PMID:29928397

  10. 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.

  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 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.

  13. Efficacy and safety of adoptive immunotherapy using anti-CD19 chimeric antigen receptor transduced T-cells: a systematic review of phase I clinical trials.

    PubMed

    Xu, Xiao-Jun; Zhao, Hai-Zhao; Tang, Yong-Min

    2013-02-01

    There remain some key questions regarding the adoptive infusion of chimeric antigen receptor (CAR) transduced T-cells in the clinical setting. This article systematically reviews the phase I clinical trials using CARs targeting CD19 in B-lineage malignancies. Twenty-nine patients were enrolled and the 6-month progression free survival for this cohort was 50.0 ± 9.9%. Univariate analysis showed that patients benefited from lymphodepletion before CAR+T-cell infusion and the administration of interleukin-2 (IL-2). Longer-term persistence (≥ 4 weeks) and stronger expansion of CAR+ T-cells in the blood and higher peak serum interferon-γ (IFN-γ) level (≥ 200 pg/mL) were also related to superior outcome. Regarding treatment-related adverse events, the most prominent toxicities were fever, rigors, chills, acute renal failure, hypotension and capillary leak syndrome. In conclusion, anti-CD19 CAR+ T-cells have shown some benefits in patients with B-lineage malignancies and are well tolerated in most patients. Preconditioning and cytokine supplement are required to improve the clinical outcome.

  14. Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

    PubMed

    Humbert, Olivier; Gisch, Don W; Wohlfahrt, Martin E; Adams, Amie B; Greenberg, Phil D; Schmitt, Tom M; Trobridge, Grant D; Kiem, Hans-Peter

    2016-08-01

    Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

  15. 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

  16. 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.

  17. FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation.

    PubMed

    Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

    2011-10-01

    AIM: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25(hi) FOXP3+ T (T(reg)) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (T(h)) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called T(reg)-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. METHODS: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. RESULTS: Although GARP-transduced T(h) cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive T(h) cells. GARP-mediated FOXP3 upregulation in T(h) cells is not associated with T(reg)-specific demethylation of the FOXP3 TSDR. CONCLUSION: Although GARP-engineered T(h) cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards T(reg) cells in vitro, these cells do not completely mimic the epigenotype of natural T(reg) cells. Thus, concepts based on the genetic modification of T(h) cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application.

  18. 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.

  19. 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

  20. Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

    PubMed

    Jin, Jianjian; Gkitsas, Nikolaos; Fellowes, Vicki S; Ren, Jiaqiang; Feldman, Steven A; Hinrichs, Christian S; Stroncek, David F; Highfill, Steven L

    2018-01-24

    Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

    PubMed Central

    Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

    1999-01-01

    Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

  7. 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.

  8. 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.

  9. 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

  10. Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2

    PubMed Central

    Morgan, Richard A; Yang, James C; Kitano, Mio; Dudley, Mark E; Laurencot, Carolyn M; Rosenberg, Steven A

    2010-01-01

    In an attempt to treat cancer patients with ERBB2 overexpressing tumors, we developed a chimeric antigen receptor (CAR) based on the widely used humanized monoclonal antibody (mAb) Trastuzumab (Herceptin). An optimized CAR vector containing CD28, 4-1BB, and CD3ζ signaling moieties was assembled in a γ-retroviral vector and used to transduce autologous peripheral blood lymphocytes (PBLs) from a patient with colon cancer metastatic to the lungs and liver, refractory to multiple standard treatments. The gene transfer efficiency into autologous T cells was 79% CAR+ in CD3+ cells and these cells demonstrated high-specific reactivity in in vitro coculture assays. Following completion of nonmyeloablative conditioning, the patient received 1010 cells intravenously. Within 15 minutes after cell infusion the patient experienced respiratory distress, and displayed a dramatic pulmonary infiltrate on chest X-ray. She was intubated and despite intensive medical intervention the patient died 5 days after treatment. Serum samples after cell infusion showed marked increases in interferon-γ (IFN-γ), granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10, consistent with a cytokine storm. We speculate that the large number of administered cells localized to the lung immediately following infusion and were triggered to release cytokine by the recognition of low levels of ERBB2 on lung epithelial cells. PMID:20179677

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  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. Stimulatory role of interleukin 10 in CD8+ T cells through STATs in gastric cancer.

    PubMed

    Xi, Jianjun; Xu, Mingzheng; Song, Zongchang; Li, Hongqiang; Xu, Shumin; Wang, Chunmei; Song, Haihan; Bai, Jianwen

    2017-05-01

    CD8 + T cells are considered to be critical in tumor surveillance and elimination. Increased CD8 + T cell frequency and function is associated with better prognosis in cancer patients. Interleukin 10 is a cytokine with controversial roles in CD8 + T cell-mediated anti-tumor immunity. We therefore examined the interleukin 10 expression and consumption in CD8 + T cells harvested from the peripheral blood and resected tumors of gastric cancer patients of stages II-IV. We found that the gastric cancer patients presented significantly elevated frequencies of interleukin 10-expressing cells in both CD4 + and CD8 + T cells compared to healthy controls. But distinctive from the interleukin 10-expressing CD4 + T cells, which increased in frequency in advanced cancer, the interleukin 10-expressing CD8 + T cells did not increase with cancer stage in the peripheral blood and actually decreased with cancer stage in resected tumor. Interleukin 10 and interleukin 10 receptor expression was also enriched in interferon gamma-expressing activated CD8 + T cells. Compared to interleukin 10-nonexpressing CD8 + T cells, interleukin 10 receptor-expressing CD8 + T cells secreted significantly elevated interferon gamma levels. Treatment of anti-CD3/CD28-stimulated, purified CD8 + T cells with interleukin 10 alone could significantly enhance CD8 + T cell survival, an effect dependent on interleukin 10 receptor expression. Interleukin 10 also increased CD8 + T cell proliferation synergistically with interferon gamma but not alone. Analysis of downstream signal transducer and activator of transcription molecules showed that interleukin 10 treatment significantly increased the phosphorylation of signal transducer and activator of transcription 3 and signal transducer and activator of transcription 1 to lesser extent. Together, these results demonstrate that interleukin 10 possessed stimulatory roles in activated CD8 + T cells from gastric cancer patients.

  3. 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

  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. Mesenchymal stem cells are efficiently transduced with adenoviruses bearing type 35-derived fibers and the transduced cells with the IL-28A gene produces cytotoxicity to lung carcinoma cells co-cultured.

    PubMed

    Suzuki, Takeo; Kawamura, Kiyoko; Li, Quanhai; Okamoto, Shinya; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Yamaguchi, Naoto; Tagawa, Masatoshi

    2014-09-25

    Transduction of human mesenchymal stem cells (MSCs) with type 5 adenoviruses (Ad5) is limited in the efficacy because of the poor expression level of the coxsackie adenovirus receptor (CAR) molecules. We examined a possible improvement of Ad-mediated gene transfer in MSCs by substituting the fiber region of type 5 Ad with that of type 35 Ad. Expression levels of CAR and CD46 molecules, which are the major receptors for type 5 and type 35 Ad, respectively, were assayed with flow cytometry. We constructed vectors expressing the green fluorescent protein gene with Ad5 or modified Ad5 bearing the type 35 fiber region (AdF35), and examined the infectivity to MSCs with flow cytometry. We investigated anti-tumor effects of MSCs transduced with interleukin (IL)-28A gene on human lung carcinoma cells with a colorimetric assay. Expression of IL-28A receptors was tested with the polymerase chain reaction. A promoter activity of transcriptional regulatory regions in MSCs was determined with a luciferase assay and a tumor growth-promoting ability of MSCs was tested with co-injection of human tumor cells in nude mice. MSCs expressed CD46 but scarcely CAR molecules, and subsequently were transduced with AdF35 but not with Ad5. Growth of MSCs transduced with the IL-28A gene remained the same as that of untransduced cells since MSCs were negative for the IL-28A receptors. The IL-28A-transduced MSCs however suppressed growth of lung carcinoma cells co-cultured, whereas MSCs transduced with AdF35 expressing the β-galactosidase gene did not. A regulatory region of the cyclooygenase-2 gene possessed transcriptional activities greater than other tumor promoters but less than the cytomegalovirus promoter, and MSCs themselves did not support tumor growth in vivo. AdF35 is a suitable vector to transduce MSCs that are resistant to Ad5-mediated gene transfer. MSCs infected with AdF35 that activate an exogenous gene by the cytomegalovirus promoter can be a vehicle to deliver the gene product

  6. 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.

  7. 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.

  8. Receptor for macrophage colony-stimulating factor transduces a signal decreasing erythroid potential in the multipotent hematopoietic EML cell line.

    PubMed

    Pawlak, G; Grasset, M F; Arnaud, S; Blanchet, J P; Mouchiroud, G

    2000-10-01

    To test the hypothesis that hematopoietic growth factors may influence lineage choice in pluripotent progenitor cells, we investigated the effects of macrophage colony-stimulating factor (M-CSF) on erythroid and myeloid potentials of multipotent EML cells ectopically expressing M-CSF receptor (M-CSFR). EML cells are stem cell factor (SCF)-dependent murine cells that give rise spontaneously to pre-B cells, burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte macrophage (CFU-GM). We determined BFU-E and CFU-GM frequencies among EML cells transduced with murine M-CSFR, human M-CSFR, or chimeric receptors, and cultivated in the presence of SCF, M-CSF, or both growth factors. Effects of specific inhibitors of signaling molecules were investigated. EML cells transduced with murine M-CSFR proliferated in response to M-CSF but also exhibited a sharp and rapid decrease in BFU-E frequency associated with an increase in CFU-GM frequency. In contrast, EML cells expressing human M-CSFR proliferated in response to M-CSF without any changes in erythroid or myeloid potential. Using chimeric receptors between human and murine M-CSFR, we showed that the effects of M-CSF on EML cell differentiation potential are mediated by a large region in the intracellular domain of murine M-CSFR. Furthermore, phospholipase C (PLC) inhibitor U73122 interfered with the negative effects of ligand-activated murine M-CSFR on EML cell erythroid potential. We propose that signaling pathways activated by tyrosine kinase receptors may regulate erythroid potential and commitment decisions in multipotent progenitor cells and that PLC may play a key role in this process.

  9. 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

  10. Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

    PubMed

    Sun, Jiali; Huye, Leslie E; Lapteva, Natalia; Mamonkin, Maksim; Hiregange, Manasa; Ballard, Brandon; Dakhova, Olga; Raghavan, Darshana; Durett, April G; Perna, Serena K; Omer, Bilal; Rollins, Lisa A; Leen, Ann M; Vera, Juan F; Dotti, Gianpietro; Gee, Adrian P; Brenner, Malcolm K; Myers, Douglas G; Rooney, Cliona M

    2015-01-01

    Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro. We developed a rapid and GMP compliant method for the early transduction of

  11. Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

    PubMed Central

    Andolfi, Grazia; Fousteri, Georgia; Rossetti, Maura; Magnani, Chiara F; Jofra, Tatiana; Locafaro, Grazia; Bondanza, Attilio; Gregori, Silvia; Roncarolo, Maria-Grazia

    2012-01-01

    Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. PMID:22692497

  12. FOXP3 Expression in GARP-Transduced Helper T Cells Is Not Associated with FOXP3 TSDR Demethylation

    PubMed Central

    Kehrmann, Jan; Zeschnigk, Michael; Buer, Jan; Probst-Kepper, Michael

    2011-01-01

    Aim: Glycoprotein A repetitions predominant (GARP or LRRC32) represents a human regulatory CD4+ CD25hi FOXP3+ T (Treg) cell-specific receptor that controls FOXP3. Ectopic expression of GARP in helper T (Th) cells has been shown to be sufficient for the induction of FOXP3 and generation of a stable regulatory phenotype. Since expression of FOXP3 in Treg cells is epigenetically controlled by a conserved motif, the so-called Treg-specific demethylated region (TSDR), we asked whether GARP-mediated upregulation of FOXP3 in Th cells is similarly accompanied by demethylation of the TSDR. Methods: DNA methylation of the FOXP3 TSDR was analyzed by direct sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA. Results: Although GARP-transduced Th cells exhibit constitutive FOXP3 expression and a regulatory phenotype, the FOXP3 TSDR is completely methylated as in naive Th cells. GARP-mediated FOXP3 upregulation in Th cells is not associated with Treg-specific demethylation of the FOXP3 TSDR. Conclusion: Although GARP-engineered Th cells exhibit stable FOXP3 expression and a phenotypic reprogramming towards Treg cells in vitro, these cells do not completely mimic the epigenotype of natural Treg cells. Thus, concepts based on the genetic modification of Th cells as cellular therapies to treat autoimmune diseases or to control transplantation tolerance should be critically tested before any clinical application. PMID:22670117

  13. 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.

  14. 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.

  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. 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.

  17. 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

  18. 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.

  19. 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.

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

    PubMed

    Budde, Lihua E; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E; Brouns, Shaunda A; Spencer, David M; Till, Brian G; Jensen, Michael C; Riddell, Stanley R; Press, Oliver W

    2013-01-01

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

  1. 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

  2. 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 .).

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

    PubMed

    Legut, Mateusz; Dolton, Garry; Mian, Afsar Ali; Ottmann, Oliver G; Sewell, Andrew K

    2018-01-18

    Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4 + and CD8 + T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. © 2018 by The American Society of Hematology.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. A Fusion Receptor as a Safety Switch, Detection, and Purification Biomarker for Adoptive Transferred T Cells.

    PubMed

    Wu, Xiuqi; Shi, Bizhi; Zhang, Jiqin; Shi, Zhimin; Di, Shengmeng; Fan, Minliang; Gao, Huiping; Wang, Hai; Gu, Jianren; Jiang, Hua; Li, Zonghai

    2017-10-04

    The incorporation of an endogenous safety switch represents a rational strategy for the control of toxicities following the administration of adoptive T cell therapies. An ideal safety switch should be capable of depleting the transferred T cells with minimal injury to normal tissues. We generated a fusion receptor by engineering a cryptic 806 epitope of human epidermal growth factor receptor (EGFR) into the N terminus of the full-length human folate receptor 1 (FOLR1), designated as FR806. The expression of FR806 allows transducedcells to be targeted with CH12, a monoclonal antibody recognizing the 806 epitope, but not wild-type EGFR in healthy tissues. FR806, therefore, constitutes a specific cell-surface marker for the elimination of transducedcells. We demonstrate that the antibody-drug conjugate (ADC) CH12-MMAF is efficiently internalized by FR806-expressing T cells and has the potential to eliminate them. Transfected T cells could, furthermore, be efficiently detected and purified using CH12 antibodies. In immuno-compromised mice, CH12-MMAF eliminated the majority of transferred T cells expressing FR806 and anti-CD19 chimeric antigen receptor (CAR). The selectivity for the 806 epitope and internalization capacity of FOLR1 makes FR806 an efficient safety switch, which may additionally be used as a detection and purification biomarker for human T cell immunotherapies. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response.

    PubMed

    Gri, Giorgia; Gallo, Elena; Di Carlo, Emma; Musiani, Piero; Colombo, Mario P

    2003-01-01

    Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

  18. 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

  19. Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma

    PubMed Central

    Budde, Lihua E.; Berger, Carolina; Lin, Yukang; Wang, Jinjuan; Lin, Xubin; Frayo, Shani E.; Brouns, Shaunda A.; Spencer, David M.; Till, Brian G.; Jensen, Michael C.; Riddell, Stanley R.; Press, Oliver W.

    2013-01-01

    Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a “suicide gene” relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned. PMID:24358223

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. © 2015 British Society for Immunology.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Intra-tumoral delivery of CXCL11 via a vaccinia virus, but not by modified T cells, enhances the efficacy of adoptive T cell therapy and vaccines.

    PubMed

    Moon, Edmund K; Wang, Liang-Chuan S; Bekdache, Kheng; Lynn, Rachel C; Lo, Albert; Thorne, Stephen H; Albelda, Steven M

    2018-01-01

    T cell trafficking into tumors depends on a "match" between chemokine receptors on effector cells (e.g., CXCR3 and CCR5) and tumor-secreted chemokines. There is often a chemokine/chemokine receptor "mismatch", with tumors producing minute amounts of chemokines, resulting in inefficient targeting of effectors to tumors. We aimed to alter tumors to produce higher levels of CXCL11, a CXCR3 ligand, to attract more effector cells following immunotherapy. Mice bearing established subcutaneous tumors were studied. In our first approach, we used modified chimeric antigen receptor (CAR)-transduced human T cells to deliver CXCL11 (CAR/CXCL11) into tumors. In our second approach, we intravenously (iv) administered a modified oncolytic vaccinia virus (VV) engineered to produce CXCL11 (VV.CXCL11). The effect of these treatments on T cell trafficking into the tumors and anti-tumor efficacy after subsequent CAR T cell injections or anti-tumor vaccines was determined. CAR/CXCL11 and VV.CXCL11 significantly increased CXCL11 protein levels within tumors. For CAR/CXCL11, injection of a subsequent dose of CAR T cells did not result in increased intra-tumoral trafficking, and appeared to decrease the function of the injected CAR T cells. In contrast, VV.CXCL11 increased the number of total and antigen-specific T cells within tumors after CAR T cell injection or vaccination and significantly enhanced anti-tumor efficacy. Both approaches were successful in increasing CXCL11 levels within the tumors; however, only the vaccinia approach was successful in recruiting T cells and augmenting anti-tumor efficacy. VV.CXCL11 should be considered as a potential approach to augment adoptive T cell transfer or vaccine immunotherapy.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Engineered T cells for pancreatic cancer treatment

    PubMed Central

    Katari, Usha L; Keirnan, Jacqueline M; Worth, Anna C; Hodges, Sally E; Leen, Ann M; Fisher, William E; Vera, Juan F

    2011-01-01

    Objective Conventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive T cell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumour-associated antigen that is frequently expressed by pancreatic cancer cells. Methods Expression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor- and patient-derived T cells were isolated, activated in vitro using CD3/CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr51) release. Results Prostate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified T cells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach. Conclusions Prostate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred T cells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer. PMID:21843265

  9. 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

  10. 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

  11. 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.

  12. Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies

    PubMed Central

    Wang, X; Rivière, I

    2015-01-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic. PMID:25721207

  13. Manufacture of tumor- and virus-specific T lymphocytes for adoptive cell therapies.

    PubMed

    Wang, X; Rivière, I

    2015-03-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TILs) and genetically engineered T lymphocytes expressing chimeric antigen receptors (CARs) or conventional alpha/beta T-cell receptors (TCRs), collectively termed adoptive cell therapy (ACT), is an emerging novel strategy to treat cancer patients. Application of ACT has been constrained by the ability to isolate and expand functional tumor-reactive T cells. The transition of ACT from a promising experimental regimen to an established standard of care treatment relies largely on the establishment of safe, efficient, robust and cost-effective cell manufacturing protocols. The manufacture of cellular products under current good manufacturing practices (cGMPs) has a critical role in the process. Herein, we review current manufacturing methods for the large-scale production of clinical-grade TILs, virus-specific and genetically modified CAR or TCR transduced T cells in the context of phase I/II clinical trials as well as the regulatory pathway to get these complex personalized cellular products to the clinic.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. Vav1 GEF activity is required for T cell mediated allograft rejection.

    PubMed

    Haubert, Dirk; Li, Jianping; Saveliev, Alexander; Calzascia, Thomas; Sutter, Esther; Metzler, Barbara; Kaiser, Daniel; Tybulewicz, Victor L J; Weckbecker, Gisbert

    2012-06-01

    The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Vav1 GEF activity is required for T cell mediated allograft rejection

    PubMed Central

    Haubert, Dirk; Li, Jianping; Saveliev, Alexander; Calzascia, Thomas; Sutter, Esther; Metzler, Barbara; Kaiser, Daniel; Tybulewicz, Victor L.J.; Weckbecker, Gisbert

    2012-01-01

    The GDP exchange factor (GEF) Vav1 is a central signal transducer downstream of the T cell receptor and has been identified as a key factor for T cell activation in the context of allograft rejection. Vav1 has been shown to transduce signals both dependent and independent of its GEF function. The most promising approach to disrupt Vav1 activity by pharmacological inhibition would be to target its GEF function. However, the contribution of Vav1 GEF activity for allogeneic T cell activation has not been clarified yet. To address this question, we used knock-in mice bearing a mutated Vav1 with disrupted GEF activity but intact GEF-independent functions. T cells from these mice showed strongly reduced proliferation and activation in response to allogeneic stimulation. Furthermore, lack of Vav1 GEF activity strongly abrogated the in vivo expansion of T cells in a systemic graft-versus-host model. In a cardiac transplantation model, mice with disrupted Vav1 GEF activity show prolonged allograft survival. These findings demonstrate a strong requirement for Vav1 GEF activity for allogeneic T cell activation and graft rejection suggesting that disruption of Vav1 GEF activity alone is sufficient to induce significant immunosuppression. PMID:22456277

  5. 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

  6. 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

  7. 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.

  8. Type17 T-cells in Central Nervous System Autoimmunity and Tumors

    PubMed Central

    Okada, Hideho; Khoury, Samia J.

    2012-01-01

    Interleukin-17 (IL-17) producing Type17 T-cells, specifically T-helper (Th)17 cells reactive to central nervous system (CNS) autoantigens, manifest a higher migratory capability to the CNS parenchyma compared with other T-cell subpopulations due to their ability to penetrate the blood brain barrier (BBB). In the field of cancer immunotherapy, there are now a number of cell therapy approaches including early studies using T-cells transduced with chimeric antigen receptors in hematologic malignancy, suggesting that the use of T-cells or genetically modified T-cells could have a significant role in effective cancer therapy. However, the successful application of this strategy in solid tumors, such as CNS tumors, requires careful consideration of critical factors to improve the tumor-homing of T-cells. The current review is dedicated to discuss recent findings on the role of Type17 T-cells in CNS autoimmunity and cancer. The insight gained from these findings may lead to the development of novel therapeutic and prophylactic strategies for CNS autoimmunity and tumors. PMID:22454247

  9. 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

  10. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells.

    PubMed

    Hurton, Lenka V; Singh, Harjeet; Najjar, Amer M; Switzer, Kirsten C; Mi, Tiejuan; Maiti, Sourindra; Olivares, Simon; Rabinovich, Brian; Huls, Helen; Forget, Marie-Andrée; Datar, Vrushali; Kebriaei, Partow; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2016-11-29

    Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (T SCM ) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR + T cells with preserved T SCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19 + leukemia. Long-lived T cells were CD45RO neg CCR7 + CD95 + , phenotypically most similar to T SCM , and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR + T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  16. Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats

    PubMed Central

    Moralejo, Daniel; Yanay, Ofer; Kernan, Kelly; Bailey, Adam; Lernmark, Ake; Osborne, William

    2011-01-01

    Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyteTM encapsulation devices, implanted subcutaneously and rats monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3±10.2 pM that was significantly elevated over control values of 32.4±2.9 pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9±2.3 ng/ml that were significantly increased over control levels of 7.3±1.5 ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with β-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of β-cells and increased islet mass. These data suggest encapsulated transduced cells may offer a potential long term treatment of patients. PMID:21216666

  17. 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.

  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. 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...

  20. Increasing functional avidity of TCR-redirected T cells by removing defined N-glycosylation sites in the TCR constant domain

    PubMed Central

    Hauptrock, Beate; Malina, Victoria; Antunes, Edite; Voss, Ralf-Holger; Wolfl, Matthias; Strong, Roland; Theobald, Matthias; Greenberg, Philip D.

    2009-01-01

    Adoptive transfer of T lymphocytes transduced with a T cell receptor (TCR) to impart tumor reactivity has been reported as a potential strategy to redirect immune responses to target cancer cells (Schumacher, T.N. 2002. Nat. Rev. Immunol. 2:512–519). However, the affinity of most TCRs specific for shared tumor antigens that can be isolated is usually low. Thus, strategies to increase the affinity of TCRs or the functional avidity of TCR-transduced T cells might be therapeutically beneficial. Because glycosylation affects the flexibility, movement, and interactions of surface molecules, we tested if selectively removing conserved N-glycoslyation sites in the constant regions of TCR α or β chains could increase the functional avidity of T cells transduced with such modified TCRs. We observed enhanced functional avidity and improved recognition of tumor cells by T cells harboring TCR chains with reduced N-glycosylation (ΔTCR) as compared with T cells with wild-type (WT) TCR chains. T cells transduced with WT or ΔTCR chains bound tetramer equivalently at 4°C, but tetramer binding was enhanced at 37°C, predominantly as a result of reduced tetramer dissociation. This suggested a temperature-dependent mechanism such as TCR movement in the cell surface or structural changes of the TCR allowing improved multimerization. This strategy was effective with mouse and human TCRs specific for different antigens and, thus, should be readily translated to TCRs with any specificity. PMID:19171765

  1. Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells

    PubMed Central

    Hurton, Lenka V.; Singh, Harjeet; Najjar, Amer M.; Switzer, Kirsten C.; Mi, Tiejuan; Maiti, Sourindra; Olivares, Simon; Rabinovich, Brian; Huls, Helen; Forget, Marie-Andrée; Datar, Vrushali; Kebriaei, Partow; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2016-01-01

    Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+ T cells with preserved TSCM potential using the Sleeping Beauty platform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+ leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+ T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials. PMID:27849617

  2. 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.

  3. 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.

  4. CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains.

    PubMed

    Shiina, Satoshi; Ohno, Masasuke; Ohka, Fumiharu; Kuramitsu, Shunichiro; Yamamichi, Akane; Kato, Akira; Motomura, Kazuya; Tanahashi, Kuniaki; Yamamoto, Takashi; Watanabe, Reiko; Ito, Ichiro; Senga, Takeshi; Hamaguchi, Michinari; Wakabayashi, Toshihiko; Kaneko, Mika K; Kato, Yukinari; Chandramohan, Vidyalakshmi; Bigner, Darell D; Natsume, Atsushi

    2016-03-01

    Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults with a 5-year overall survival rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported to present the worst prognosis among GBM subtypes. Chimeric antigen receptor (CAR)-transduced T cells can recognize predefined tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructed a lentiviral vector expressing a third-generation CAR comprising a PDPN-specific antibody (NZ-1-based single-chain variable fragment) with CD28, 4-1BB, and CD3ζ intracellular domains. CAR-transduced peripheral blood monocytes were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets PDPN would be a promising adoptive immunotherapy to treat mesenchymal GBM. ©2016 American Association for Cancer Research.

  5. 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.

  6. 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

  7. 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

  8. In Vivo Selection of CD4+ T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

    PubMed Central

    Braun, Stephen E.; Taube, Ran; Zhu, Quan; Wong, Fay Eng; Murakami, Akikazu; Kamau, Erick; Dwyer, Markryan; Qiu, Gang; Daigle, Janet; Carville, Angela; Johnson, R. Paul

    2012-01-01

    Abstract We evaluated the potential of an anti–human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4+ cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4+ T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4+ cell. Upon reinfusion of engineered autologous CD4+ cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4+ T cells progressively decreased, concurrent with loss of CD4+ cells and elevated viral loads in both animals. However, CD4+ T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4+ cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo. PMID:22734618

  9. 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.

  10. Adenoviral-transduced dendritic cells are susceptible to suppression by T regulatory cells and promote interleukin 17 production.

    PubMed

    Wang, Adele Y; Crome, Sarah Q; Jenkins, Kristina M; Medin, Jeffrey A; Bramson, Jonathan L; Levings, Megan K

    2011-03-01

    Dendritic cell (DC) vaccines offer a robust platform for the development of cancer vaccines, but their effectiveness is thought to be limited by T regulatory cells (Tregs). Recombinant adenoviruses (RAdV) have been used successfully to engineer tumor antigen expression in DCs, but the impact of virus transduction on susceptibility to suppression by Tregs is unknown. We investigated the functional consequences of exposure to adenovirus on interactions between human monocyte-derived DCs and Tregs. Since the development of Tregs is linked to that of pro-inflammatory Th17 cells, the role of Th17 cells and IL-17-producing Tregs in the context of DC-based immunotherapies was also investigated. We found that Tregs potently suppressed the co-stimulatory capacity of RAdV-transduced DCs, regardless of whether the DCs were maturated by inflammatory cytokines or by exposure to Th1 or Th17 cells. Furthermore, exposure of Tregs to RAdV-exposed DCs increased IL-17 production and suppressive capacity, and correlated with enhanced secretion of IL-1β and IL-6 by DCs. The findings that DCs exposed to RAdV are suppressed by Tregs, promote Treg plasticity, and enhance Treg suppression indicates that strategies to limit Tregs will be required to enhance the efficacy of such DC-based immunotherapies.

  11. 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

  12. 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.

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

    PubMed Central

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

    2016-01-01

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

  14. 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

  15. 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.

  16. 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.

  17. A transduced living hyaline cartilage graft releasing transgenic stromal cell-derived factor-1 inducing endogenous stem cell homing in vivo.

    PubMed

    Zhang, Feng; Leong, Wenyan; Su, Kai; Fang, Yu; Wang, Dong-An

    2013-05-01

    Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4(+)(SDF-1 receptor) cell population, accompanied by upregulation of the CD34(+), CD44(+), and Sca-1(+) cell populations as well as a downregulation of the CD11b(+) cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.

  18. Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats.

    PubMed

    Moralejo, Daniel; Yanay, Ofer; Kernan, Kelly; Bailey, Adam; Lernmark, Ake; Osborne, William

    2011-04-01

    Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyte(TM) encapsulation devices, implanted subcutaneously and rats were monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3 ± 10.2pM that was significantly elevated over control values of 32.4 ± 2.9pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9 ± 2.3ng/ml that were significantly increased over control levels of 7.3±1.5ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with α-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of α-cells and increased islet mass. These data suggest that encapsulated transduced cells may offer a potential long term treatment of patients. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Force Generation upon T Cell Receptor Engagement

    PubMed Central

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

    2011-01-01

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

  7. 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

  8. 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

  9. 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

  10. Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

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

    Sarafian, Theodore; Montes, Cindy; Harui, Airi

    Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that {delta}{sup 9}-tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelialmore » cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p < 0.05) and were 80- to 100-fold more sensitive to the inhibitory effects of THC. Studies using SR144528, a selective CB2R antagonist, verified that these effects were mediated by the CB2R. Marijuana smoke extract, but not smoke extracts from tobacco or placebo marijuana cigarettes, reproduced these effects (p < 0.05). THC decreased ATP level and mitochondrial membrane potential ({psi}{sub m}) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against

  11. 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

  12. 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.

  13. 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

  14. Effects of protein tyrosine phosphatase-PEST are reversed by Akt in T cells.

    PubMed

    Arimura, Yutaka; Shimizu, Kazuhiko; Koyanagi, Madoka; Yagi, Junji

    2014-12-01

    T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PTP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PTP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PTP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PTP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bcl-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Akt interferes with PTP-PEST. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

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

    PubMed

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

    2016-04-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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

  4. Targeting Fibroblast Activation Protein in Tumor Stroma with Chimeric Antigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity Without Severe Toxicity

    PubMed Central

    Wang, Liang-Chuan S; Lo, Albert; Scholler, John; Sun, Jing; Majumdar, Rajrupa S; Kapoor, Veena; Antzis, Michael; Cotner, Cody E.; Johnson, Laura A; Durham, Amy C; Solomides, Charalambos C.; June, Carl H; Puré, Ellen; Albelda, Steven M

    2013-01-01

    The majority of chimeric antigen receptor (CAR) T cell research has focused on attacking cancer cells. Here we show that targeting the tumor-promoting, non-transformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single chain Fv FAP (mAb 73.3) with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFNγ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective suggesting that further clinical development of anti-human FAP-CAR is warranted. PMID:24778279

  5. PLAC1-specific TCR-engineered T cells mediate antigen-specific antitumor effects in breast cancer

    PubMed Central

    Li, Qiongshu; Liu, Muyun; Wu, Man; Zhou, Xin; Wang, Shaobin; Hu, Yuan; Wang, Youfu; He, Yixin; Zeng, Xiaoping; Chen, Junhui; Liu, Qubo; Xiao, Dong; Hu, Xiang; Liu, Weibin

    2018-01-01

    Placenta-specific 1 (PLAC1), a novel cancer-testis antigen (CTA), is expressed in a number of different human malignancies. It is frequently produced in breast cancer, serving a function in tumorigenesis. Adoptive immunotherapy using T cell receptor (TCR)-engineered T cells against CTA mediates objective tumor regression; however, to the best of our knowledge, targeting PLAC1 using engineered T cells has not yet been attempted. In the present study, the cDNAs encoding TCRα- and β-chains specific for human leukocyte antigen (HLA)-A*0201-restricted PLAC1 were cloned from a cytotoxic T-lymphocyte, generated by in vitro by the stimulation of CD8+ T cells using autologous HLA-A2+ dendritic cells loaded with a PLAC1-specific peptide (p28-36, VLCSIDWFM). The TCRα/β-chains were linked by a 2A peptide linker (TCRα-Thosea asigna virus-TCRβ), and the constructs were cloned into the lentiviral vector, followed by transduction into human cytotoxic (CD8+) T cells. The efficiency of transduction was up to 25.16%, as detected by PLAC1 multimers. TCR-transduced CD8+ T cells, co-cultured with human non-metastatic breast cancer MCF-7 cells (PLAC1+, HLA-A2+) and triple-negative breast cancer MDAMB-231 cells (PLAC1+, HLA-A2+), produced interferon γ and tumor necrosis factor α, suggesting TCR activation. Furthermore, the PLAC1 TCR-transduced CD8+ T cells efficiently and specifically identified and annihilated the HLA-A2+/PLAC1+ breast cancer cell lines in a lactate dehydrogenase activity assay. Western blot analysis demonstrated that TCR transduction stimulated the production of mitogen-activated protein kinase signaling molecules, extracellular signal-regulated kinases 1/2 and nuclear factor-κB, through phosphoinositide 3-kinase γ-mediated phosphorylation of protein kinase B in CD8+ T cells. Xenograft mouse assays revealed that PLAC1 TCR-transduced CD8+T cells significantly delayed the tumor progression in mice-bearing breast cancer compared with normal saline or negative

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2013-01-01

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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

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

    PubMed Central

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

    2016-01-01

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

  3. 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.

  4. Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses.

    PubMed

    Wu, Gregory F; Corbo, Evann; Schmidt, Michelle; Smith-Garvin, Jennifer E; Riese, Matthew J; Jordan, Martha S; Laufer, Terri M; Brown, Eric J; Maltzman, Jonathan S

    2011-07-01

    The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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

  6. PTPROt maintains T cell immunity in the microenvironment of hepatocellular carcinoma.

    PubMed

    Hou, Jiajie; Deng, Lei; Zhuo, Han; Lin, Zhe; Chen, Yun; Jiang, Runqiu; Chen, Dianyu; Zhang, Xudong; Huang, Xingxu; Sun, Beicheng

    2015-08-01

    Intratumoral T cells play a central role in anti-tumor immunity, and the balance between T effector cells (Teff) and regulatory T cells (Treg) affects the prognosis of cancer patients. However, educated by tumor microenvironment, T cells frequently fail in their responsibility. In this study, we aimed to investigate the role of truncated isoform of protein tyrosine phosphatase receptor-type O (PTPROt) in T cell-mediated anti-tumor immunity. We recruited 70 hepatocellular carcinoma (HCC) patients and 30 healthy volunteers for clinical investigation, and analyzed cellular tumor immunity by using ptpro(-/-) C57BL/6 mice and NOD/SCID mice. PTPROt expression was significantly downregulated in human HCC-infiltrating T cells due to the hypoxia microenvironment; PTPROt expression highly correlated with the intratumoral Teff/Treg ratio and clinicopathologic characteristics. Moreover, PTPROt deficiency attenuated T cell-mediated anti-tumor immunity and remarkably promoted mouse HCC growth. Mechanistically, deletion of PTPROt decreased Teff quantity and quality through phosphorylation of lymphocyte-specific tyrosine kinase, but increased Treg differentiation through phosphorylation of signal transducer and activator of transcription 5. In support of the Teff/Treg homeostasis, PTPROt serves as an important tumor suppressor in HCC microenvironment. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. 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.

  19. 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

  20. Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

    PubMed Central

    Owens, Gemma L.; Sheard, Victoria E.; Kalaitsidou, Milena; Blount, Daniel; Lad, Yatish; Cheadle, Eleanor J.; Edmondson, Richard J.; Kooner, Gurdeep; Gilham, David E.

    2018-01-01

    Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells. PMID:29239915

  1. 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

  2. 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

  3. 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.

  4. 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

  5. Cloning and expansion of antigen-specific T cells using iPS cell technology: development of "off-the-shelf" T cells for the use in allogeneic transfusion settings.

    PubMed

    Kawamoto, Hiroshi; Masuda, Kyoko; Nagano, Seiji; Maeda, Takuya

    2018-03-01

    Recent advances in adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) have led to moderate therapeutic anti-cancer effects in clinical trials. However, a critical issue, namely that CTLs collected from patients are easily exhausted during expansion culture, has yet to be solved. To address this issue, we have been developing a strategy which utilizes induced pluripotent stem cell (iPSC) technology. This strategy is based on the idea that when iPSCs are produced from antigen-specific CTLs, CTLs regenerated from such iPSCs should show the same antigen specificity as the original CTLs. Pursuing this idea, we previously succeeded in regenerating melanoma antigen MART1-specific CTLs, and more recently in producing potent CTLs expressing CD8αβ heterodimer. We are now developing a novel method by which non-T derived iPSCs are transduced with exogenous T cell receptor genes. If this method is applied to Human Leukocyte Antigen (HLA) haplotype-homozygous iPSC stock, it will be possible to prepare "off-the-shelf" T cells. As a first-in-human trial, we are planning to apply our strategy to relapsed acute myeloid leukemia patients by targeting the WT1 antigen.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Recent thymic emigrants are preferentially incorporated only into the depleted T-cell pool.

    PubMed

    Houston, Evan G; Higdon, Lauren E; Fink, Pamela J

    2011-03-29

    Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, and they differ functionally and phenotypically from the rest of the naïve T-cell pool. RTEs are present in the peripheral T-cell pool throughout life but are the most common subset of T cells in neonates and adults recovering from lymphoablation. Using a murine model to study the homeostasis of RTEs, we show that under lymphoreplete conditions, RTEs are at a competitive disadvantage to already established mature naïve (MN) T cells. This disadvantage may be caused by a defect in survival, because RTEs may transduce homeostatic signals inefficiently, and their ability to survive is enhanced with increased expression of IL-7 receptor or B-cell lymphoma 2 (Bcl-2). Conversely, under lymphopenic conditions, enhanced proliferation by RTEs allows them to out-compete their MN T-cell counterparts. These results suggest that in times of need, such as in neonates or lymphopenic adults, RTEs perform well to fill the gaps in the peripheral T-cell pool, but when the periphery already is full, many RTEs are not incorporated into the pool of recirculating lymphocytes.

  12. 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

  13. Signal Transduction in T Cell Activation and Tolerance

    DTIC Science & Technology

    1993-01-01

    chains and ’ chains may transduce different signals in intact T cells. These studies demonstrate that while c- deficient and c-containing TCR complexes...three independently derived pairs of CD45- and CD45+ murine T cell lymphomas, the CD45- expressing cells were consistently deficient in...D.B., Larsen, A. and Wilson, C.B. (1986) Reduced interferon-gamma mRNA levels in human neonates: Evidence for an intrinsic T cell deficiency yi 114

  14. 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.

  15. Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses1

    PubMed Central

    Wu, Gregory F.; Corbo, Evann; Schmidt, Michelle; Smith-Garvin, Jennifer E.; Riese, Matthew J.; Jordan, Martha S.; Laufer, Terri M.; Brown, Eric J.; Maltzman, Jonathan S.

    2011-01-01

    SUMMARY The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of wild-type SLP-76 protein in thymocytes. Following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T cell mediated autoimmune disease experimental autoimmune encephalomyelitis. Our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects. PMID:21469089

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. TCRγ4δ1-Engineered αβT Cells Exhibit Effective Antitumor Activity

    PubMed Central

    He, Kangxia; You, Hongqin; Li, Yuxia; Cui, Lianxian; Zhang, Jianmin; He, Wei

    2016-01-01

    T cell engineering with T cell receptors (TCRs) specific for tumors plays an important role in adoptive T cell transfer (ATC) therapy for cancer. Here, we present a novel strategy to redirect peripheral blood-derived αβT cells against tumors via TCRγ4δ1 gene transduction. The broad-spectrum antitumor activity of TCRδ1 cells in innate immunity is dependent on CDR3δ1. TCRγ4δ1-engineered αβT cells were prepared by lentiviral transduction and characterized by analyzing in vitro and in vivo cytotoxicity to tumors, ability of proliferation and cytokine production, and potential role in autoimmunity. Results show that TCRγ4δ1 genes were transduced to approximately 36% of polyclonal αβT cells. TCRγ4δ1-engineered αβT cells exhibited effective in vitro TCRγδ-dependent cytotoxicity against various tumor cells via the perforin-granzyme pathway. They also showed a strong proliferative capacity and robust cytokine production. TCRγ4δ1-engineered αβT cells neither expressed mixed TCR dimers nor bound/killed normal cells in vitro. More important, adoptive transfer of TCRγ4δ1-engineered αβT cells into nude mice bearing a human HepG2 cell line significantly suppressed tumor growth. Our results demonstrate a novel role for TCRγ4δ1 in gene therapy and ATC for cancer. PMID:27463149

  8. 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

  9. Effective and persistent antitumor activity of HER2-directed CAR-T cells against gastric cancer cells in vitro and xenotransplanted tumors in vivo.

    PubMed

    Song, Yanjing; Tong, Chuan; Wang, Yao; Gao, Yunhe; Dai, Hanren; Guo, Yelei; Zhao, Xudong; Wang, Yi; Wang, Zizheng; Han, Weidong; Chen, Lin

    2017-03-10

    Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. Adoptive immunotherapy for B-cell malignancies using CD19-targeted chimeric antigen receptor T-cells: A systematic review of efficacy and safety.

    PubMed

    Hao, Lu; Li, Tongtong; Chang, Lung-Ji; Chen, Xiaochuan

    2017-08-01

    Adoptive infusion of chimeric antigen receptor transduced T-cells (CAR-T) is a powerful tool of immunotherapy for hematological malignancies, as evidenced by recently published and unpublished clinical results. In this report, we performed a meta-analysis to evaluate the efficacy and side effects of CAR-T on relapsed B-cell malignancies, including leukemia and lymphoma. Clinical studies investigating efficacy and safety of CAR-T in acute and chronic lymphocytic leukemia and lymphoma were identified by searching PubMed and EMBASE. Outcomes of efficacy subjected to analysis were the rates of complete remission (CR) and partial remission (PR). The safety parameters were the prevalence of adverse effects including fever, hypotension, and acute renal failure. Meta analyses were performed using R software. Weighted hazard ratio (HR) with 95% confidence intervals was calculated for each outcome. Fixed or random-effects models were employed depending on the heterogeneity across the included studies. Nineteen published clinical studies, with a total of 391 patients were included for the meta-analysis. The pooled rate of complete remission was 55% (95% CI 41%-69%); the pooled rate of partial remission was 25% (95% CI: 19%-33%). The prevalence of fever was 62% (95% CI: 41%-79%); the hypotension was 22% (95% CI: 15%-31%); acute renal failure was 24% (95% CI: 16%-34%). All adverse effects were manageable and no death was reported due to toxicity. CD19-targeted CAR-T is an effective modality in treating refractory B-cell malignancies including acute and chronic lymphatic leukemia, Hodjkin's and non-Hodjkin's lymphoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. 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).

  18. Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

    PubMed Central

    Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

    1995-01-01

    The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU

  19. The Glycosylphosphatidylinositol-Anchored Variable Region of Llama Heavy Chain-Only Antibody JM4 Efficiently Blocks both Cell-Free and T Cell-T Cell Transmission of Human Immunodeficiency Virus Type 1

    PubMed Central

    Liu, Lihong; Wang, Weiming; Matz, Julie; Ye, Chaobaihui; Bracq, Lucie; Delon, Jerome; Kimata, Jason T.; Chen, Zhiwei

    2016-01-01

    ABSTRACT The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. IMPORTANCE Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we

  20. Regulation of T cell homeostasis by JAKs and STATs.

    PubMed

    Ross, Jeremy A; Nagy, Zsuzsanna S; Cheng, Hanyin; Stepkowski, Stanislaw M; Kirken, Robert A

    2007-01-01

    Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Effect of memory CD4+ T cells' signal transducer and activator of transcription (STATs) functional shift on cytokine-releasing properties in asthma.

    PubMed

    Chen, Zhihong; Pan, Jue; Jia, Yi; Li, Dandan; Min, Zhihui; Su, Xiaoqiong; Yuan, Honglei; Shen, Geng; Cao, Shengxuan; Zhu, Lei; Wang, Xiangdong

    2017-02-01

    Recent data have demonstrated that long-lived memory T cells are present in the human lung and can play significant roles in the pathogenesis of specific allergic and autoimmune diseases. However, most evidence has been obtained from mouse studies, and the potential roles of memory T cells in human allergic diseases, such as asthma, remain largely unknown. Thirty-three asthmatics, 26 chronic obstructive pulmonary disease (COPD) patients, and 22 healthy volunteers were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood, and cell surface staining (CD4, CD45RO, CRTH2, CD62L, and CCR7) was performed for the detection of memory CD4 + T cells in blood. After stimulation with interleukin-27 (IL-27) or IL-4 for 15 min, the STAT1/STAT6 phosphorylation of memory CD4 + T cells was measured separately by flow cytometric techniques. The cytokine-releasing profiles after 6 days of culture under neutralization, T H 2, T H 2 + lipopolysaccharide (LPS), and T H 2 + house dust mite (HDM) conditions were detected by intracellular protein (IL-5, IL-17, and interferon (IFN)-γ) staining. Correlation analyses between the profile of memory CD4 + T cells and clinical characteristics of asthma were performed. The number of circulating memory CD4 + T (CD4 + Tm) cells in asthmatics was increased compared with that in the healthy subjects (48 ± 5.7 % vs. 32 ± 4.1 %, p < 0.05). Compared with COPD and healthy subjects, the phosphorylation of signal transducer and activator of transcription 1 (STAT1-py) was impaired in asthmatics, whereas the phosphorylation of signal transducer and activator of transcription 6 (STAT6-py) was slightly enhanced. This imbalance of STAT1-py/STAT6-py was attributed to T H 2 memory cells but not non-T H 2 memory cells in blood. The cytokine-releasing profiles of asthmatics was unique, specifically IL-5 high , IL-17 high , and IFN-r low , compared with those of COPD patients and healthy subjects

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Eradication of Large Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a Single Cancer-Specific Point Mutation.

    PubMed

    Leisegang, Matthias; Engels, Boris; Schreiber, Karin; Yew, Poh Yin; Kiyotani, Kazuma; Idel, Christian; Arina, Ainhoa; Duraiswamy, Jaikumar; Weichselbaum, Ralph R; Uckert, Wolfgang; Nakamura, Yusuke; Schreiber, Hans

    2016-06-01

    Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734-43. ©2015 AACRSee related commentary by Liu, p. 2602. ©2015 American Association for Cancer Research.

  12. Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors.

    PubMed

    Handa, A; Muramatsu, S; Qiu, J; Mizukami, H; Brown, K E

    2000-08-01

    Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID(50) of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.

  13. 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.

  14. 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

  15. 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

  16. Soluble TNF receptor 1-secreting ex vivo-derived dendritic cells reduce injury after stroke.

    PubMed

    Works, Melissa G; Koenig, Jenny B; Sapolsky, Robert M

    2013-09-01

    Inflammation is a major factor in the progression of damage after stroke and in the clinic, current therapies treat the clot, not the resulting damage. We have developed a novel method of protein delivery that exploits the migration ability of leukocytes after ischemic stroke (transient middle cerebral artery occlusion; tMCAO). In our studies, ex vivo-derived dendritic cells (exDCs) migrate to the inflamed rat brain soon after tMCAO onset and the number of cells that remain in the brain after injection is significantly correlated with the amount of local inflammation at the injury site. In addition, exDCs transduced to overexpress soluble tumor necrosis factor (TNF) receptor1 (sTNFR1) produce functional cargo that is secreted and that blocks TNF-α bioavailability in vitro. When delivered at 6 hours post-tMCAO reperfusion, sTNFR1-exDC-treated rats show significantly smaller infarct size and decreased inflammation compared with animals treated with exDCs transduced with GFP lentivirus. These studies indicate that cell-mediated delivery of proteins may be a promising new approach to reduce brain damage after acute neurologic insult.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

    PubMed Central

    Ueda, Yukiko; Neel, Nicole F.; Schutyser, Evemie; Raman, Dayanidhi; Richmond, Ann

    2009-01-01

    The CXC chemokine receptor 4 (CXCR4) contributes to the metastasis of human breast cancer cells. The CXCR4 COOH-terminal domain (CTD) seems to play a major role in regulating receptor desensitization and down-regulation. We expressed either wild-type CXCR4 (CXCR4-WT) or CTD-truncated CXCR4 (CXCR4-ΔCTD) in MCF-7 human mammary carcinoma cells to determine whether the CTD is involved in CXCR4-modulated proliferation of mammary carcinoma cells. CXCR4-WT-transduced MCF-7 cells (MCF-7/CXCR4-WT cells) do not differ from vector-transduced MCF-7 control cells in morphology or growth rate. However, CXCR4-ΔCTD-transduced MCF-7 cells (MCF-7/CXCR4-ΔCTD cells) exhibit a higher growth rate and altered morphology, potentially indicating an epithelial-to-mesenchymal transition. Furthermore, extracellular signal-regulated kinase (ERK) activation and cell motility are increased in these cells. Ligand induces receptor association with β-arrestin for both CXCR4-WT and CXCR4-ΔCTD in these MCF-7 cells. Overexpressed CXCR4-WT localizes predominantly to the cell surface in unstimulated cells, whereas a significant portion of overexpressed CXCR4-ΔCTD resides intracellularly in recycling endosomes. Analysis with human oligomicroarray, Western blot, and immunohistochemistry showed that E-cadherin and Zonula occludens are down-regulated in MCF-7/CXCR4-ΔCTD cells. The array analysis also indicates that mesenchymal marker proteins and certain growth factor receptors are up-regulated in MCF-7/CXCR4-ΔCTD cells. These observations suggest that (a) the overexpression of CXCR4-ΔCTD leads to a gain-of-function of CXCR4-mediated signaling and (b) the CTD of CXCR4-WT may perform a feedback repressor function in this signaling pathway. These data will contribute to our understanding of how CXCR4-ΔCTD may promote progression of breast tumors to metastatic lesions. PMID:16740704

  9. FADD and the NF-κB family member Bcl-3 regulate complementary pathways to control T-cell survival and proliferation

    PubMed Central

    Rangelova, Svetla; Kirschnek, Susanne; Strasser, Andreas; Häcker, Georg

    2008-01-01

    Fas-associated protein with death domain/mediator of receptor induced toxicity (FADD/MORT1) was first described as a transducer of death receptor signalling but was later recognized also to be important for proliferation of T cells. B-cell lymphoma 3 (Bcl-3) is a relatively little understood member of the nuclear factor (NF)-κB family of transcription factors. We recently found that Bcl-3 is up-regulated in T cells from mice where FADD function is blocked by a dominant negative transgene (FADD-DN). To understand the importance of this, we generated FADD-DN/bcl-3−/− mice. Here, we report that T cells from these mice show massive cell death and severely reduced proliferation in response to T-cell receptor (TCR) stimulation in vitro. Transgenic co-expression of Bcl-2 (FADD-DN/bcl-3−/−/vav-bcl-2 mice) rescued the survival but not the proliferation of T cells. FADD-DN/bcl-3−/− mice had normal thymocyte numbers but reduced numbers of peripheral T cells despite an increase in cycling T cells in vivo. However, activation of the classical NF-κB and extracellular regulated kinase (ERK) pathways and expression of interleukin (IL)-2 mRNA upon stimulation were normal in T cells from FADD-DN/bcl-3−/− mice. These data suggest that FADD and Bcl-3 regulate separate pathways that both contribute to survival and proliferation in mouse T cells. PMID:18557791

  10. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.

    PubMed

    Lee, Daniel W; Kochenderfer, James N; Stetler-Stevenson, Maryalice; Cui, Yongzhi K; Delbrook, Cindy; Feldman, Steven A; Fry, Terry J; Orentas, Rimas; Sabatino, Marianna; Shah, Nirali N; Steinberg, Seth M; Stroncek, Dave; Tschernia, Nick; Yuan, Constance; Zhang, Hua; Zhang, Ling; Rosenberg, Steven A; Wayne, Alan S; Mackall, Crystal L

    2015-02-07

    Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10

  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. 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.

  13. 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

  14. 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

  15. 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.

  16. Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

    PubMed Central

    Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

    2013-01-01

    Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

  17. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    PubMed

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP + memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP + memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation. © 2016 John Wiley & Sons Ltd.

  18. 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

  19. [131I]FIAU labeling of genetically transduced, tumor-reactive lymphocytes: cell-level dosimetry and dose-dependent toxicity.

    PubMed

    Zanzonico, Pat; Koehne, Guenther; Gallardo, Humilidad F; Doubrovin, Mikhail; Doubrovina, Ekaterina; Finn, Ronald; Blasberg, Ronald G; Riviere, Isabelle; O'Reilly, Richard J; Sadelain, Michel; Larson, Steven M

    2006-09-01

    Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium. Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay. At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    PubMed

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  5. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

  6. 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

  7. 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

  8. 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

  9. Function of the nucleotide exchange activity of vav1 in T cell development and activation.

    PubMed

    Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J; Rittinger, Katrin; Tybulewicz, Victor L J

    2009-12-15

    The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.

  10. Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation*

    PubMed Central

    Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J.; Rittinger, Katrin; Tybulewicz, Victor L. J.

    2012-01-01

    The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions. PMID:20009105

  11. 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

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

    PubMed

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

    2016-11-01

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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

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

    Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu

    2010-11-26

    Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in amore » leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

    PubMed

    Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

    2016-01-01

    The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

  3. 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

  4. In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance.

    PubMed

    Lei, Fengyang; Zhao, Baohua; Haque, Rizwanul; Xiong, Xiaofang; Budgeon, Lynn; Christensen, Neil D; Wu, Yuzhang; Song, Jianxun

    2011-07-15

    Adoptive T-cell immunotherapy has garnered wide attention, but its effective use is limited by the need of multiple ex vivo manipulations and infusions that are complex and expensive. In this study, we show how highly reactive antigen (Ag)-specific CTLs can be generated from induced pluripotent stem (iPS) cells to provide an unlimited source of functional CTLs for adoptive immunotherapy. iPS cell-derived T cells can offer the advantages of avoiding possible immune rejection and circumventing ethical and practical issues associated with other stem cell types. iPS cells can be differentiated into progenitor T cells in vitro by stimulation with the Notch ligand Delta-like 1 (DL1) overexpressed on bone marrow stromal cells, with complete maturation occurring upon adoptive transfer into Rag1-deficient mice. Here, we report that these iPS cells can be differentiated in vivo into functional CTLs after overexpression of MHC I-restricted Ag-specific T-cell receptors (TCR). In this study, we generated murine iPS cells genetically modified with ovalbumin (OVA)-specific and MHC-I restricted TCR (OT-I) by retrovirus-mediated transduction. After their adoptive transfer into recipient mice, the majority of OT-I/iPS cells underwent differentiation into CD8+ CTLs. TCR-transduced iPS cells developed in vivo responded in vitro to peptide stimulation by secreting interleukin 2 and IFN-γ. Most importantly, adoptive transfer of TCR-transduced iPS cells triggered infiltration of OVA-reactive CTLs into tumor tissues and protected animals from tumor challenge. Taken together, our findings offer proof of concept for a potentially more efficient approach to generate Ag-specific T lymphocytes for adoptive immunotherapy. ©2011 AACR.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Advanced generation anti-prostate specific membrane antigen designer T cells for prostate cancer immunotherapy.

    PubMed

    Ma, Qiangzhong; Gomes, Erica M; Lo, Agnes Shuk-Yee; Junghans, Richard P

    2014-02-01

    Adoptive immunotherapy by infusion of designer T cells (dTc) engineered with chimeric antigen receptors (CARs) for tumoricidal activity represents a potentially highly specific modality for the treatment of cancer. In this study, 2nd generation (gen) anti-prostate specific membrane antigen (PSMA) dTc were developed for improving the efficacy of previously developed 1st gen dTc for prostate cancer immunotherapy. The 1st gen dTc are modified with chimeric immunoglobulin-T cell receptor (IgTCR) while the 2nd gen dTc are engineered with an immunoglobulin-CD28-T cell receptor (IgCD28TCR), which incorporates a CD28 costimulatory signal for optimal T cell activation. A 2nd gen anti-PSMA IgCD28TCR CAR was constructed by inserting the CD28 signal domain into the 1st gen CAR. 1st and 2nd gen anti-PSMA dTc were created by transducing human T cells with anti-PSMA CARs and their antitumor efficacy was compared for specific activation on PSMA-expressing tumor contact, cytotoxicity against PSMA-expressing tumor cells in vitro, and suppression of tumor growth in an animal model. The 2nd gen dTc can be optimally activated to secrete larger amounts of cytokines such as IL2 and IFNγ than 1st gen and to proliferate more vigorously on PSMA-expressing tumor contact. More importantly, the 2nd gen dTc preserve the PSMA-specific cytotoxicity in vitro and suppress tumor growth in animal models with significant higher potency. Our results demonstrate that 2nd gen anti-PSMA designer T cells exhibit superior antitumor functions versus 1st gen, providing a rationale for advancing this improved agent toward clinical application in prostate cancer immunotherapy. © 2013 Wiley Periodicals, Inc.

  12. An inducible caspase 9 safety switch for T-cell therapy

    PubMed Central

    Straathof, Karin C.; Pulè, Martin A.; Yotnda, Patricia; Dotti, Gianpietro; Vanin, Elio F.; Brenner, Malcolm K.; Heslop, Helen E.; Spencer, David M.; Rooney, Cliona M.

    2005-01-01

    The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a “safety switch” that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies. PMID:15728125

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

    PubMed Central

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

    2017-01-01

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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

    PubMed Central

    Ponnazhagan, Selvarangan; Weigel, Kirsten A.; Raikwar, Sudhanshu P.; Mukherjee, Pinku; Yoder, Mervin C.; Srivastava, Arun

    1998-01-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited

  20. Adoptive transfer of MART-1 T cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma

    PubMed Central

    Chodon, Thinle; Comin-Anduix, Begonya; Chmielowski, Bartosz; Koya, Richard C; Wu, Zhongqi; Auerbach, Martin; Ng, Charles; Avramis, Earl; Seja, Elizabeth; Villanueva, Arturo; McCannel, Tara A.; Ishiyama, Akira; Czernin, Johannes; Radu, Caius G.; Wang, Xiaoyan; Gjertson, David W.; Cochran, Alistair J.; Cornetta, Kenneth; Wong, Deborah J.L.; Kaplan-lefko, Paula; Hamid, Omid; Samlowski, Wolfram; Cohen, Peter A.; Daniels, Gregory A.; Mukherji, Bijay; Yang, Lili; Zack, Jerome A.; Kohn, Donald B.; Heath, James R.; Glaspy, John A.; Witte, Owen N.; Baltimore, David; Economou, James S.; Ribas, Antoni

    2014-01-01

    Purpose It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short one-week manufacture protocol to determine the feasibility, safety and antitumor efficacy of this double cell therapy. Experimnetal Design A clinical trial (NCT00910650) adoptively transferring MART-1 T cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and re-infused with (n = 10) or without (n = 3) prior cryopreservation. Results 14 patients with metastatic melanoma were enrolled and nine out of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within two weeks of ACT indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared to cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using non-cryopreserved T cells. Conclusion Double cell therapy with ACT of TCR engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. PMID:24634374

  1. CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

    PubMed Central

    Aguado, Enrique; Garcia-Cozar, Francisco

    2014-01-01

    Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein. PMID:24465502

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

    PubMed Central

    Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

    2012-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

  12. 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

  13. Simplified process for the production of anti-CD19-CAR engineered T cells

    PubMed Central

    Tumaini, Barbara; Lee, Daniel W.; Lin, Tasha; Castiello, Luciano; Stroncek, David F.; Mackall, Crystal; Wayne, Alan; Sabatino, Marianna

    2014-01-01

    Background Adoptive Immunotherapy using chimeric antigen receptor (CAR) engineered T cells specific for CD19 has shown promising results for the treatment of B cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. Here, we describe a new, simplified method to produce anti-CD19-CAR T cells. Methods T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 hours and then a second transduction was performed. No spinoculation was used. Cells were then expanded for an additional 9 days. Results The method was validated using 2 PBMC products from a patient with B-CLL and one PBMC product from a healthy subject. The 2 PBMC products from the B-CLL patient contained 11.4% and 12.9% T cells. The manufacture process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6 fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first 4 ALL patients treated at our institution. Discussion We developed a simplified semi-closed system for the initial selection, activation, transduction and expansion of T cells using anti-CD3/anti-CD28 beads and bags, to produce autologous anti-CD19 CAR transduced T cells to support an ongoing clinical trial. PMID:23992830

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

    PubMed

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M; Litvinov, Ivan V; Fredholm, Simon; Petersen, David L; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P; Sasseville, Denis; Wasik, Mariusz A; Bonefeld, Charlotte M; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B; Odum, Niels

    2016-03-10

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. © 2016 by The American Society of Hematology.

  3. 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

  4. 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.

  5. 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

  6. Ex vivo Akt inhibition promotes the generation of potent CD19CAR T cells for adoptive immunotherapy.

    PubMed

    Urak, Ryan; Walter, Miriam; Lim, Laura; Wong, ChingLam W; Budde, Lihua E; Thomas, Sandra; Forman, Stephen J; Wang, Xiuli

    2017-01-01

    Insufficient persistence and effector function of chimeric antigen receptor (CAR)-redirected T cells have been challenging issues for adoptive T cell therapy. Generating potent CAR T cells is of increasing importance in the field. Studies have demonstrated the importance of the Akt pathway in the regulation of T cell differentiation and memory formation. We now investigate whether inhibition of Akt signaling during ex vivo expansion of CAR T cells can promote the generation of CAR T cells with enhanced antitumor activity following adoptive therapy in a murine leukemia xenograft model. Various T cell subsets including CD8+ T cells, bulk T cells, central memory T cells and naïve/memory T cells were isolated from PBMC of healthy donors, activated with CD3/CD28 beads, and transduced with a lentiviral vector encoding a second-generation CD19CAR containing a CD28 co-stimulatory domain. The transduced CD19CAR T cells were expanded in the presence of IL-2 (50U/mL) and Akt inhibitor (Akti) (1 μM) that were supplemented every other day. Proliferative/expansion potential, phenotypical characteristics and functionality of the propagated CD19CAR T cells were analyzed in vitro and in vivo after 17-21 day ex vivo expansion. Anti-tumor activity was evaluated after adoptive transfer of the CD19CAR T cells into CD19+ tumor-bearing immunodeficient mice. Tumor signals were monitored with biophotonic imaging, and survival rates were analyzed by the end of the experiments. We found that Akt inhibition did not compromise CD19CAR T cell proliferation and expansion in vitro, independent of the T cell subsets, as comparable CD19CAR T cell expansion was observed after culturing in the presence or absence of Akt inhibitor. Functionally, Akt inhibition did not dampen cell-mediated effector function, while Th1 cytokine production increased. With respect to phenotype, Akti-treated CD19CAR T cells expressed higher levels of CD62L and CD28 as compared to untreated CD19CAR T cells. Once

  7. 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

  8. 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

  9. 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

  10. 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.

  11. Asymmetric cell division during T cell development controls downstream fate

    PubMed Central

    Pham, Kim; Shimoni, Raz; Charnley, Mirren; Ludford-Menting, Mandy J.; Hawkins, Edwin D.; Ramsbottom, Kelly; Oliaro, Jane; Izon, David; Ting, Stephen B.; Reynolds, Joseph; Lythe, Grant; Molina-Paris, Carmen; Melichar, Heather; Robey, Ellen; Humbert, Patrick O.; Gu, Min

    2015-01-01

    During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal. PMID:26370500

  12. 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

  13. 4-1BB and CD28 Signaling Plays a Synergistic Role in Redirecting Umbilical Cord Blood T Cells Against B-Cell Malignancies

    PubMed Central

    Tammana, Syam; Huang, Xin; Wong, Marianna; Milone, Michael C.; Ma, Linan; Levine, Bruce L.; June, Carl H.; Wagner, John E.; Blazar, Bruce R.

    2010-01-01

    Abstract Umbilical cord blood (UCB) T cells can be redirected to kill leukemia and lymphoma cells by engineering with a single-chain chimeric antigen receptor (CAR) and thus may have general applications in adoptive cell therapy. However, the role of costimulatory molecules in UCB T-cell activation and effector functions in context with CAR remains elusive. To investigate the effect of costimulatory molecules (4-1BB and CD28) on UCB T cells, we transduced UCB T cells with lentiviral vectors expressing Green Fluorescent Protein (GFP) and CAR for CD19 containing an intracellular domain of the CD3ζ chain and either a 4-1BB (UCB-19BBζ) or a CD28 intracellular domain (UCB-1928ζ), both (UCB-1928BBζ), or neither (UCB-19ζ). We found that UCB-19BBζ and UCB-28BBζ T cells exhibited more cytotoxicity to CD19+ leukemia and lymphoma cell lines than UCB-19ζ and UCB-1928ζ, although differences in secretion of interleukin-2 and interferon-γ by these T cells were not evident. In vivo adoptive transfer of these T cells into intraperitoneal tumor-bearing mice demonstrated that UCB-19BBζ and UCB-1928BBζ T cells mounted the most potent antitumor response. The mice adoptively transferred with UCB-1928BBζ cells survived longer than the mice with UCB-19BBζ. Moreover, UCB-1928BBζ T cells mounted a more robust antitumor response than UCB-19BBζ in a systemic tumor model. Our data suggest a synergistic role of 4-1BB and CD28 costimulation in engineering antileukemia UCB effector cells and implicate a design for redirected UCB T-cell therapy for refractory leukemia. PMID:19719389

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Optimization of methods for the genetic modification of human T cells.

    PubMed

    Bilal, Mahmood Y; Vacaflores, Aldo; Houtman, Jon Cd

    2015-11-01

    CD4(+) T cells are not only critical in the fight against parasitic, bacterial and viral infections, but are also involved in many autoimmune and pathological disorders. Studies of protein function in human T cells are confined to techniques such as RNA interference (RNAi) owing to ethical reasons and relative simplicity of these methods. However, introduction of RNAi or genes into primary human T cells is often hampered by toxic effects from transfection or transduction methods that yield cell numbers inadequate for downstream assays. Additionally, the efficiency of recombinant DNA expression is frequently low because of multiple factors including efficacy of the method and strength of the targeting RNAs. Here, we describe detailed protocols that will aid in the study of primary human CD4(+) T cells. First, we describe a method for development of effective microRNA/shRNAs using available online algorithms. Second, we illustrate an optimized protocol for high efficacy retroviral or lentiviral transduction of human T-cell lines. Importantly, we demonstrate that activated primary human CD4(+) T cells can be transduced efficiently with lentiviruses, with a highly activated population of T cells receiving the largest number of copies of integrated DNA. We also illustrate a method for efficient lentiviral transduction of hard-to-transduce un-activated primary human CD4(+) T cells. These protocols will significantly assist in understanding the activation and function of human T cells and will ultimately aid in the development or improvement of current drugs that target human CD4(+) T cells.

  20. 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

  1. 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

  2. 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

  3. Neuron-derived orphan receptor 1 transduces survival signals in neuronal cells in response to hypoxia-induced apoptotic insults.

    PubMed

    Chio, Chung-Ching; Wei, Li; Chen, Tyng Guey; Lin, Chien-Min; Shieh, Ja-Ping; Yeh, Poh-Shiow; Chen, Ruei-Ming

    2016-06-01

    OBJECT Hypoxia can induce cell death or trigger adaptive mechanisms to guarantee cell survival. Neuron-derived orphan receptor 1 (NOR-1) works as an early-response protein in response to a variety of environmental stresses. In this study, the authors evaluated the roles of NOR-1 in hypoxia-induced neuronal insults. METHODS Neuro-2a cells were exposed to oxygen/glucose deprivation (OGD). Cell viability, cell morphology, cas-pase-3 activity, DNA fragmentation, and cell apoptosis were assayed to determine the mechanisms of OGD-induced neuronal insults. RNA and protein analyses were carried out to evaluate the effects of OGD on expressions of NOR-1, cAMP response element-binding (CREB), and cellular inhibitor of apoptosis protein 2 (cIAP2) genes. Translations of these gene expressions were knocked down using RNA interference. Mice subjected to traumatic brain injury (TBI) and NOR-1 was immunodetected. RESULTS Exposure of neuro-2a cells to OGD decreased cell viability in a time-dependent manner. Additionally, OGD led to cell shrinkage, DNA fragmentation, and cell apoptosis. In parallel, treatment of neuro-2a cells with OGD time dependently increased cellular NOR-1 mRNA and protein expressions. Interestingly, administration of TBI also augmented NOR-1 levels in the impacted regions of mice. As to the mechanism, exposure to OGD increased nuclear levels of the transcription factor CREB protein. Downregulating CREB expression using RNA interference simultaneously inhibited OGD-induced NOR-1 mRNA expression. Also, levels of cIAP2 mRNA and protein in neuro-2a cells were augmented by OGD. After reducing cIAP2 translation, OGD-induced cell death was reduced. Sequentially, application of NOR-1 small interfering RNA to neuro-2a cells significantly inhibited OGD-induced cIAP2 mRNA expression and concurrently alleviated hypoxia-induced alterations in cell viability, caspase-3 activation, DNA damage, and cell apoptosis. CONCLUSIONS This study shows that NOR-1 can transduce survival

  4. A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

    PubMed Central

    Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

    2015-01-01

    Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. Adoptive T-cell Therapy Using Autologous Tumor-infiltrating Lymphocytes for Metastatic Melanoma: Current Status and Future Outlook

    PubMed Central

    Wu, Richard; Forget, Marie-Andree; Chacon, Jessica; Bernatchez, Chantale; Haymaker, Cara; Chen, Jie Qing; Hwu, Patrick; Radvanyi, Laszlo

    2012-01-01

    Immunotherapy using autologous T-cells has emerged to be a powerful treatment option for patients with metastatic melanoma. These include the adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL), T-cells transduced with high-affinity T-cell receptors (TCR) against major melanosomal tumor antigens, and T cells transduced with chimeric antigen receptors (CAR) composed of hybrid immunoglobulin light chains with endo-domains of T-cell signaling molecules. Among these and other options for T-cell therapy, TIL together with high-dose IL-2 has had the longest clinical history with multiple clinical trials in centers across the world consistently demonstrating durable clinical response rates near 50% or more. A distinct advantage of TIL therapy making it still the T-cell therapy of choice is the broad nature of the T-cell recognition against both defined as well as un-defined tumors antigens against all possible MHC, rather than the single specificity and limited MHC coverage of the newer TCR and CAR transduction technologies. In the past decade, significant inroads have been made in defining the phenotypes of T cells in TIL mediating tumor regression. CD8+ T cells are emerging to be critical, although the exact subset of CD8+ T cells exhibiting the highest clinical activity in terms of memory and effector markers is still controversial. We present a model in which both effector-memory and more differentiated effector T cells ultimately may need to cooperate to mediate long-term tumor control in responding patients. Although TIL therapy has shown great potential to treat metastatic melanoma, a number of issues have emerged that need to be addressed to bring it more into the mainstream of melanoma care. First, we have a reached the point where a pivotal phase II or phase III trials are needed in an attempt to gain regulatory approval of TIL as standard-of-care. Second, improvements in how we expand TIL for therapy are needed, that minimize the time the T-cells

  12. 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.

  13. 17beta-estradiol promotes breast cancer cell proliferation-inducing stromal cell-derived factor-1-mediated epidermal growth factor receptor transactivation: reversal by gefitinib pretreatment.

    PubMed

    Pattarozzi, Alessandra; Gatti, Monica; Barbieri, Federica; Würth, Roberto; Porcile, Carola; Lunardi, Gianluigi; Ratto, Alessandra; Favoni, Roberto; Bajetto, Adriana; Ferrari, Angelo; Florio, Tullio

    2008-01-01

    The coordinated activity of estrogens and epidermal growth factor receptor (EGFR) family agonists represents the main determinant of breast cancer cell proliferation. Stromal cell-derived factor-1 (SDF-1) enhances extracellular signal-regulated kinases 1 and 2 (ERK1/2) activity via the transactivation of EGFR and 17beta-estradiol (E2) induces SDF-1 production to exert autocrine proliferative effects. On this basis, we evaluated whether the inhibition of the tyrosine kinase (TK) activity of EGFR may control different mitogenic stimuli in breast tumors using the EGFR-TK inhibitor gefitinib to antagonize the proliferation induced by E2 in T47D human breast cancer cells. EGF, E2, and SDF-1 induced a dose-dependent T47D cell proliferation, that being nonadditive suggested the activation of common intracellular pathways. Gefitinib treatment inhibited not only the EGF-dependent proliferation and ERK1/2 activation but also the effects of SDF-1 and E2, suggesting that these activities were mediated by EGFR transactivation. Indeed, both SDF-1 and E2 caused EGFR tyrosine phosphorylation. The molecular link between E2 and SDF-1 proliferative effects was identified because 1,1'-(1,4-phenylenebis(methylene))-bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist, inhibited SDF-1- and E2-dependent proliferation and EGFR and ERK1/2 phosphorylation. EGFR transactivation was dependent on c-Src activation. E2 treatment caused a powerful SDF-1 release from T47D cells. Finally, in SKBR3, E2-resistant cells, EGFR was constitutively activated, and AMD3100 reduced EGFR phosphorylation and cell proliferation, whereas HER2-neu was transactivated by SDF-1 in SKBR3 but not in T47D cells. In conclusion, we show that activation of CXCR4 transduces proliferative signals from the E2 receptor to EGFR, whose inhibition is able to revert breast cancer cell proliferation induced by multiple receptor activation.

  14. 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.

  15. 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.

  16. 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.

  17. Pasteurella multocida Toxin Manipulates T Cell Differentiation

    PubMed Central

    Hildebrand, Dagmar; Heeg, Klaus; Kubatzky, Katharina F.

    2015-01-01

    Pasteurella multocida causes various diseases in a broad range of wild and domestic animals. Toxigenic strains of the serotypes A and D produce an AB protein toxin named Pasteurella multocida toxin (PMT). PMT constitutively activates the heterotrimeric G protein subunits Gαq, Gα13, and Gαi through deamidation of a glutamine residue, which results in cytoskeletal rearrangements as well as increased proliferation and survival of the host cell. In human monocytes, PMT alters the lipopolysaccharide (LPS)-induced activation toward a phenotype that suppresses T cell activation. Here we describe that the toxin also modulates CD4-positive T helper (Th) cells directly. PMT amplifies the expansion of Th cells through enhanced cell cycle progression and suppression of apoptosis and manipulates the differentiation of Th subclasses through activation of Signal Transducers and Activators of Transcription (STAT) family members and induction of subtype-specific master transcription factors. A large population of toxin-treated T cells is double-positive for Foxp3 and RORγt, the transcription factors expressed by Treg and Th17 cells, respectively. This suggests that these cells could have the potential to turn into Th17 cells or suppressive Treg cells. However, in terms of function, the PMT-differentiated cells behave as inflammatory Th17 cells that produce IL-17 and trigger T cell proliferation. PMID:26635744

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

    PubMed

    Hombach, Andreas A; Abken, Hinrich

    2017-02-01

    Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30 + hematopoietic stem and progenitor cells while eliminating CD30 + lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-β and CD123, is of risk to destruct tissue stem cells.

  4. Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

    PubMed

    Hege, Kristen M; Bergsland, Emily K; Fisher, George A; Nemunaitis, John J; Warren, Robert S; McArthur, James G; Lin, Andy A; Schlom, Jeffrey; June, Carl H; Sherwin, Stephen A

    2017-01-01

    T cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s. Patients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 10 10 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72. Fourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111 Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72

  5. 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.

  6. 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.

  7. 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

  8. An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells

    PubMed Central

    Shimizu, Nobuaki; Soda, Yasushi; Kanbe, Katsuaki; Liu, Hui-Yu; Jinno, Atsushi; Kitamura, Toshio; Hoshino, Hiroo

    1999-01-01

    Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro. PMID:10233994

  9. Identification of novel gammadelta T-cell subsets following bacterial infection in the absence of Vgamma1+ T cells: homeostatic control of gammadelta T-cell responses to pathogen infection by Vgamma1+ T cells.

    PubMed

    Newton, Darren J; Andrew, Elizabeth M; Dalton, Jane E; Mears, Rainy; Carding, Simon R

    2006-02-01

    Although gammadelta T cells are a common feature of many pathogen-induced immune responses, the factors that influence, promote, or regulate the response of individual gammadelta T-cell subsets to infection is unknown. Here we show that in the absence of Vgamma1+ T cells, novel subsets of gammadelta T cells, expressing T-cell receptor (TCR)-Vgamma chains that normally define TCRgammadelta+ dendritic epidermal T cells (DETCs) (Vgamma5+), intestinal intraepithelial lymphocytes (iIELs) (Vgamma7+), and lymphocytes associated with the vaginal epithelia (Vgamma6+), are recruited to the spleen in response to bacterial infection in TCR-Vgamma1-/- mice. By comparison of phenotype and structure of TCR-Vgamma chains and/or -Vdelta chains expressed by these novel subsets with those of their epithelium-associated counterparts, the Vgamma6+ T cells elicited in infected Vgamma1-/- mice were shown to be identical to those found in the reproductive tract, from where they are presumably recruited in the absence of Vgamma1+ T cells. By contrast, Vgamma5+ and Vgamma7+ T cells found in infected Vgamma1-/- mice were distinct from Vgamma5+ DETCs and Vgamma7+ iIELs. Functional analyses of the novel gammadelta T-cell subsets identified for infected Vgamma1-/- mice showed that whereas the Vgamma5+ and Vgamma7+ subsets may compensate for the absence of Vgamma1+ T cells by producing similar cytokines, they do not possess cytocidal activity and they cannot replace the macrophage homeostasis function of Vgamma1+ T cells. Collectively, these findings identify novel subsets of gammadelta T cells, the recruitment and activity of which is under the control of Vgamma1+ T cells.

  10. Transcriptional network profile on synovial fluid T cells in psoriatic arthritis.

    PubMed

    Fiocco, Ugo; Martini, Veronica; Accordi, Benedetta; Caso, Francesco; Costa, Luisa; Oliviero, Francesca; Scanu, Anna; Facco, Monica; Boso, Daniele; Gatto, Mariele; Felicetti, Mara; Frallonardo, Paola; Ramonda, Roberta; Piva, Lucia; Zambello, Renato; Agostini, Carlo; Scarpa, Raffaele; Basso, Giuseppe; Semenzato, Gianpietro; Dayer, Jean-Michel; Punzi, Leonardo; Doria, Andrea

    2015-09-01

    The objective of the study was to quantify the transcriptional profile, as the main T cell lineage-transcription factors on synovial fluid (SF) T cells, in relation to SF cytokines and T cell frequencies (%) of psoriatic arthritis (PsA) patients. Reverse phase protein array was employed to identify interleukin (IL)-23Rp19-, FOXP3- and related orphan receptor gamma T (RORγt)- protein and Janus associated tyrosine kinases 1 (JAK1), signal transducer and activator and transcription 1 (STAT1), STAT3 and STAT5 phosphoproteins in total T cell lysates from SF of PsA patients. IL-1β, IL-2, IL-6, IL-21 and interferon (INF)-γ were measured using a multiplex bead immunoassay in SF from PsA patients and peripheral blood (PB) from healthy controls (HC). Frequencies of CD4(+)CD25(-), CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg, and either mean fluorescence intensity (MFI) of FOXP3(+) on CD4(+) Treg or MFI of classic IL-6 receptor (IL-6R) α expression on CD4(+)CD25(-) helper/effector T cells (Th/eff) and Treg cells, were quantified in SF of PsA patients and in PB from HC by flow cytometry (FC). In PsA SF samples, IL-2, IL-21 and IFN-γ were not detectable, whereas IL-6 and IL-1β levels were higher than in SF of non-inflammatory osteoarthritis patients. Higher levels of IL-23R-, FOXP3- and RORγt proteins and JAK1, STAT1, STAT3 and STAT5 were found in total T cells from SF of PsA patients compared with PB from HC. Direct correlations between JAK1 Y1022/Y1023 and STAT5 Y694, and STAT3 Y705 and IL6, were found in SF of PsA patients. Increased proportion of CD4(+)CD25(high) FOXP3(+) and CD4(+)CD25(high) CD127(low) Treg cells and brighter MFI of IL-6Rα were observed both on CD4(+)CD25(high)- and CD4(+)CD25(-) T cells in PsA SF. The study showed a distinctive JAK1/STAT3/STAT5 transcriptional network on T cells in the joint microenvironment, outlining the interplay of IL-6, IL-23, IL-1β and γC cytokines in the polarization and plasticity of Th17 and Treg cells

  11. 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.

  12. BJ-3105, a 6-Alkoxypyridin-3-ol Analog, Impairs T Cell Differentiation and Prevents Experimental Autoimmune Encephalomyelitis Disease Progression

    PubMed Central

    Timilshina, Maheshwor; Kang, Youra; Dahal, Ishmit; You, Zhiwei; Nam, Tae-gyu; Kim, Keuk-Jun

    2017-01-01

    CD4+ T cells are essential in inflammation and autoimmune diseases. Interferon-γ (IFN-γ) secreting T helper (Th1) and IL-17 secreting T helper (Th17) cells are critical for several autoimmune diseases. To assess the inhibitory effect of a given compound on autoimmune disease, we screened many compounds with an in vitro Th differentiation assay. BJ-3105, a 6-alkoxypyridin-3-ol analog, inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells which were activated by T cell receptor (TCR) engagement. BJ-3105 ameliorated the experimental autoimmune encephalomyelitis (EAE) model by reducing Th1 and Th17 generation. Notably, Th cell differentiation was significantly suppressed by BJ-3105 treatment without inhibiting in vitro proliferation of T cells or inducing programmed cell death. Mechanistically, BJ-3105 inhibited the phosphorylation of JAK and its downstream signal transducer and activator of transcription (STAT) that is critical for Th differentiation. These results demonstrated that BJ-3105 inhibits the phosphorylation of STAT in response to cytokine signals and subsequently suppressed the differentiation of Th cell responses. PMID:28095433

  13. 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.

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

    PubMed

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

    2016-10-15

    Pre-emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease. CIK cells are a heterogeneous effector cell population including T cells (CD3(+) CD56(-) ), natural killer (NK) cells (CD3(-) CD56(+) ) and natural killer T (T-NK) cells (CD3(+) CD56(+) ) that exhibit non-major histocompatibility complex (MHC)-restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)-γ, anti-CD3 antibody, interleukin-2 (IL-2) and interleukin-15 (IL-15). To facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19-targeted CIK/63.28.z cells against otherwise CIK-resistant cancer cell lines and primary B-ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre-B-ALL. Our results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL. © 2016 UICC.

  15. 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.

  16. B cells as accessory cells in a Con A response of a T cell clone.

    PubMed

    Takeuchi, M; Kakiuchi, T; Taira, S; Nariuchi, H

    1987-12-01

    Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. Fish T cells: recent advances through genomics

    USGS Publications Warehouse

    Laing, Kerry J.; Hansen, John D.

    2011-01-01

    This brief review is intended to provide a concise overview of the current literature concerning T cells, advances in identifying distinct T cell functional subsets, and in distinguishing effector cells from memory cells. We compare and contrast a wealth of recent progress made in T cell immunology of teleost, elasmobranch, and agnathan fish, to knowledge derived from mammalian T cell studies. From genome studies, fish clearly have most components associated with T cell function and we can speculate on the presence of putative T cell subsets, and the ability to detect their differentiation to form memory cells. Some recombinant proteins for T cell associated cytokines and antibodies for T cell surface receptors have been generated that will facilitate studying the functional roles of teleost T cells during immune responses. Although there is still a long way to go, major advances have occurred in recent years for investigating T cell responses, thus phenotypic and functional characterization is on the near horizon.

  7. Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells.

    PubMed

    Pérez-Cota, Fernando; Smith, Richard J; Moradi, Emilia; Marques, Leonel; Webb, Kevin F; Clark, Matt

    2015-10-01

    At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

  8. 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/.

  9. Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

    PubMed

    Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

    2017-10-01

    Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

  10. Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

    PubMed Central

    Powell, Daniel J.; Dudley, Mark E.; Robbins, Paul F.; Rosenberg, Steven A.

    2007-01-01

    In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen–specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA− CD62 ligand− (CD62L−) CC chemokine receptor 7− (CCR7−) interleukin-7 receptor αLo (IL-7RαLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Rα in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen–specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L− CCR7− profile, which may explain in part their ability to mediate tumor destruction. PMID:15345595

  11. 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

  12. CAR-T cells and allogeneic hematopoietic stem cell transplantation for relapsed/refractory B-cell acute lymphoblastic leukemia.

    PubMed

    Liu, Jun; Zhang, Xi; Zhong, Jiang F; Zhang, Cheng

    2017-10-01

    Relapsed/refractory acute lymphoblastic leukemia (ALL) has a low remission rate after chemotherapy, a high relapse rate and poor long-term survival even when allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed. Chimeric antigen receptors redirected T cells (CAR-T cells) can enhance disease remission with a favorable outcome for relapsed/refractory ALL, though some cases quickly relapsed after CAR-T cell treatment. Thus, treatment with CAR-T cells followed by allo-HSCT may be the best way to treat relapsed/refractory ALL. In this review, we first discuss the different types of CAR-T cells. We then discuss the treatment of relapsed/refractory ALL using only CAR-T cells. Finally, we discuss the use of CAR-T cells, followed by allo-HSCT, for the treatment of relapsed/refractory ALL.

  13. 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

  14. The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

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

    Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se; Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se; Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se

    2011-07-08

    Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects aremore » however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.« less

  15. 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

  16. 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.

  17. 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

  18. Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

    PubMed

    Gerriets, Valerie A; Danzaki, Keiko; Kishton, Rigel J; Eisner, William; Nichols, Amanda G; Saucillo, Donte C; Shinohara, Mari L; MacIver, Nancie J

    2016-08-01

    Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, 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-cell 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 cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

    PubMed

    Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

    2017-09-01

    Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

  20. [Regulatory T cells].

    PubMed

    Marinić, Igor; Gagro, Alenka; Rabatić, Sabina

    2006-12-01

    Regulatory T-cells are a subset of T cells that have beene extensively studied in modern immunology. They are important for the maintenance of peripheral tolerance, and have an important role in various clinical conditions such as allergy, autoimmune disorders, tumors, infections, and in transplant medicine. Basically, this population has a suppressive effect on the neighboring immune cells, thus contributing to the local modulation and control of immune response. There are two main populations of regulatory T cells - natural regulatory T cells, which form a distinct cellular lineage, develop in thymus and perform their modulatory action through direct intercellular contact, along with the secreted cytokines; and inducible regulatory T cells, which develop in the periphery after contact with the antigen that is presented on the antigen presenting cell, and their primary mode of action is through the interleukin 10 (IL-10) and transforming growth factor beta (TGF-alpha) cytokines. Natural regulatory T cells are activated through T cell receptor after contact with specific antigen and inhibit proliferation of other T cells in an antigen independent manner. One of the major difficulties in the research of regulatory T cells is the lack of specific molecular markers that would identify these cells. Natural regulatory T cells constitutively express surface molecule CD25, but many other surface and intracellular molecules (HLA-DR, CD122, CD45RO, CD62, CTLA-4, GITR, PD-1, Notch, FOXP3, etc.) are being investigated for further phenotypic characterization of these cells. Because regulatory T cells have an important role in establishing peripheral tolerance, their importance is manifested in a number of clinical conditions. In the IPEX syndrome (immunodysregulation, polyendocrinopathy and enteropathy, X-linked), which is caused by mutation in Foxp3 gene that influences the development and function of regulatory T cells, patients develop severe autoimmune reactions that

  1. Termination of T cell priming relies on a phase of unresponsiveness promoting disengagement from APCs and T cell division.

    PubMed

    Bohineust, Armelle; Garcia, Zacarias; Beuneu, Hélène; Lemaître, Fabrice; Bousso, Philippe

    2018-05-07

    T cells are primed in secondary lymphoid organs by establishing stable interactions with antigen-presenting cells (APCs). However, the cellular mechanisms underlying the termination of T cell priming and the initiation of clonal expansion remain largely unknown. Using intravital imaging, we observed that T cells typically divide without being associated to APCs. Supporting these findings, we demonstrate that recently activated T cells have an intrinsic defect in establishing stable contacts with APCs, a feature that was reflected by a blunted capacity to stop upon T cell receptor (TCR) engagement. T cell unresponsiveness was caused, in part, by a general block in extracellular calcium entry. Forcing TCR signals in activated T cells antagonized cell division, suggesting that T cell hyporesponsiveness acts as a safeguard mechanism against signals detrimental to mitosis. We propose that transient unresponsiveness represents an essential phase of T cell priming that promotes T cell disengagement from APCs and favors effective clonal expansion. © 2018 Bohineust et al.

  2. 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.

  3. T cell ignorance is bliss: T cells are not tolerized by Langerhans cells presenting human papillomavirus antigens in the absence of costimulation.

    PubMed

    Woodham, Andrew W; Yan, Lisa; Skeate, Joseph G; van der Veen, Daniel; Brand, Heike H; Wong, Michael K; Da Silva, Diane M; Kast, W Martin

    2016-12-01

    Human papillomavirus type 16 (HPV16) infections are intra-epithelial, and thus, HPV16 is known to interact with Langerhans cells (LCs), the resident epithelial antigen-presenting cells (APCs). The current paradigm for APC-mediated induction of T cell anergy is through delivery of T cell receptor signals via peptides on MHC molecules (signal 1), but without costimulation (signal 2). We previously demonstrated that LCs exposed to HPV16 in vitro present HPV antigens to T cells without costimulation, but it remained uncertain if such T cells would remain ignorant, become anergic, or in the case of CD4+ T cells, differentiate into Tregs. Here we demonstrate that Tregs were not induced by LCs presenting only signal 1, and through a series of in vitro immunizations show that CD8 + T cells receiving signal 1 + 2 from LCs weeks after consistently receiving signal 1 are capable of robust effector functions. Importantly, this indicates that T cells are not tolerized but instead remain ignorant to HPV, and are activated given the proper signals.

  4. 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.

  5. Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy

    PubMed Central

    Chen, Can; Tan, Wee-Kiat; Chi, Zhixia; Xu, Xue-Hu; Wang, Shu

    2016-01-01

    Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer. PMID:27598655

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. The Transcription Factor STAT3 and Type I Interferons Are Mutually Repressive Insulators for Differentiation of Follicular Helper and T Helper_1 Cells

    PubMed Central

    Ray, John P.; Marshall, Heather D.; Laidlaw, Brian J.; Staron, Matthew M.; Kaech, Susan M.; Craft, Joe

    2014-01-01

    Summary Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4+ T cells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of interferoninducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper-1 (Th1) cell-like effector phenotype. Conversely, IFNαβ receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4+ T cells. These data suggest mutually repressive roles for STAT3 and type I IFN signaling pathways in the differentiation of Tfh cells following viral infection. PMID:24631156

  14. Gammadelta receptor bearing T cells in scleroderma: enhanced interaction with vascular endothelial cells in vitro.

    PubMed

    Kahaleh, M B; Fan, P S; Otsuka, T

    1999-05-01

    In view of the documented perivascular mononuclear cell infiltration in the involved organs in scleroderma (SSc) and the reported accumulation of gammadelta-T cells in SSc skin and lung, we evaluated gammadelta-T cell interaction with endothelial cells (EC) in vitro. gammadelta- and alphabeta-T cells were isolated from BPMN of SSc patients with early diffuse disease and of matched control subjects by an immunomagnetic method after stimulation with mycobacterium lysate and interleukin-2 for 2 weeks. Lymphocyte adhesion, proliferation, and cytotoxicity to EC were investigated. SSc gammadelta-T cells adhered to cultured EC and proliferated at higher rates than control cells. Furthermore, significant EC cytotoxicity by SSc gammadelta was seen. The cytotoxicity was blocked by addition of anti-gammadelta-TCR antibody and by anti-granzyme A antibody but not by anti-MHC class I and II antibodies. Expression of granzyme A mRNA was seen in five/five SSc gammadelta-T cells and in one/five control cells. alphabeta-T cells from both SSc and control subjects were significantly less interactive with EC than gammadelta-T cells. The data demonstrate EC recognition by SSc gammadelta-T cells and propose gammadelta-T cells as a possible effector cell type in the immune pathogenesis of SSc. Copyright 1999 Academic Press.

  15. Perspectives on Regulatory T Cell Therapies.

    PubMed

    Probst-Kepper, Michael; Kröger, Andrea; Garritsen, Henk S P; Buer, Jan

    2009-01-01

    Adoptive transfer in animal models clearly indicate an essential role of CD4+ CD25+ FOXP3+ regulatory T (T(reg)) cells in prevention and treatment of autoimmune and graft-versus-host disease. Thus, T(reg) cell therapies and development of drugs that specifically enhance T(reg) cell function and development represent promising tools to establish dominant tolerance. So far, lack of specific markers to differentiate human T(reg) cells from activated CD4+ CD25+ effector T cells, which also express FOXP3 at different levels, hampered such an approach. Recent identification of the orphan receptor glycoprotein-A repetitions predominant (GARP or LRRC32) as T(reg) cell-specific key molecule that dominantly controls FOXP3 via a positive feedback loop opens up new perspectives for molecular and cellular therapies. This brief review focuses on the role of GARP as a safeguard of a complex regulatory network of human T(reg) cells and its implications for regulatory T cell therapies in autoimmunity and graft-versus-host disease.

  16. Immunosuppression after Sepsis: Systemic Inflammation and Sepsis Induce a Loss of Naïve T-Cells but No Enduring Cell-Autonomous Defects in T-Cell Function

    PubMed Central

    Markwart, Robby; Condotta, Stephanie A.; Requardt, Robert P.; Borken, Farina; Schubert, Katja; Weigel, Cynthia; Bauer, Michael; Griffith, Thomas S.; Förster, Martin; Brunkhorst, Frank M.; Badovinac, Vladimir P.; Rubio, Ignacio

    2014-01-01

    Sepsis describes the life-threatening systemic inflammatory response (SIRS) of an organism to an infection and is the leading cause of mortality on intensive care units (ICU) worldwide. An acute episode of sepsis is characterized by the extensive release of cytokines and other mediators resulting in a dysregulated immune response leading to organ damage and/or death. This initial pro-inflammatory burst often transits into a state of immune suppression characterised by loss of immune cells and T-cell dysfunction at later disease stages in sepsis survivors. However, despite these appreciations, the precise nature of the evoked defect in T-cell immunity in post-acute phases of SIRS remains unknown. Here we present an in-depth functional analysis of T-cell function in post-acute SIRS/sepsis. We document that T-cell function is not compromised on a per cell basis in experimental rodent models of infection-free SIRS (LPS or CpG) or septic peritonitis. Transgenic antigen-specific T-cells feature an unaltered cytokine response if challenged in vivo and ex vivo with cognate antigens. Isolated CD4+/CD8+ T-cells from post-acute septic animals do not exhibit defects in T-cell receptor-mediated activation at the the level of receptor-proximal signalling, activation marker upregulation or expansion. However, SIRS/sepsis induced transient lymphopenia and gave rise to an environment of immune attenuation at post acute disease stages. Thus, systemic inflammation has an acute impact on T-cell numbers and adaptive immunity, but does not cause major cell-autonomous enduring functional defects in T-cells. PMID:25541945

  17. 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

  18. 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

  19. 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.

  20. Cord-Blood-Derived Mesenchymal Stromal Cells Downmodulate CD4+ T-Cell Activation by Inducing IL-10-Producing Th1 Cells

    PubMed Central

    Selleri, Silvia; Dieng, Mame Massar; Nicoletti, Simon; Louis, Isabelle; Beausejour, Christian; Le Deist, Françoise

    2013-01-01

    The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response. PMID:23167734

  1. 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

  2. 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.

  3. A Cell-Permeable Phospholipase C[gamma]1-Binding Peptide Transduces Neurons and Impairs Long-Term Spatial Memory

    ERIC Educational Resources Information Center

    Blum, Sonja; Dash, Pramod K.

    2004-01-01

    Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLC[gamma]1) binding, cell-penetrating peptide to sequester PLC[gamma]1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons…

  4. Modification of Hematopoietic Stem/Progenitor Cells with CD19-Specific Chimeric Antigen Receptors as a Novel Approach for Cancer Immunotherapy

    PubMed Central

    Ryan, Christine; Giannoni, Francesca; Hardee, Cinnamon L.; Tremcinska, Irena; Katebian, Behrod; Wherley, Jennifer; Sahaghian, Arineh; Tu, Andy; Grogan, Tristan; Elashoff, David; Cooper, Laurence J.N.; Hollis, Roger P.; Kohn, Donald B.

    2013-01-01

    Abstract Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1–2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity. PMID:23978226

  5. 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

  6. 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

  7. Calcium signaling properties of a thyrotroph cell line, mouse TαT1 cells.

    PubMed

    Tomić, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    T1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in TαT1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. TαT1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that TαT1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. Published by Elsevier Ltd.

  8. 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.

  9. 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

  10. Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

    PubMed Central

    Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J.; Rivière, Isabelle

    2009-01-01

    Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor. PMID:19238016

  11. 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

  12. 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.

  13. Origin and fate of lymphocytic choriomeningitis virus-specific CD8+ T cells coexpressing the inhibitory NK cell receptor Ly49G2.

    PubMed

    Peacock, Craig D; Welsh, Raymond M

    2004-07-01

    CD8+ T cells that coexpress the inhibitory NK cell receptor, Ly49G2 (G2), are present in immunologically naive C57BL/6 mice but display Ags found on memory T cells. To assess how G2+CD8+ cells relate to bona fide memory cells, we examined the origin and fate of lymphocytic choriomeningitis virus (LCMV)-induced G2+CD8+ cells. During early (day 4) acute LCMV infection, both G2+ and G2-CD8+ T cell subsets underwent an attrition in number and displayed an activation (CD69(high)1B11(high)CD62L(low)) phenotype. By day 8, both subsets synthesized IFN-gamma in response to immunodominant LCMV peptides, though the expansion of G2+ cells was less than that of G2- cells. Adoptive transfer experiments with purified G2- or G2+CD8+ cells from naive mice indicated that the LCMV-specific G2+ subset was derived from a pre-existing G2+ population and not generated from G2- cells responding to LCMV infection. Their participation in the LCMV-specific T cell response increased with age, reflecting an increase in the size of the pre-existing G2+ pool. Following establishment of stable LCMV memory, the proportion of CD8+ cells coexpressing G2 was reduced in comparison to naive controls, presumably due to displacement by G2- LCMV-specific memory cells. LCMV-specific G2+ cells were present in the memory pool, but at low frequencies, and they did not exhibit the typical phenotypic changes of reactivation during secondary challenge. We suggest that G2+CD8+ cells represent a cell lineage distinct from bona fide memory T cells, but that they can participate in an acute virus-specific T cell response.

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

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

    Ryu, Min Sook; Woo, Min-Yeong; Department of Biomedical Sciences, The Graduate School, Ajou University

    2014-10-01

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

  15. 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.

  16. 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.

  17. Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells

    PubMed Central

    Wang, Jueqiong; Lu, Liu; Kok, Chung H.; Saunders, Verity A.; Goyne, Jarrad M.; Dang, Phuong; Leclercq, Tamara M.; Hughes, Timothy P.; White, Deborah L.

    2017-01-01

    Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in

  18. Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19 Chimeric Antigen Receptor

    PubMed Central

    Kochenderfer, James N.; Dudley, Mark E.; Kassim, Sadik H.; Somerville, Robert P.T.; Carpenter, Robert O.; Stetler-Stevenson, Maryalice; Yang, James C.; Phan, Giao Q.; Hughes, Marybeth S.; Sherry, Richard M.; Raffeld, Mark; Feldman, Steven; Lu, Lily; Li, Yong F.; Ngo, Lien T.; Goy, Andre; Feldman, Tatyana; Spaner, David E.; Wang, Michael L.; Chen, Clara C.; Kranick, Sarah M.; Nath, Avindra; Nathan, Debbie-Ann N.; Morton, Kathleen E.; Toomey, Mary Ann; Rosenberg, Steven A.

    2015-01-01

    Purpose T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19+ B-cell malignancies. Patients and Methods We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells. Results Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/μL. Conclusion This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach. PMID:25154820

  19. 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

  20. Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity.

    PubMed

    Shanker, Anil; Pellom, Samuel T; Dudimah, Duafalia F; Thounaojam, Menaka C; de Kluyver, Rachel L; Brooks, Alan D; Yagita, Hideo; McVicar, Daniel W; Murphy, William J; Longo, Dan L; Sayers, Thomas J

    2015-12-15

    Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy. ©2015 American Association for Cancer Research.