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    T-cells "à la CAR-T(e)" - Genetically engineering T-cell response against cancer. Eisenberg Vasyl,Hoogi Shiran,Shamul Astar,Barliya Tilda,Cohen Cyrille J Advanced drug delivery reviews The last decade will be remembered as the dawn of the immunotherapy era during which we have witnessed the approval by regulatory agencies of genetically engineered CAR T-cells and of checkpoint inhibitors for cancer treatment. Understandably, T-lymphocytes represent the essential player in these approaches. These cells can mediate impressive tumor regression in terminally-ill cancer patients. Moreover, they are amenable to genetic engineering to improve their function and specificity. In the present review, we will give an overview of the most recent developments in the field of T-cell genetic engineering including TCR-gene transfer and CAR T-cells strategies. We will also elaborate on the development of other types of genetic modifications to enhance their anti-tumor immune response such as the use of co-stimulatory chimeric receptors (CCRs) and unconventional CARs built on non-antibody molecules. Finally, we will discuss recent advances in genome editing and synthetic biology applied to T-cell engineering and comment on the next challenges ahead. 10.1016/j.addr.2019.01.007
    TCR gene therapy for HPV-associated epithelial cancers. Burki Talha Khan The Lancet. Oncology 10.1016/S1470-2045(19)30541-8
    TCR Gene Therapy Improves AML Prognosis. Cancer discovery T cells engineered to express a receptor specific for Wilms tumor antigen 1 helped prevent relapse in a small trial of patients with acute myeloid leukemia who had received an allogeneic stem-cell transplant. 10.1158/2159-8290.CD-NB2019-083
    Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Baeuerle Patrick A,Ding Jian,Patel Ekta,Thorausch Niko,Horton Holly,Gierut Jessica,Scarfo Irene,Choudhary Rashmi,Kiner Olga,Krishnamurthy Janani,Le Bonnie,Morath Anna,Baldeviano G Christian,Quinn Justin,Tavares Patrick,Wei Qi,Weiler Solly,Maus Marcela V,Getts Daniel,Schamel Wolfgang W,Hofmeister Robert Nature communications T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner. 10.1038/s41467-019-10097-0
    B7-H7 (HHLA2) inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling. Rieder Sadiye Amcaoglu,Wang Jingya,White Natalie,Qadri Ariful,Menard Catherine,Stephens Geoffrey,Karnell Jodi L,Rudd Christopher E,Kolbeck Roland Cellular & molecular immunology Modulation of T-cell responses has played a key role in treating cancers and autoimmune diseases. Therefore, understanding how different receptors on T cells impact functional outcomes is crucial. The influence of B7-H7 (HHLA2) and CD28H (TMIGD2) on T-cell activation remains controversial. Here we examined global transcriptomic changes in human T cells induced by B7-H7. Stimulation through TCR with OKT3 and B7-H7 resulted in modest fold changes in the expression of select genes; however, these fold changes were significantly lower than those induced by OKT3 and B7-1 stimulation. The transcriptional changes induced by OKT3 and B7-H7 were insufficient to provide functional stimulation as measured by evaluating T-cell proliferation and cytokine production. Interestingly, B7-H7 was coinhibitory when simultaneously combined with TCR and CD28 stimulation. This inhibitory activity was comparable to that observed with PD-L1. Finally, in physiological assays using T cells and APCs, blockade of B7-H7 enhanced T-cell activation and proliferation, demonstrating that this ligand acts as a break signal. Our work defines that the transcriptomic changes induced by B7-H7 are insufficient to support full costimulation with TCR signaling and, instead, B7-H7 inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling. 10.1038/s41423-020-0361-7
    Harnessing innate immunity in cancer therapy. Demaria Olivier,Cornen Stéphanie,Daëron Marc,Morel Yannis,Medzhitov Ruslan,Vivier Eric Nature New therapies that promote antitumour immunity have been recently developed. Most of these immunomodulatory approaches have focused on enhancing T-cell responses, either by targeting inhibitory pathways with immune checkpoint inhibitors, or by targeting activating pathways, as with chimeric antigen receptor T cells or bispecific antibodies. Although these therapies have led to unprecedented successes, only a minority of patients with cancer benefit from these treatments, highlighting the need to identify new cells and molecules that could be exploited in the next generation of immunotherapy. Given the crucial role of innate immune responses in immunity, harnessing these responses opens up new possibilities for long-lasting, multilayered tumour control. 10.1038/s41586-019-1593-5
    Precursor exhausted T cells: key to successful immunotherapy? Kallies Axel,Zehn Dietmar,Utzschneider Daniel T Nature reviews. Immunology Cytotoxic T cell immunity in response to chronic infections and tumours is maintained by a specialized population of CD8 T cells that exhibit hallmarks of both exhausted and memory cells and give rise to terminally differentiated exhausted effector cells that contribute to viral or tumour control. Importantly, recent work suggests these cells, which we refer to as 'precursor exhausted' T (T) cells, are responsible for the proliferative burst that generates effector T cells in response to immune checkpoint blockade targeting programmed cell death 1 (PD1), and increased T cell frequencies have recently been linked to increased patient survival. We believe the recent discovery of T cells not only represents a paradigm shift in our understanding of the mechanisms that maintain CD8 T cell responses in chronic infections and tumours but also opens up unexpected avenues for the development of new and innovative therapeutic approaches. In this Opinion article, we discuss the differentiation and function of T cells and suggest that targeting these cells may be key for successful immunotherapy. 10.1038/s41577-019-0223-7
    Combination Immunotherapy with CAR T Cells and Checkpoint Blockade for the Treatment of Solid Tumors. Grosser Rachel,Cherkassky Leonid,Chintala Navin,Adusumilli Prasad S Cancer cell Checkpoint blockade (CPB) therapy can elicit durable clinical responses by reactivating an exhausted immune response. However, response rates remain limited, likely secondary to a lack of a tumor-reactive immune infiltrate. Chimeric antigen receptor (CAR) T cells may provide the necessary tumor-targeting immune infiltrate and a highly specific antitumor immune response. This can be further amplified by the addition of CPB agents, which serve to counteract the immune inhibitory environment undermining optimal CAR T cell efficacy. Herein, we review preclinical and clinical combination therapy with CAR T cells and CPB agents, with a focus on solid tumor malignancies. 10.1016/j.ccell.2019.09.006
    Synthetic T cell receptor-based lymphocytes for cancer therapy. Getts Daniel,Hofmeister Robert,Quintás-Cardama Alfonso Advanced drug delivery reviews Chimeric antigen receptor (CAR) T cells have been remarkably successful in patients with hematological malignancies expressing the CD19 surface antigen, but such level of success is far from being replicated in solid tumors. Engineered T cell receptor (TCR) T cells targeting cancer antigens were first developed over two decades ago and represent an alternative adoptive T cell approach that has produced provocative clinical data in solid cancers. However, several factors may hinder this technology from realizing its full potential, including the need for HLA matching, HLA downregulation by cancer cells, the suppressive tumor microenvironment, and tissue liabilities resulting from targeting antigens shared with normal tissues. Efforts therefore continue to engineer enhanced versions of CAR T and TCR T therapies that can overcome current barriers. Furthermore, emergent novel TCR-based, HLA-unrestricted platforms may also provide unique tools that integrate the complexity of the TCR signaling cascade that can be applied to treat solid tumors. This article reviews the current state of development of TCR T cell approaches and discusses next generation improvements to overcome their current limitations. 10.1016/j.addr.2019.04.002
    Tissue-specific functions of invariant natural killer T cells. Crosby Catherine M,Kronenberg Mitchell Nature reviews. Immunology Invariant natural killer T cells (iNKT cells) are an innate-like T cell subset that expresses an invariant T cell receptor (TCR) α-chain and recognizes lipids presented on CD1d. They secrete diverse cytokines and can influence many types of immune responses. Despite having highly similar TCR specificities, iNKT cells differentiate in the thymus into distinct subsets that are analogous to T helper 1 (T1), T2 and T17 cell subsets. Additional iNKT cell subsets that may require peripheral activation have also been described, including one that produces IL-10. In general, iNKT cells are non-circulating, tissue-resident lymphocytes, but the prevalence of different iNKT cell subsets differs markedly between tissues. Here, we summarize the functions of iNKT cells in four tissues in which they are prevalent, namely, the liver, the lungs, adipose tissue and the intestine. Importantly, we explain how local iNKT cell responses at each site contribute to tissue homeostasis and protection from infection but can also contribute to tissue inflammation and damage. 10.1038/s41577-018-0034-2
    Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1 T Cells in Synovial Sarcoma. D'Angelo Sandra P,Melchiori Luca,Merchant Melinda S,Bernstein Donna,Glod John,Kaplan Rosandra,Grupp Stephan,Tap William D,Chagin Karen,Binder Gwendolyn K,Basu Samik,Lowther Daniel E,Wang Ruoxi,Bath Natalie,Tipping Alex,Betts Gareth,Ramachandran Indu,Navenot Jean-Marc,Zhang Hua,Wells Daniel K,Van Winkle Erin,Kari Gabor,Trivedi Trupti,Holdich Tom,Pandite Lini,Amado Rafael,Mackall Crystal L Cancer discovery We evaluated the safety and activity of autologous T cells expressing NY-ESO-1, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1T cells exhibited an effector memory phenotype following expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8 NY-ESO-1T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects. Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8 NY-ESO-1T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. . 10.1158/2159-8290.CD-17-1417
    T cell receptor-based cancer immunotherapy: Emerging efficacy and pathways of resistance. Chandran Smita S,Klebanoff Christopher A Immunological reviews Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)-modified T cells can induce durable remissions in patients with refractory B-lymphoid cancers. By contrast, results applying CAR-modified T cells to solid malignancies have been comparatively modest. Alternative strategies to redirect T cell specificity and cytolytic function are therefore necessary if ACT is to serve a greater role in human cancer treatments. T cell receptors (TCRs) are antigen recognition structures physiologically expressed by all T cells that have complementary, and in some cases superior, properties to CARs. Unlike CARs, TCRs confer recognition to epitopes derived from proteins residing within any subcellular compartment, including the membrane, cytoplasm and nucleus. This enables TCRs to detect a broad universe of targets, such as neoantigens, cancer germline antigens, and viral oncoproteins. Moreover, because TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors respond to epitope densities many fold smaller than required for CAR-signaling. Herein, we summarize recent clinical data demonstrating that TCR-based immunotherapies can mediate regression of solid malignancies, including immune-checkpoint inhibitor refractory cancers. These trials simultaneously highlight emerging mechanisms of TCR resistance. We conclude by discussing how TCR-based immunotherapies can achieve broader dissemination through innovations in cell manufacturing and non-viral genome integration techniques. 10.1111/imr.12772
    Engineering an Artificial T-Cell Stimulating Matrix for Immunotherapy. Hickey John W,Dong Yi,Chung Jae Wook,Salathe Sebastian F,Pruitt Hawley C,Li Xiaowei,Chang Calvin,Fraser Andrew K,Bessell Catherine A,Ewald Andrew J,Gerecht Sharon,Mao Hai-Quan,Schneck Jonathan P Advanced materials (Deerfield Beach, Fla.) T cell therapies require the removal and culture of T cells ex vivo to expand several thousand-fold. However, these cells often lose the phenotype and cytotoxic functionality for mediating effective therapeutic responses. The extracellular matrix (ECM) has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies. Here, a hyaluronic acid (HA)-based hydrogel is engineered to present the two stimulatory signals required for T-cell activation-termed an artificial T-cell stimulating matrix (aTM). It is found that biophysical properties of the aTM-stimulatory ligand density, stiffness, and ECM proteins-potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM results in a rapid and robust expansion of rare, antigen-specific CD8+ T cells. Adoptive transfer of these tumor-specific cells significantly suppresses tumor growth and improves animal survival compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM-mimetic materials for therapeutic immune stimulation in the future. 10.1002/adma.201807359
    Reprogramming human T cell function and specificity with non-viral genome targeting. Roth Theodore L,Puig-Saus Cristina,Yu Ruby,Shifrut Eric,Carnevale Julia,Li P Jonathan,Hiatt Joseph,Saco Justin,Krystofinski Paige,Li Han,Tobin Victoria,Nguyen David N,Lee Michael R,Putnam Amy L,Ferris Andrea L,Chen Jeff W,Schickel Jean-Nicolas,Pellerin Laurence,Carmody David,Alkorta-Aranburu Gorka,Del Gaudio Daniela,Matsumoto Hiroyuki,Morell Montse,Mao Ying,Cho Min,Quadros Rolen M,Gurumurthy Channabasavaiah B,Smith Baz,Haugwitz Michael,Hughes Stephen H,Weissman Jonathan S,Schumann Kathrin,Esensten Jonathan H,May Andrew P,Ashworth Alan,Kupfer Gary M,Greeley Siri Atma W,Bacchetta Rosa,Meffre Eric,Roncarolo Maria Grazia,Romberg Neil,Herold Kevan C,Ribas Antoni,Leonetti Manuel D,Marson Alexander Nature Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic sites. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells. 10.1038/s41586-018-0326-5
    Effective NY-ESO-1-specific MHC II-restricted T cell receptors from antigen-negative hosts enhance tumor regression. Poncette Lucia,Chen Xiaojing,Lorenz Felix Km,Blankenstein Thomas The Journal of clinical investigation Adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells is a promising approach in cancer therapy but needs improvement for more effective treatment of solid tumors. While most clinical approaches have focused on CD8+ T cells, the importance of CD4+ T cells in mediating tumor regression has become apparent. Regarding shared (self) tumor antigens, it is unclear whether the human CD4+ T cell repertoire has been shaped by tolerance mechanisms and lacks highly functional TCRs suitable for therapy. Here, TCRs against the tumor-associated antigen NY-ESO-1 were isolated either from human CD4+ T cells or from mice that express a diverse human TCR repertoire with HLA-DRA/DRB1*0401 restriction and are NY-ESO-1 negative. NY-ESO-1-reactive TCRs from the mice showed superior recognition of tumor cells and higher functional activity compared with TCRs from humans. We identified a candidate TCR, TCR-3598_2, which was expressed in CD4+ T cells and caused tumor regression in combination with NY-ESO-1-redirected CD8+ T cells in a mouse model of adoptive T cell therapy. These data suggest that MHC II-restricted TCRs against NY-ESO-1 from a nontolerant nonhuman host are of optimal affinity and that the combined use of MHC I- and II-restricted TCRs against NY-ESO-1 can make adoptive T cell therapy more effective. 10.1172/JCI120391
    Framework engineering to produce dominant T cell receptors with enhanced antigen-specific function. Thomas Sharyn,Mohammed Fiyaz,Reijmers Rogier M,Woolston Annemarie,Stauss Theresa,Kennedy Alan,Stirling David,Holler Angelika,Green Louisa,Jones David,Matthews Katherine K,Price David A,Chain Benjamin M,Heemskerk Mirjam H M,Morris Emma C,Willcox Benjamin E,Stauss Hans J Nature communications TCR-gene-transfer is an efficient strategy to produce therapeutic T cells of defined antigen specificity. However, there are substantial variations in the cell surface expression levels of human TCRs, which can impair the function of engineered T cells. Here we demonstrate that substitutions of 3 amino acid residues in the framework of the TCR variable domains consistently increase the expression of human TCRs on the surface of engineered T cells.The modified TCRs mediate enhanced T cell proliferation, cytokine production and cytotoxicity, while reducing the peptide concentration required for triggering effector function up to 3000-fold. Adoptive transfer experiments in mice show that modified TCRs control tumor growth more efficiently than wild-type TCRs. Our data indicate that simple variable domain modifications at a distance from the antigen-binding loops lead to increased TCR expression and improved effector function. This finding provides a generic platform to optimize the efficacy of TCR gene therapy in humans. 10.1038/s41467-019-12441-w
    Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Schober Kilian,Müller Thomas R,Gökmen Füsun,Grassmann Simon,Effenberger Manuel,Poltorak Mateusz,Stemberger Christian,Schumann Kathrin,Roth Theodore L,Marson Alexander,Busch Dirk H Nature biomedical engineering Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and β-chains combined with orthotopic TCR placement leads to accurate αβ-pairing and results in TCR regulation similar to that of physiological T cells. 10.1038/s41551-019-0409-0
    Nonlytic Lymphocytes Engineered to Express Virus-Specific T-Cell Receptors Limit HBV Infection by Activating APOBEC3. Koh Sarene,Kah Janine,Tham Christine Y L,Yang Ninghan,Ceccarello Erica,Chia Adeline,Chen Margaret,Khakpoor Atefeh,Pavesi Andrea,Tan Anthony T,Dandri Maura,Bertoletti Antonio Gastroenterology BACKGROUND & AIMS:Strategies to develop virus-specific T cells against hepatic viral infections have been hindered by safety concerns. We engineered nonlytic human T cells to suppress replication of hepatitis B virus (HBV) and hepatitis C virus (HCV) without overt hepatotoxicity and investigated their antiviral activity. METHODS:We electroporated resting T cells or T cells activated by anti-CD3 with mRNAs encoding HBV or HCV-specific T-cell receptors (TCRs) to create 2 populations of TCR-reprogrammed T cells. We tested their ability to suppress HBV or HCV replication without lysis in 2-dimensional and 3-dimensional cultures of HepG2.2.15 cells and HBV-infected HepG2-hNTCP cells. We also injected TCR-reprogrammed resting and activated T cells into HBV-infected urokinase-type plasminogen activator/severe combined immunodeficiency disease/interleukin 2γ mice with humanized livers and measured levels of intrahepatic and serological viral parameters and serum alanine aminotransferase. Livers were collected for analysis of gene expression patterns to determine effects of the TCR-reprogrammed T cells. RESULTS:TCR-reprogrammed resting T cells produced comparable levels of interferon gamma but lower levels of perforin and granzyme than activated T cells and did not lyse HCV- or HBV-infected hepatoma cells. Although T-cell secretion of interferon gamma was required to inhibit HCV replication, the HBV-specific TCR-reprogrammed resting T cells reduced HBV replication also through intracellular activation of apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3). The mechanism of APOBEC3 intracellular activation involved temporal expression of lymphotoxin-β receptor ligands on resting T cells after TCR-mediated antigen recognition and activation of lymphotoxin-β receptor in infected cells. CONCLUSIONS:We developed TCR-reprogrammed nonlytic T cells capable of activating APOBEC3 in hepatoma cells and in HBV-infected human hepatocytes in mice, limiting viral infection. These cells with limited hepatotoxicity might be developed for treatment of chronic HBV infection. 10.1053/j.gastro.2018.03.027
    TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps. Yi Jason,Balagopalan Lakshmi,Nguyen Tiffany,McIntire Katherine M,Samelson Lawrence E Nature communications Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation. 10.1038/s41467-018-08064-2
    Attenuation of TCR-induced transcription by Bach2 controls regulatory T cell differentiation and homeostasis. Sidwell Tom,Liao Yang,Garnham Alexandra L,Vasanthakumar Ajithkumar,Gloury Renee,Blume Jonas,Teh Peggy P,Chisanga David,Thelemann Christoph,de Labastida Rivera Fabian,Engwerda Christian R,Corcoran Lynn,Kometani Kohei,Kurosaki Tomohiro,Smyth Gordon K,Shi Wei,Kallies Axel Nature communications Differentiation and homeostasis of Foxp3 regulatory T (Treg) cells are strictly controlled by T-cell receptor (TCR) signals; however, molecular mechanisms that govern these processes are incompletely understood. Here we show that Bach2 is an important regulator of Treg cell differentiation and homeostasis downstream of TCR signaling. Bach2 prevents premature differentiation of fully suppressive effector Treg (eTreg) cells, limits IL-10 production and is required for the development of peripherally induced Treg (pTreg) cells in the gastrointestinal tract. Bach2 attenuates TCR signaling-induced IRF4-dependent Treg cell differentiation. Deletion of IRF4 promotes inducible Treg cell differentiation and rescues pTreg cell differentiation in the absence of Bach2. In turn, loss of Bach2 normalizes eTreg cell differentiation of IRF4-deficient Treg cells. Mechanistically, Bach2 counteracts the DNA-binding activity of IRF4 and limits chromatin accessibility, thereby attenuating IRF4-dependent transcription. Thus, Bach2 balances TCR signaling induced transcriptional activity of IRF4 to maintain homeostasis of thymically-derived and peripherally-derived Treg cells. 10.1038/s41467-019-14112-2
    TCR-based therapy for multiple myeloma and other B-cell malignancies targeting intracellular transcription factor BOB1. Jahn Lorenz,Hombrink Pleun,Hagedoorn Renate S,Kester Michel G D,van der Steen Dirk M,Rodriguez Tania,Pentcheva-Hoang Tsvetelina,de Ru Arnoud H,Schoonakker Marjolein P,Meeuwsen Miranda H,Griffioen Marieke,van Veelen Peter A,Falkenburg J H Frederik,Heemskerk Mirjam H M Blood Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1 but HLA-B*07:02 nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma. 10.1182/blood-2016-09-737536
    Defining virus-specific CD8+ TCR repertoires for therapeutic regeneration of T cells against chronic hepatitis E. Soon Chai Fen,Behrendt Patrick,Todt Daniel,Manns Michael Peter,Wedemeyer Heiner,Sällberg Chen Margaret,Cornberg Markus Journal of hepatology BACKGROUND & AIMS:Immunosuppressed patients with chronic hepatitis E virus infection (cHEV), who are ineligible or have failed current treatment with off-label ribavirin, are a potential target population for T cell-based therapy. T cell responses are important for viral control. Herein, we aimed to identify human leukocyte antigen (HLA)-A2 restricted HEV-specific CD8+ T cell epitopes and T cell receptors (TCR) targeting these epitopes, as the basis for a redirected TCR treatment approach for patients with cHEV. METHODS:HEV genotype 3 overlapping peptide pools were used to screen HEV-specific CD8+ T cell immune responses in HLA-A2+ patients with acute HEV infection and healthy donors, by intracellular cytokine staining. CD8+ T cells targeting the identified epitopes were sorted for sequencing of the TCR repertoires by next generation sequencing. Messenger RNA encoding these TCRs were introduced into lymphocytes of healthy donors and patients with cHEV through TCR redirection. TCR-engineered lymphocytes were evaluated for Dextramer®-binding capacity, target sensitivity and cytotoxicity against peptide-loaded T2 cells. RESULTS:HEV-specific responses were observed across open reading frame (ORF)1 and ORF2 of the HEV genome in patients with acute resolving HEV infection. HLA-A2-restricted HEV-specific CD8+ T cell epitopes targeting the HEV RNA helicase and RNA-dependent RNA polymerase were selected for functional studies. Introduction of HEV-specific TCRs into lymphocytes of immunocompetent donors and patients with chronic hepatitis E enabled the lymphocytes to bind HEV Dextramers, secrete multiple cytokines and exert cytotoxicity in a target-specific manner. CONCLUSION:We identified TCRs that target HEV-specific CD8+ T cell epitopes, and characterized their immune properties, which may have clinical potential in future T cell-based therapy. LAY SUMMARY:Patients who are immunosuppressed are vulnerable to developing chronic liver disease following infection with hepatitis E virus (HEV). To-date, there is no approved therapy for chronic hepatitis E. Interferon-α and ribavirin are off-label treatment options, but their applications are limited by side effects. Thus, immunotherapy, more specifically T cell-based therapy, may be an alternative approach. We designed T cell receptor-engineered T cells that effectively conferred immune cells, taken from patients with chronic hepatitis E, with the ability to recognize virus-specific epitopes and mediate killing of target cells in vitro. 10.1016/j.jhep.2019.06.005
    Assessment of TCR signal strength of antigen-specific memory CD8 T cells in human blood. Wu Hanchih,Witzl Ashley,Ueno Hideki Blood advances Assessment of the quality and the breadth of antigen (Ag)-specific memory T cells in human samples is of paramount importance to elucidate the pathogenesis and to develop new treatments in various diseases. T-cell receptor (TCR) signal strength, primarily controlled by TCR affinity, affects many fundamental aspects of T-cell biology; however, no current assays for detection of Ag-specific CD8 T cells can assess their TCR signal strength in human samples. Here, we provide evidence that interferon regulatory factor 4 (IRF4), a transcription factor rapidly upregulated in correlation with TCR signal strength, permits the assessment of the TCR signal strength of Ag-specific CD8 T cells in human peripheral blood mononuclear cells (PBMCs). Coexpression of IRF4 and CD137 sensitively detected peptide-specific CD8 T cells with extremely low background in PBMCs stimulated for 18 hours with MHC class I peptides. Our assay revealed that human memory CD8 T cells with high-affinity TCRs have an intrinsic ability to highly express CD25. Furthermore, HIV-specific CD8 T cells in chronic HIV subjects were found to display primarily low-affinity TCRs with low CD25 expression capacity. Impairment in the functions of HIV-specific CD8 T cells might be associated with their suboptimal TCR signals, as well as impaired responsiveness to interleukin-2. 10.1182/bloodadvances.2019000292
    NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease. Mastaglio Sara,Genovese Pietro,Magnani Zulma,Ruggiero Eliana,Landoni Elisa,Camisa Barbara,Schiroli Giulia,Provasi Elena,Lombardo Angelo,Reik Andreas,Cieri Nicoletta,Rocchi Martina,Oliveira Giacomo,Escobar Giulia,Casucci Monica,Gentner Bernhard,Spinelli Antonello,Mondino Anna,Bondanza Attilio,Vago Luca,Ponzoni Maurilio,Ciceri Fabio,Holmes Michael C,Naldini Luigi,Bonini Chiara Blood Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1 targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy. 10.1182/blood-2016-08-732636
    CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Legut Mateusz,Dolton Garry,Mian Afsar Ali,Ottmann Oliver G,Sewell Andrew K Blood 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. 10.1182/blood-2017-05-787598
    TCR signal strength controls the differentiation of CD4 effector and memory T cells. Snook Jeremy P,Kim Chulwoo,Williams Matthew A Science immunology CD4 T cell responses are composed of heterogeneous T cell receptor (TCR) signals that influence the acquisition of effector and memory characteristics. We sought to define early TCR-dependent activation events that control T cell differentiation. A polyclonal panel of TCRs specific for the same viral antigen demonstrated substantial variability in TCR signal strength, expression of CD25, and activation of nuclear factor of activated T cells and nuclear factor κB. After viral infection, strong TCR signals corresponded to T helper cell (T1) differentiation, whereas T follicular helper cell and memory T cell differentiation were most efficient when TCR signals were comparatively lower. We observed substantial heterogeneity in TCR-dependent CD25 expression in vivo, and the vast majority of CD4 memory T cells were derived from CD25 effector cells that displayed decreased TCR signaling in vivo. Nevertheless, memory T cells derived from either CD25 or CD25 effector cells responded vigorously to rechallenge, indicating that, although early clonal differences in CD25 expression predicted memory T cell numbers, they did not predict memory T cell function on a per cell basis. Gene transcription analysis demonstrated expression clustering based on CD25 expression and enrichment of transcripts associated with enhanced T follicular helper cell and memory development within CD25 effector cells. Direct enhancement of TCR signaling via knockdown of Src homology region 2 domain-containing phosphatase 1, a tyrosine phosphatase that suppresses early TCR signaling events, favored the differentiation of T1 effector and memory cells. We conclude that strong TCR signals during early T cell activation favor terminal T1 differentiation over long-term T1 and T follicular helper cell memory responses. 10.1126/sciimmunol.aas9103
    Mechano-regulation of Peptide-MHC Class I Conformations Determines TCR Antigen Recognition. Wu Peng,Zhang Tongtong,Liu Baoyu,Fei Panyu,Cui Lei,Qin Rui,Zhu Huaying,Yao Danmei,Martinez Ryan J,Hu Wei,An Chenyi,Zhang Yong,Liu Junwei,Shi Jiawei,Fan Juan,Yin Weiwei,Sun Jie,Zhou Chun,Zeng Xun,Xu Chenqi,Wang Jianan,Evavold Brian D,Zhu Cheng,Chen Wei,Lou Jizhong Molecular cell TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy. 10.1016/j.molcel.2018.12.018
    Targeting human melanoma neoantigens by T cell receptor gene therapy. Leisegang Matthias,Kammertoens Thomas,Uckert Wolfgang,Blankenstein Thomas The Journal of clinical investigation In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy. 10.1172/JCI83465
    Treatment of Patients With Metastatic Cancer Using a Major Histocompatibility Complex Class II-Restricted T-Cell Receptor Targeting the Cancer Germline Antigen MAGE-A3. Lu Yong-Chen,Parker Linda L,Lu Tangying,Zheng Zhili,Toomey Mary Ann,White Donald E,Yao Xin,Li Yong F,Robbins Paul F,Feldman Steven A,van der Bruggen Pierre,Klebanoff Christopher A,Goff Stephanie L,Sherry Richard M,Kammula Udai S,Yang James C,Rosenberg Steven A Journal of clinical oncology : official journal of the American Society of Clinical Oncology Purpose Adoptive transfer of genetically modified T cells is being explored as a treatment for patients with metastatic cancer. Most current strategies use genes that encode major histocompatibility complex (MHC) class I-restricted T-cell receptors (TCRs) or chimeric antigen receptors to genetically modify CD8 T cells or bulk T cells for treatment. Here, we evaluated the safety and efficacy of an adoptive CD4 T-cell therapy using an MHC class II-restricted, HLA-DPB1*0401-restricted TCR that recognized the cancer germline antigen, MAGE-A3 (melanoma-associated antigen-A3). Patients and Methods Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4 T cells, retrovirally transduced with MAGE-A3 TCR plus systemic high-dose IL-2. A cell dose escalation was conducted, starting at 10 total cells and escalating at half-log increments to approximately 10 cells. Nine patients were treated at the highest dose level (0.78 to 1.23 × 10 cells). Results Seventeen patients were treated. During the cell dose-escalation phase, an objective complete response was observed in a patient with metastatic cervical cancer who received 2.7 × 10 cells (ongoing at ≥ 29 months). Among nine patients who were treated at the highest dose level, objective partial responses were observed in a patient with esophageal cancer (duration, 4 months), a patient with urothelial cancer (ongoing at ≥ 19 months), and a patient with osteosarcoma (duration, 4 months). Most patients experienced transient fevers and the expected hematologic toxicities from lymphodepletion pretreatment. Two patients experienced transient grade 3 and 4 transaminase elevations. There were no treatment-related deaths. Conclusion These results demonstrate the safety and efficacy of administering autologous CD4 T cells that are genetically engineered to express an MHC class II-restricted antitumor TCR that targets MAGE-A3. This clinical trial extends the reach of TCR gene therapy for patients with metastatic cancer. 10.1200/JCO.2017.74.5463
    Optimizing T-cell receptor gene therapy for hematologic malignancies. Morris Emma C,Stauss Hans J Blood Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients. 10.1182/blood-2015-11-629071
    T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant. Chapuis Aude G,Egan Daniel N,Bar Merav,Schmitt Thomas M,McAfee Megan S,Paulson Kelly G,Voillet Valentin,Gottardo Raphael,Ragnarsson Gunnar B,Bleakley Marie,Yeung Cecilia C,Muhlhauser Petri,Nguyen Hieu N,Kropp Lara A,Castelli Luca,Wagener Felecia,Hunter Daniel,Lindberg Marcus,Cohen Kristen,Seese Aaron,McElrath M Juliana,Duerkopp Natalie,Gooley Ted A,Greenberg Philip D Nature medicine Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens. We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCR) from HLA-A2 normal donor repertoires, inserted TCR into Epstein-Bar virus-specific donor CD8 T cells (T) to minimize graft-versus-host disease risk and enhance transferred T cell survival, and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). T maintained TCR expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse. 10.1038/s41591-019-0472-9