Transgenic T-cell Receptor Research Articles

Simultaneous Deletion of Endogenous TCRαβ for TCR Gene Therapy Creates an Improved and Safe Cellular Therapeutic.

Generation of an optimal Tcell therapeutic expressing high frequencies of transgenic Tcell receptor (tgTCR) is essential for improving TCR gene therapy. Upon TCR gene transfer, presence of endogenous TCRαβ reduces expression of tgTCR due to TCR mixed-dimer formation and competition for binding CD3. Knockout (KO) of endogenous TCRαβ was recently achieved using CRISPR/Cas9 editing of the TRAC or TRBC loci, resulting in increased expression and function of tgTCR. Here, we adopt this approach into current protocols for generating Tcell populations expressing tgTCR to validate this strategy in the context of four clinically relevant TCRs. First, simultaneous editing of TRAC and TRBC loci was reproducible and resulted in high double KO efficiencies in bulk CD8 Tcells. Next, tgTCR expression was significantly higher in double TRAC/BC KO conditions for all TCRs tested, including those that contained structural modifications to encourage preferential pairing. Finally, increased expression of tgTCR in edited Tcell populations allowed for increased recognition of antigen expressing tumor targets and prolonged control of tumor outgrowth in a preclinical model of multiple myeloma. In conclusion, CRISPR/Cas9-mediated KO of both endogenous TCRαβ chains can be incorporated in current Tcell production protocols and is preferential to ensure an improved and safe clinical therapeutic.

Class-switch recombination to IgA in the Peyer's patches requires natural thymus-derived Tregs and appears to be antigen independent.

Our understanding of how class-switch recombination (CSR) to IgA occurs in the gut is still incomplete. Earlier studies have indicated that Tregs are important for IgA CSR and these cells were thought to transform into follicular helper T cells (Tfh), responsible for germinal center formation in the Peyer's patches (PP). Following adoptive transfer of T-cell receptor-transgenic (TCR-Tg) CD4 T cells into nude mice, we unexpectedly found that oral immunization did not require an adjuvant to induce strong gut IgA and systemic IgG responses, suggesting an altered regulatory environment in the PP. After sorting of splenic TCR-Tg CD4 T cells into CD25+ or CD25- cells we observed that none of these fractions supported a gut IgA response, while IgG responses were unperturbed in mice receiving the CD25- cell fraction. Hence, while Tfh functions resided in the CD25- fraction the IgA CSR function in the PP was dependent on CD25+ Foxp3+ Tregs, which were found to be Helios+ neuropilin-1+ thymus-derived Tregs. This is the first study to demonstrate that Tfh and IgA CSR functions are indeed, unique, and separate functions in the PP with the former being TCR-dependent while the latter appeared to be antigen independent.

Low mutational load in pediatric medulloblastoma still translates into neoantigens as targets for specific T-cell immunotherapy

BackgroundMedulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity. MethodsWe identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients’ human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro. ResultsDespite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants. ConclusionDetection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.

Loss of gut barrier integrity triggers activation of islet-reactive T cells and autoimmune diabetes

Low-grade intestinal inflammation and alterations of gut barrier integrity are found in patients affected by extraintestinal autoimmune diseases such as type 1 diabetes (T1D), but a direct causal link between enteropathy and triggering of autoimmunity is yet to be established. Here, we found that onset of autoimmunity in preclinical models of T1D is associated with alterations of the mucus layer structure and loss of gut barrier integrity. Importantly, we showed that breakage of the gut barrier integrity in BDC2.5XNOD mice carrying a transgenic T cell receptor (TCR) specific for a beta cell autoantigen leads to activation of islet-reactive T cells within the gut mucosa and onset of T1D. The intestinal activation of islet-reactive T cells requires the presence of gut microbiota and is abolished when mice are depleted of endogenous commensal microbiota by antibiotic treatment. Our results indicate that loss of gut barrier continuity can lead to activation of islet-specific T cells within the intestinal mucosa and to autoimmune diabetes and provide a strong rationale to design innovative therapeutic interventions in "at-risk" individuals aimed at restoring gut barrier integrity to prevent T1D occurrence.

NEXT GENERATION CAR T CELLS FOR LYMPHOMA AND BEYOND

Engineered T cells that encode transgenic T cell receptors (TCR) or chimeric antigen receptors (CAR) are increasingly being tested is a new modality for cancer immunotherapy. In this presentation we will review emerging patterns of efficacy and toxicity after adoptive cell transfer (ACT), with a focus on blood cancers. Striking efficacy has been observed in acute and chronic B cell leukemia and lymphoma. More unexpectedly, strong antitumor effects are being observed in multiple trials in patients with advanced refractory and relapsed myeloma. In some cases, toxicity has been predicted in preclinical models. In other cases, toxicity was only uncovered after results from Phase 1 clinical trial results were in hand. Onset of toxicity can be immediately observed following infusion of cells or maybe delayed. Toxicity usually occurs coincident with peak (Cmax) levels of adoptively transferred cells. Today with more than 1,000 patients having been infused with genetically engineered human T cells, genotoxicity has not been reported. To date, the induction of irAE appears to be less than with checkpoint therapies, although more experience with ACT is required to determine the ultimate incidence of autoimmune toxicities. Keywords: T-cell receptor (TCR). Disclosures: June, C: Research Funding: Parker Institute for Cancer Immunotherapy, NIH/NCI, Tmunity Therapeutics.

T-Cell Receptor-Based Immunotherapy for Hematologic Malignancies.

Adoptive immunotherapy with engineered T cells is at the forefront of cancer treatment. T cells can be engineered to express T-cell receptors (TCRs) specific for tumor-associated antigens (TAAs) derived from intracellular or cell surface proteins. T cells engineered with TCRs (TCR-T) allow for targeting diverse types of TAAs, including proteins overexpressed in malignant cells, those with lineage-restricted expression, cancer-testis antigens, and neoantigens created from abnormal, malignancy-restricted proteins. Minor histocompatibility antigens can also serve as TAAs for TCR-T to treat relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. Moreover, TCR constructs can be modified to improve safety and enhance function and persistence of TCR-T. Transgenic T-cell receptor therapies targeting 3 different TAAs are in early-phase clinical trials for treatment of hematologic malignancies. Preclinical studies of TCR-T specific for many other TAAs are underway and offer great promise as safe and effective therapies for a wide range of cancers.

Abstract A188: T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity

Abstract Introduction: Interleukin-15 (IL-15), provides strong activation of both CD8+ T-cells and NK cells, without regulatory T-cells activation, making it an attractive immune modulator in cancer therapy. Systemic delivery of IL-15 to patients has revealed dose-limited toxicities resulting primarily in expansion of NK cells. Preclinical data suggest that IL-15 immunotoxicity is mediated by hyperproliferation and activation of NK cells (Guo Y, J Immunol 2015). In this study, we investigate safety and efficacy of T-cells loaded with Deep IL-15 (Deep IL-15 Primed T-cells), in a syngeneic mouse model. Deep IL-15 is a multimer of chemically crosslinked IL-15/IL-15 Rα/Fc heterodimers (IL15-Fc) that is designed for T-cell loading prior to adoptive cell transfer with the aim of improving the therapeutic window by autocrine signaling to the primed cells without causing the immunotoxicologic effects normally associated with IL-15. Deep IL-15 is loaded on the T-cells and, upon crosslinker cleavage, releases IL15-Fc to stimulate the primed cells. This novel T-cell-based therapeutic approach enables autocrine T-cell activation and expansion, and limits systemic exposure to IL15-Fc, thus reducing associated toxicities. Methods: Deep IL-15 was synthesized by incubation of IL15-Fc with a crosslinking reagent. PMEL CD8+ T (PMEL) cells were isolated from B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J mice. PMEL cells carry a transgenic T-cell receptor specific for gp100, a protein expressed by B16-F10 melanoma cells. PMEL cells were activated, expanded, and loaded with Deep IL-15 to generate Deep IL-15 Primed PMEL (Deep-15 PMEL) cells. Deep-15 PMEL cells were transferred into naïve or B16-F10 tumor-bearing mice (10 x 106; 15 ug Deep IL-15/106 cells), and the toxicity of Deep-15 PMEL was compared with PMEL (10 x 106) co-injected with soluble IL15-Fc at the maximum tolerated dose of 10 μg/mouse (PMEL + IL15-Fc). Readouts included IL15-Fc systemic exposure (ELISA), cytokine release (Luminex), and changes in endogenous T-cells (complete blood counts, CBC; flow cytometry). In addition, the biodistribution and the antitumor activity of Deep-15 PMEL was evaluated in B16-F10 tumor-bearing mice. Results: Deep-15 PMEL cells, carrying 15-fold more IL15-Fc than PMEL + IL15-Fc, resulted in >300-fold lower systemic exposure to IL15-Fc and 30-fold lower circulating IFN-γ. Deep-15 PMEL did not affect CBCs and did not expand endogenous CD8+ nor NK cells. Conversely, IL15-Fc induced significant changes in CBCs and promoted expansion of both transferred and endogenous CD8+ (6.7-fold) and NK (18.2-fold) cells. Similar results were observed in both naïve and tumor-bearing mice. Deep-15 PMEL improved persistence of transferred cells across multiple tissues (15-44-fold rel. to PMEL co-injected with IL15-Fc; day 16): blood, spleen, lymph nodes (tumor draining and non-draining) and tumor. The tumor presence affected the biodistribution of Deep-15 PMEL cells, resulting in lower (0.5-fold) accumulation of cells in the spleen, and increased (2.1-fold) cell numbers in the tumor-draining lymph node. Furthermore, Deep-15 PMEL showed significantly improved anti-tumor activity compared to PMEL in the B16-F10 model. Conclusions: The Deep Priming technology developed by Torque offers the advantage of loading Deep IL-15 prior to cell infusion in ACT, at concentrations unachievable with systemic injection of IL15-Fc, thus resulting in controlled, local delivery and cell-specific activation and proliferation. In vivo transfer of Deep-15 PMEL cells was well tolerated. Importantly, Deep-15 PMEL cells persisted longer across multiple organs compared to PMEL cells exposed to systemic IL15-Fc, while not inducing expansion of endogenous cells, including NK cells, which are reported to be the primary mediators of IL-15 toxicity. Torque is preparing to initiate clinical trials for Deep IL-15 Primed multitargeted human T-cells, TRQ15-01, in both hematologic and solid tumors. Citation Format: Elena Geretti, Philip Bardwell, Xiaoyan Liang, Santina Caruso, De-Kuan Chang, Jesse Lyons, Austin Boesch, Aaron Handler, Carlos Tassa, Sanela Bilic, Janice Lancita, Becker Hawes, Jonathan Fitzgerald, Thomas Andresen. T-cell priming with Deep IL-15 improves preclinical safety compared to systemic IL-15, and increases in vivo persistence and activity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A188.

Efficient and Non-genotoxic RNA-Based Engineering of Human T Cells Using Tumor-Specific T Cell Receptors With Minimal TCR Mispairing.

Genetic engineering of T cells with tumor specific T-cell receptors (TCR) is a promising strategy to redirect their specificity against cancer cells in adoptive T cell therapy protocols. Most studies are exploiting integrating retro- or lentiviral vectors to permanently introduce the therapeutic TCR, which can pose serious safety issues when treatment-related toxicities would occur. Therefore, we developed a versatile, non-genotoxic transfection method for human unstimulated CD8+ T cells. We describe an optimized double sequential electroporation platform whereby Dicer-substrate small interfering RNAs (DsiRNA) are first introduced to suppress endogenous TCR α and β expression, followed by electroporation with DsiRNA-resistant tumor-specific TCR mRNA. We demonstrate that double sequential electroporation of human primary unstimulated T cells with DsiRNA and TCR mRNA leads to unprecedented levels of transgene TCR expression due to a strongly reduced degree of TCR mispairing. Importantly, superior transgenic TCR expression boosts epitope-specific CD8+ T cell activation and killing activity. Altogether, DsiRNA and TCR mRNA double sequential electroporation is a rapid, non-integrating and highly efficient approach with an enhanced biosafety profile to engineer T cells with antigen-specific TCRs for use in early phase clinical trials.

Isolation and Characterization of an HLA-DRB1*04-Restricted HPV16-E7 T Cell Receptor for Cancer Immunotherapy.

High-risk human papillomavirus (HPV) infection is a causal factor in oropharyngeal and gynecological malignancies, and development of HPV-targeted immunotherapy could be used to treat patients with these cancers. T cell-mediated adoptive immunotherapy targeting E6 and E7, two HPV16 proteins consistently expressed in tumor cells, appears to be both attractive and safe. However, isolation of HPV-specific T cells is difficult owing to the low frequency of these cell precursors in the peripheral blood. In addition, HPV-positive cancer cells often down-regulate major histocompatibility complex (MHC) class I expression ex vivo, limiting the efficacy of MHC class I-restricted approaches. Of particular interest is that both CD4 and CD8 T cells can mediate the responses. Given that CD4 T cells play a critical role in coordinating effective antitumor responses, the generation of a T helper response in patients with HPV16-associated malignancies would unleash the ultimate potential of immunotherapy. In this view, T-cell receptor (TCR) gene transfer could be a relevant strategy to generate HPV16-E7-specific and MHC class II-restricted T cells in sufficient numbers. An HPV16-E7/HLA-DRB1*04 TCR has been isolated from a cancer patient with complete response, and retroviral particles encoding this TCR have been produced. The transgenic TCR is highly expressed in transduced T cells, with a functional inducible caspase-9 suicide gene safety cassette. TCR transgenic T cells are HPV16-E770-89 specific and HLA-DRB1*04 restricted, as determined by interferon (IFN)-γ secretion. CD8 and CD4 T cells are equivalently transduced and secrete interleukin-2 and IFN-γ when cultured with appropriate targets. We also demonstrate that TCR transgenic T cells recognize the endogenously processed and presented HPV16-E770-89 peptide. In conclusion, our data indicate that the production of MHC class II-restricted HPV16-E7-specific T cells is feasible through TCR gene transfer and could be used for immunotherapy.

Autotransplantation of T-lymphocytes as a tool for antigen-specific immunotherapy of oncological diseases

Autotransplantation of immune cells to induce immunological rejection of tumors can be a useful approach in tumor treatment. Despite the low efficiency of this approach demonstrated in the past, the active development of cell technologies and genetic engineering has led to the significant improvement in clinical outcomes. A great success was achieved in adoptive immunotherapy with the T-lymphocytes specific to tumor antigens. This review describes the clinical experience in application of various antigen-specific adoptive immunotherapy methods including tumor-infiltrating T-lymphocytes, T-lymphocytes with redirected transgenic T-cell and chimeric antigenrecognizing receptors. The possibilities and limitations of these biomedical technologies are also analyzed in the article.

Polarity of CD4+ T cells towards the antigen presenting cell is regulated by the Lck adapter TSAd

Polarization of T cells towards the antigen presenting cell (APC) is critically important for appropriate activation and differentiation of the naïve T cell. Here we used imaging flow cytometry (IFC) and show that the activation induced Lck and Itk adapter T cell specific adapter protein (TSAd), encoded by SH2D2A, modulates polarization of T cells towards the APC. Upon exposure to APC presenting the cognate antigen Id, Sh2d2a−/− CD4+ T cells expressing Id-specific transgenic T cell receptor (TCR), displayed impaired polarization of F-actin and TCR to the immunological synapse (IS). Sh2d2a−/− T-cells that did polarize F-actin and TCR still displayed impaired polarization of PKCξ, PAR3 and the microtubule-organizing center (MTOC). In vitro differentiation of activated Sh2d2a−/− T cells was skewed towards an effector memory (Tem) rather than a central memory (Tcm) phenotype. A similar trend was observed for Id-specific TCR Sh2d2a−/− T cells stimulated with APC and cognate antigen. Taken together our data suggest that TSAd modulates differentiation of experienced T cells possibly through polarization of CD4+ T cells towards the APC.

Identification of the transgenic integration site in 2C T cell receptor transgenic mice

2C T cell receptor (TCR) transgenic mice have been long used to study the molecular basis of TCR binding to peptide/major compatibility complexes and the cytotoxicity mechanism of cytotoxic T lymphocytes (CTLs). To study the role of variable gene promoters in allelic exclusion, we previously constructed mutant mice in which the Vβ13 promoter was deleted (P13 mice). Introduction of 2C transgene into P13 mice accelerated the onset of systemic CD8 T cell lymphoma between 14 and 27weeks of age, although parental P13 mice appeared to be normal. This observation suggests that the lymphoma development may be linked to features of 2C transgene. To identify the integration site of 2C transgene, Southern blotting identified a 2C-specific DNA fragment by 3' region probe of 2C TCR α transgene, and digestion-circularization-polymerase chain reaction (DC-PCR) amplified the 2C-specific DNA fragment with inverse primers specific to the southern probe. Sequence analysis revealed that DC-PCR product contained the probe sequences and the junction sequences of integration site, indicating that 2C TCR α transgene is integrated into chromosome 1. Further genomic analysis revealed cytosolic phospholipase A2 group IVA (cPLA2) as the nearest gene to the integration site. cPLA2 expression was upregulated in the normal thymi and T cell lymphomas from 2C transgenic mice, although it was not altered in the lymph nodes of 2C transgenic mice. The result is the first report demonstrating the integration site of 2C TCR transgene, and will facilitate the proper use of 2C transgenic mice in studies of CTLs.

Abstract 2980: Single cell RNA sequencing reveals AML immunoediting under pressure from engineered T cell therapy

Abstract Targeting relapsed acute myeloid leukemia (AML) with engineered T cells holds exceptional promise, but obstacles to efficacy exist. Understanding resistance mechanisms is essential to improve therapy and may broadly inform cancer immune escape. We and others have developed transgenic T cell receptor (TCR) therapies targeting the intracellular WT1 oncoprotein that promotes growth of most high-risk AMLs. TCR therapies generally target epitopes processed by the proteasome for Class I presentation. As mutation or loss of WT1 or loss of HLA expression is infrequent in AML, we hypothesized resistant clones may have defective antigen processing. A 25-year-old man with AML recurrent after a 2nd allogeneic stem cell transplant received reinduction chemotherapy and transgenic donor-derived T cells recognizing the HLA-A2 restricted WT1126-134 epitope (NCT01640301). The patient experienced a 12-month remission followed by relapse despite robust T cell persistence (>6% of CD8+ T cells). The relapsed leukemia expressed WT1 and HLA-A2 but was refractory to retreatment with additional WT1126-134-restricted T cells. Single cell RNA sequencing (scRNAseq) was performed on cryopreserved PBMCs from remission (n=4976 cells) and relapsed (n=2780) time points (10x Genomics platform). During apparent remission, transgenic T cells had a transcriptome distinct from native CD8s, expressing activation markers including HLA-DR, IL32, CD69 and CTLA4, suggesting recent TCR triggering from antigen encounter. However, at the time AML progression was observed, transgenic cells clustered with native quiescent CD8s, implying they were no longer encountering the targeted epitope. Transcriptome analysis of relapsed AML demonstrated downregulation of a single subunit of the immune proteasome (β1i), which we recently demonstrated in cell lines to be critical for processing the targeted WT1126-134 epitope. This patient's refractory AML was killed by T cells restricted to an alternate HLA-A2 epitope of WT1 processed independently of β1i, confirming the functional impact of immune proteasome downregulation. At an early responding timepoint, scRNAseq detected clinically unrecognized rare circulating AML cells already β1i-low, suggesting β1i loss represented immunoediting/outgrowth of a pre-existing clone as a consequence of T cell immunotherapy pressure. ScRNAseq is poised to become an important diagnostic in immunotherapy, with sufficient depth to profile both transgenic T cells and low burden/minimal residual AML, and, in our case, successfully identified a potentially targetable mechanism of immune escape. Our data suggest T cell therapy in AML and other tumors may be improved by in depth analysis of individual leukemic cells, and from selecting target epitopes processed by both standard and immunoproteasomes and/or early combination therapy combining TCRs recognizing differentially processed epitopes. Citation Format: Kelly G. Paulson, Thomas M. Schmitt, Daniel Egan, Valentin Voillet, Miranda C. Lahman, Felecia D. Wagener, Daniel S. Hunter, Petri R. Muhlhauser, Paul C. Hendrie, Cecilia Yeung, Nathalie Vigneron, Benoit Van den Eynde, Jason H. Bielas, Merav Bar, Raphael Gottardo, Aude G. Chapuis, Philip D. Greenberg. Single cell RNA sequencing reveals AML immunoediting under pressure from engineered T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2980.

Efficient Non-Viral T-Cell Engineering bySleeping BeautyMinicircles Diminishing DNA Toxicity and miRNAs Silencing the Endogenous T-Cell Receptors

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells, which were found to cause adverse effects. T-cell viability was decreased in a dose-dependent manner, and DNA-transfected T cells showed a delayed response upon T-cell receptor (TCR) stimulation with regard to blast formation, proliferation, and surface expression of CD25 and CD28. Gene expression analysis demonstrated a DNA-dependent induction of a type I interferon response and interferon-β upregulation. By combining Sleeping Beauty transposon minicircle vectors with SB100X transposase-encoding RNA, it was possible to reduce the amount of total DNA required, and stable expression of therapeutic TCRs was achieved in >50% of human T cells without enrichment. The TCR-engineered T cells mediated effective tumor cell killing and cytokine secretion upon antigen-specific stimulation. Additionally, the Sleeping Beauty transposon system was further improved by miRNAs silencing the endogenous TCR chains. These miRNAs increased the surface expression of the transgenic TCR, diminished mispairing with endogenous TCR chains, and enhanced antigen-specific T-cell functionality. This approach facilitates the rapid non-viral generation of highly functional, engineered T cells for immunotherapy.

Brain Ischemia Induces Diversified Neuroantigen-Specific T-Cell Responses That Exacerbate Brain Injury.

Autoimmune responses can occur when antigens from the central nervous system are presented to lymphocytes in the periphery or central nervous system in several neurological diseases. However, whether autoimmune responses emerge after brain ischemia and their impact on clinical outcomes remains controversial. We hypothesized that brain ischemia facilitates the genesis of autoimmunity and aggravates ischemic brain injury. Using a mouse strain that harbors a transgenic T-cell receptor to a central nervous system antigen, MOG35-55 (myelin oligodendrocyte glycoprotein) epitope (2D2), we determined the anatomic location and involvement of antigen-presenting cells in the development of T-cell reactivity after brain ischemia and how T-cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion and photothrombotic stroke models were used in this study. We also quantified the presence and status of T cells from brain slices of ischemic patients. By coupling transfer of labeled MOG35-55-specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG91-108 and MOG103-125 in transient middle cerebral artery occlusion and photothrombotic stroke models. This reactivity and T-cell activation first occur locally in the brain after ischemia. Also, microglia act as antigen-presenting cells that effectively present MOG antigens, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T-cell activation and MOG-specific T cells are present in the brain of patients with ischemic stroke. Our findings suggest that brain ischemia activates and diversifies T-cell responses locally, which exacerbates ischemic brain injury.

TCR deep sequencing of transgenic RAG-1-deficient mice reveals endogenous TCR recombination: a cause for caution.

The utility of T-cell receptor (TCR) transgenic mice in medical research has been considerable, with applications ranging from basic biology all the way to translational and clinical investigations. Crossing of TCR transgenic mice with either recombination-activating gene (RAG)-1 or RAG-2 knockouts is frequently used to generate mice with a monoclonal T-cell repertoire. However, low level productive TCR rearrangement has been reported in RAG-deficient mice expressing transgenic TCRs. Using deep sequencing, we set out to directly examine and quantify the presence of these endogenous TCRs. Our demonstration that functional nontransgenic TCRs are present in nonmanipulated mice has wide reaching ramifications worthy of critical consideration.

Fit αβ T-cell receptor suppresses leukemogenesis of Pten-deficient thymocytes.

Signaling through the αβT cell receptor (TCR) is a crucial determinant of T-cell fate and can induce two opposite outcomes during thymocyte development: cell death or survival and differentiation. To date, the role played by T-cell receptor in the oncogenic transformation of developing T cells remains unclear. Here we show that human primary T-cell acute lymphoblastic leukemias expressing an αβT cell receptor are frequently deficient for phosphatase and tensin homolog protein (PTEN), and fail to respond strongly to T-cell receptor activation. Using Pten-deficient T-cell acute lymphoblastic leukemia mouse models, we confirm that T-cell receptor signaling is involved in leukemogenesis. We show that abrogation of T-cell receptor expression accelerated tumor onset, while enforced expression of a fit transgenic T-cell receptor led to the development of T-cell receptor-negative lymphoma and delayed tumorigenesis. We further demonstrate that pre-tumoral Pten-deficient thymocytes harboring fit T-cell receptors undergo early clonal deletion, thus preventing their malignant transformation, while cells with unfit T-cell receptors that should normally be deleted during positive selection, pass selection and develop T-cell acute lymphoblastic leukemias. Altogether, our data show that fit T-cell receptor signaling suppresses tumor development mediated by Pten loss-of-function and point towards a role of Pten in positive selection.

Ag85-focused T-cell immune response controls Mycobacterium avium chronic infection.

CD4+ T cells are essential players for the control of mycobacterial infections. Several mycobacterial antigens have been identified for eliciting a relevant CD4+ T cell mediated-immune response, and numerous studies explored this issue in the context of Mycobacterium tuberculosis infection. Antigen 85 (Ag85), a highly conserved protein across Mycobacterium species, is secreted at the early phase of M. tuberculosis infection leading to the proliferation of Ag85-specific CD4+ T cells. However, in the context of Mycobacterium avium infection, little is known about the expression of this antigen and the elicited immune response. In the current work, we investigated if a T cell receptor (TCR) repertoire mostly, but not exclusively, directed at Ag85 is sufficient to mount a protective immune response against M. avium. We show that P25 mice, whose majority of T cells express a transgenic TCR specific for Ag85, control M. avium infection at the same level as wild type (WT) mice up to 20 weeks post-infection (wpi). During M. avium infection, Ag85 antigen is easily detected in the liver of 20 wpi mice by immunohistochemistry. In spite of the propensity of P25 CD4+ T cells to produce higher amounts of interferon-gamma (IFNγ) upon ex vivo stimulation, no differences in serum IFNγ levels are detected in P25 compared to WT mice, nor enhanced immunopathology is detected in P25 mice. These results indicate that a T cell response dominated by Ag85-specific T cells is appropriate to control M. avium infection with no signs of immunopathology.

Abstract WMP78: Brain Ischemia Induces Diversified Neuroantigen-Specific T Cell Responses That Exacerbate Brain Injury

Background: Autoimmune responses can occur when antigens from the central nervous system (CNS) are presented to lymphocytes in the periphery or CNS. However, whether autoimmune responses emerge after brain ischemia remains controversial. Similarly, it is debated whether genesis of autoimmune responses can impact outcome of ischemic stroke. We hypothesized that that brain ischemia can diversify T cell responses against CNS antigens, and that expanded CNS antigen-specific T cells can promote ischemic brain injury. Methods: We quantified the presence and status of T cells from brain slices of ischemic patients. Using a mouse strain that harbor a transgenic T cell receptor (TCR) to a CNS antigen, the myelin oligodendrocyte glycoprotein (MOG 35-55 ) epitope (2D2), we determined the anatomic location and involvement of antigen presentation cells in the development of T cell reactivity after brain ischemia and how T cell reactivity impacts stroke outcome. Transient middle cerebral artery occlusion (MCAO) and photothrombotic stroke models were used in this study. Results: By coupling transfer of labeled myelin oligodendrocyte glycoprotein (MOG) 35-55 -specific (2D2) T cells with tetramer tracking, we show an expansion in reactivity of 2D2 T cells to MOG 97-108 and MOG 103-125 in transient MCAO and photothrombotic stroke models. This reactivity and T cell activation occur in the brain and not in the peripheral lymphoid organs after ischemia. Also, microglia act as antigen-presenting cells (APCs) that effectively present MOG antigen, and depletion of microglia ablates expansion of 2D2 reactive T cells. Notably, the adoptive transfer of neuroantigen-experienced 2D2 T cells exacerbates Th1/Th17 responses and brain injury. Finally, T cell activation and MOG-specific T cells can be seen in the brain of ischemic stroke patients. Conclusion: Our findings suggest that brain ischemia activates and diversifies T cell responses locally, which exacerbates ischemic brain injury.

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells

AbstractAdoptive 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.