Specific TCR Research Articles

Reprogrammed CD4+ T Cells That Express FoxP3+ Effectively Control Inhibitory Antibody Development in Hemophilic Mice

Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) can suppress a range of immune functions, including the activation and proliferation of conventional CD4+ T cells. Inhibitory antibody development to clotting factor replacement therapy in the X-linked disorder hemophilia is dependent on T cell help. Therefore, Tregs play a crucial role in tolerance to the therapeutic replacement protein. Adoptive immunotherapy with autologous or donor Tregs have shown promise in several clinical trials for autoimmune disorders and in transplant conditions. However, antigen-specific Tregs are extremely rare and difficult to isolate. Ectopic overexpression of FoxP3 in conventional CD4+ T cells from hemophilia patients can potentially create a large pool of Tregs, enriched for antigen specificity. Here we show that adoptive immunotherapy with FoxP3 overexpressing CD4+ T cells can control immune responses to clotting factor replacement therapy in a mouse model of hemophilia A. Methods:BALB/c F8 e16−/− hemophilia A mice were repeatedly injected with recombinant B-domain deleted (BDD) FVIII to generate high inhibitory antibody (inhibitor) titers and FVIII-specific T cells. CD4+CD25- conventional T cells were isolated and transduced with pMIGR-eGFP retroviral vector encoding the murine FoxP3 gene (pMIGR-FoxP3-eGFP). 2x106 GFP+ FoxP3+ sorted cells were adoptively transferred into F8 e16−/− mice, and recipients were challenged 1 day later with weekly IV injections of 1 IU BDD-FVIII for 4 weeks. Plasma was tested for inhibitor/antibody formation at 4 and 8 weeks using the Bethesda assay and ELISA. Furthermore, mice with pre-existing inhibitors received adoptive transfer of a single or multiple doses of 2x106 FoxP3+ sorted cells to assess inhibitor reversal. FoxP3+GFP+ sorted cells were adoptively transferred into naïve mice and re-isolated 2 days later to test for phenotypic marker expression, namely CD25, FoxP3, GITR, CD39, CTLA-4, Neuropilin-1, TNFR2, CD127, and CD62L. Antigen-specific suppressive activity was assayed in vitro using FoxP3 transduced CD4+ T cells from transgenic DO11.10 Rag-/- mice with TCR specificity for ovalbumin (OVA). The proliferation of CellTrace Violet labeled DO11.10 Rag-/- responder splenocyteswas calculated in response to stimulation with OVA323-339 peptide. Results:Retroviral transduction of CD4+CD25- cells with the murine FoxP3 gene resulted in a population of cells that phenotypically resemble peripheral Tregs. FoxP3 transduced Tregs underwent antigen specific proliferation in vivo and stably maintained FoxP3 expression in proliferating cells. FoxP3 transduced OVA-specific CD4+ T from DO11.10 Rag-/- mice suppressed the proliferation of transgenic responder cells in response to OVA323-339 peptide antigen. CD4+ T cells transduced with empty vector or freshly isolated natural Tregs were unable to suppress proliferation. Adoptively transferred FoxP3 transduced cells from BDD-FVIII immunized donor mice completely prevented the induction of inhibitors following challenge with repeat injections of BDD-FVIII. FoxP3 transduction of naïve donors (i.e. not FVIII antigen-experienced) were inferior in suppressing inhibitor formation. We are currently testing for reversal / reduction of established inhibitory antibodies with a single or repeat doses of FoxP3 transduced BDD-FVIII immunized donor mice. Conclusions and future directions: Treg based therapies using ex vivo expanded recipient-derived Tregs are currently in clinical trials as primary or adjunct treatment for allograft rejection and autoimmune disease. Here, forced expression of FoxP3 into antigen-specific conventional CD4+ T cells by retroviral gene transfer resulted in a population of antigen-specific cells with phenotypic characteristics of Tregs. These cells effectively suppressed formation of inhibitors in BALB/c F8 e16−/− hemophilia A mice for a sustained 2-month period. We will further explore the mechanism of suppression, including the possibility of endogenous Treg induction via infectious tolerance. DisclosuresBiswas:Bayer Hemophilia Awards Program: Research Funding. Herzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties.

T-cell receptor repertoire of mice with organ-specific autoimmunity resulting from a partial defect in T cell negative selection and dendritic cell enhancement

Abstract Previously, we reported a new genetic model of spontaneous autoimmune disease in which mice with a hypomorphic mutation of Aire (AireGW/+) combined with deletion of Lyn in dendritic cells develop autoimmune uveitis with a 50% penetrance. Here we demonstrate that a key autoantigen in this model is interphotoreceptor retinoid-binding protein (IRBP) as AireGW/+ Lyn−/− IRBP−/− mice failed to develop uveitis. The expansion of CD4+ T cells recognizing amino acids 271–290 (“P2”) of IRBP correlated strongly with autoimmune disease in mice with intact IRBP. To study the role of these T cells, we did single-cell RNA-seq with TCR sequencing for P2-specific CD4+ T cells isolated from eye-draining lymph nodes (LN) of AireGW/+ Lyn−/− mice with and without uveitis. Both mice showed an expansion of these T cells in eye-draining LNs, but it was greater in mice with disease. A remarkable clonal diversity of TCRs was seen, with some sharing of TCR Vα or Vβ sequences. Expansions of particular T cell clonotypes were more evident in LN of mice with disease, and even greater in the retinas of mice with disease. We reconstituted several P2–specific TCR clonotypes in a T cell hybridoma line and verified their specificity. One of the TCR clonotypes was expressed in developing thymocytes by retroviral transduction of bone marrow stem cells from Rag2−/− mice and transplanted into WT or AireGW/+ recipient mice. Remarkably, the small residual expression of IRBP in the thymus of AireGW/+ mice was sufficient to induce substantial negative selection of thymocytes with this TCR clonotype. Thus, deficiency of negative selection of P2 tetramer–specific TCR promotes the development of uveitis in AireGW/+ Lyn−/− mice, even if negative selection is only partly compromised.

Myosin specific TRM cells mediate increased severity of immune checkpoint inhibitor myocarditis

Abstract Immune checkpoint blockade has revolutionized the field of cancer immunotherapy; however, it has been also associated with immune-related adverse events (IRAE). Immune checkpoint inhibitor (ICI)-myocarditis occurs in approximately 1% of patients, however, it has a high fatality rate of up to 50%. To prevent life-threatening toxicities, it is imperative to understand what predisposes patients to ICI-myocarditis. We examined if previous cardiac injury increases an individual’s susceptibility to develop ICI-myocarditis. We demonstrated that ischemic injury substantially changes the phenotype of cardiac lymphocytes, causing them to upregulate the well-characterized tissue resident markers such as CD69 and CD103. We were excited to find that these cardiac tissue resident T (TRM) cells express prominent levels of the checkpoint molecule PD-1 and have high TCR specificity for cardiac myosin. TRM cells have been described in the literature to establish residency in non-lymphoid organs where they provide a rapid functional response more efficiently than circulating memory T cells. To investigate the role that cardiac TRM cells play in the occurrence of ICI-myocarditis, we developed a novel two-hit model. In this model cardiac injury is induced through ischemic injury (first hit), mice are allowed to recover before we begin the administration of immune checkpoint antibodies (second hit). Our data suggest that after the administration of PD-1 blocking antibodies, mice with myosin specific TRM cells are more susceptible to ICI-myocarditis. Further characterization of cardiac TRM cells could provide insight into why checkpoint mediated cardiac toxicities are particularly rapid and have high fatality rates.

Expansion of donor-unrestricted MAIT cells with enhanced cytolytic function suitable for TCR redirection.

Progress in our understanding of MR1-restricted mucosa-associated invariant T (MAIT) cells has raised interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, while preserving their functional response toward their natural ligand and increasing their cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation and expressed a more diverse set of chemokine receptors, potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I–restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.

Abstract PO030: A Novel T-cell Population Expressing the Ectoenzymes CD38 and CD39 is Associated with Melanoma Patient Non-Response to Immunotherapy

Abstract Cancer immunotherapy has emerged as one of the most potent therapeutic tools in treating melanoma and a variety of other malignancies. Immunotherapy includes inhibition of the modulators of T cell antitumor function, the immune checkpoints, by using antibodies against them. In advanced melanoma patients, immune checkpoint inhibitors ipilimumab (IPI) and nivolumab (NIVO) have had unprecedented efficacy. Although many patients respond well to this therapy, many patients remain unresponsive. The mechanism underlying treatment failures remains poorly understood. Using a novel high-dimensional flow cytometry analysis methodology, CytoBrute, we found that in the cohort of advanced melanoma patients receiving sequential NIVO-IPI, high baseline levels of a novel immunophenotype, CD4+CD38+CD39+CD127-GARP- T-cells (median frequency 0.3%), was associated with poor outcomes (disease progression p=0.008, and decreased overall survival p<0.0001 HR = 6.8). Both CD38 and CD39 are ectoenzymes with CD38 hydrolyzing NAD+ to cyclic-ADP ribose and CD39 converting extracellular ATP to immunosuppressive adenosine nucleosides. Both of these proteins are being evaluated as targets to increase the efficacy of immunotherapies. The association of this novel phenotype, which we have termed T-cells expressing ectoenzymes (Tee), was validated in two independent cohorts of melanoma patients treated with NIVO monotherapy, with progressors showing a higher frequency of this immunophenotype at baseline (p=0.047). In IPI+NIVO adjuvant treated melanoma patients, an increase in this population of cells was observed during treatment in relapsing patients, leading to a higher frequency of these cells in relapsing patients relative to patients with no evidence of disease (p=002). Preliminary data obtained from single cell RNA sequencing from the ectoenzyme expressing peripheral blood T cell population, demonstrated predominant expression of IL-32 and CD99 (a membrane glycoprotein associated with lymphocyte diapedesis) in this population, along with several additional clusters distinguished by expression of specific TcR V beta genes. Collectively, we have identified a novel population of ectoenzyme expressing T-cells (CD3+CD4+CD38+CD39+CD127-GARP-) associated with poor melanoma patient outcomes in response to checkpoint immunotherapy. Further investigation of the mechanisms by which this novel T-cell phenotype is associated with poor patient outcomes is under way. Citation Format: Ankita Mitra, David M. Woods, Anjali Rao, Andressa S. Laino, Aidan Winters, Itai Yanai, Pratip K. Chattopadhyay, Jeffrey S. Weber. A Novel T-cell Population Expressing the Ectoenzymes CD38 and CD39 is Associated with Melanoma Patient Non-Response to Immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO030.

The order and logic of CD4 versus CD8 lineage choice and differentiation in mouse thymus

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.

Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+ T Lymphocyte Signature.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRβ) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRβ signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients’ repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.

Abstract 1516: Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy

Abstract Chemotherapy/ targeted therapy are both known to trigger evolution of treatment resistant clones that can lead to relapse. Allogeneic stem cell transplant (alloSCT) for refractory Chronic Lymphocytic Leukemia (CLL) patients is associated with better outcomes. We hypothesized that allogeneic T-cell immunotherapies, including alloSCT and donor lymphocyte infusion (DLI) would impact tumor evolution through the application of selective immunologic pressure with reciprocal changes in the T-cell compartment. Here, we tested a cohort of 24 heavily pre-treated CLL patients treated. Treatments consisted of alloSCT alone, or with follow-up DLI, which are two established mediators of effective Graft versus Leukemia (GVL). Our cohort included 11 patients who relapsed (denoted as non-responder, NR) after alloSCT and 13 patients who had complete response (CR) after alloSCT, with 11/13 patients showing durable CR with a median post-transplant overall survival (OS) of 9.8 years. We mapped the evolutionary trajectories of tumor cells by whole exome sequencing (WES) of sort purified CLL in post-transplant relapsed patients. To investigate changes in immune repertoire and gene expression post-transplant, CD3 positive T-cells from peripheral blood and bone marrows of CLL patients at complete donor chimerism were analyzed both at bulk and at the single cell level. We found evidence of subclonal leukemic evolution in the majority of our CLL patient cohort after nonmyeloablative HLA-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. In all of the 11 patients with longitudinal post-alloSCT samples available, we observed branched CLL evolution in 4 patients, linear evolution in 4 patients, and no evolution in 3 patients. These data suggest that differential sensitivity of leukemic subclones to allogeneic T cell killing may underlie the branched and linear evolution that we observed, and therefore can shape leukemic subclonal architecture after transplant. Of note, we found that clonal CLL was more responsive to alloSCT in comparison to CLL with subclonal disease architecture.To identify T-cells with GVL potential, we first cataloged potential neoantigens by screening mutated regions in CLL with in silico HLA binding prediction models. Neoantigen specific T-cells were then sorted from longitudinal peripheral blood samples using tetramers, followed by identification of GVL specific TCR in both bulk and single cell setting. We were able to identify T-cells that coevolved with specific tumorigenic lesions in a subset of CLL patients. Taken together, our results suggest that donor-derived antigen-specific T-cells mediate clonal selection of CLL with concurrent changes in allogeneic T-cells, and that these changes can be monitored in longitudinal patient samples. Citation Format: Celine Kerros, John P. Miller, Xizeng Mao, Haven R. Garber, Hannah C. Beird, Jianhua Zhang, Jason Roszik, Paul Leonard, Li Zhao, Sahil Seth, Pei Lin, Huandong Sun, William G. Wierda, Issa F. Khouri, Karen Clise-Dwyer, Andrew Futreal, Shoudan Liang, Koppikar Priya, Jeffrey Molldrem. Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1516.

Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity.

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

Interferons in Treg development and function

Abstract Thymus-derived regulatory T-cells (Tregs) are critical for controlling immune responses and preventing autoimmunity. In the setting of viral infection, Tregs are important modulators of anti-viral immune responses and help resolve inflammation. The subset of Tregs responsible for responding to viral infections and the types of antigens they recognize are not well understood. Using single-cell RNA sequencing we identified a unique subset of thymic Treg progenitors and mature Tregs that express a strong interferon stimulated gene (ISG) signature, as well as a comparable population in the spleen. Importantly, the ISG signature is much stronger than that expressed by thymocytes receiving tonic IFN stimulation in the final stages of maturation prior to thymic egress. Using flow cytometry we established that APCs in the thymus, including mTECs, as well as SIRPα+ and CD8α+ dendritic cells, require IFNα/β or IFNλ, acting via STAT1 signaling, for the expression of the canonical ISG BST2. TCR specificity was important for selection into this subset as SM1 TCR transgenic mice, which express a TCR that recognizes a non-ISG encoded antigen, have a reduced compartment of ISG-expressing CD4SP T-cells in the thymus. We are currently generating mice that specifically lack the ISG-signature Treg subset to test their function in vivo. Based on our current findings, we propose that thymic ISG antigens serve as TCR ligands for inducing the differentiation of ISG-signature Tregs that are in close physical proximity to the ISG-producing APCs. Furthermore, we hypothesize that ISG-signature Tregs are specialized for responding to ISGs induced during antiviral immune responses and play a key role in controlling IFN-driven inflammation.

Single-cell TCR analysis of mouse lung Vγ4 γδ T cells reveals a link between TCR clonotypes, SLAM family receptor expression, and effector function

Abstract γδ T cells are unusual T cells that are highly enriched in mucosal tissues where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. The TCR usage of tissue-specific γδ T cells is often associated with specific effector functions. We have recently found that IL-17- and IFN-γ-producing Vγ4 γδ T cells in the mouse lung are marked by the expression of SLAMF1 and SLAMF6 receptors, respectively. The objective of this study was to investigate a possible link between SLAM-associated effector function and γδ TCR usage. We first identified the major Vγ4 γδ TCR clonotypes in the mouse lung (n=13 mice). Vγ4 γδ T cells were single cell-sorted and paired TCR clonotypes were identified using next-gen sequencing of TCR amplicon libraries. The data revealed that TRDV5, TRDV2, and TRDV7 chains accounted for 51.18%, 29.61%, and 14.96% of the productive TCRδ chain rearrangements, respectively. While TRDV5 and TRDV2 CDR3 sequences were limited in diversity, TRDV7 CDR3 sequences were highly diverse. A significant fraction (30.46%) of the TRDV5 sequences were characterized by an invariant germline-encoded Vδ5Dδ2Jδ1 sequence. A comparison between sorted SLAMF1+ and SLAMF6+lung Vγ4 γδ T cells revealed that TRDV5 and TRDV2 chains were predominantly associated with SLAMF1+IL-17+ γδ T cells while the TRDV7 chain was predominantly associated with SLAMF6+IFN-γ+ γδ T cells. Moreover, the invariant germline-encoded TRDV5 sequence was primarily associated with SLAMF1+IL-17+ cells. These data indicate that the lung Vγ4 γδ TCR repertoire is limited in diversity and that specific lung Vγ4 γδ TCR clonotypes segregate with the expression of discrete SLAM family receptors.

T cell receptor repertoire features display universal rules for selection and plasticity in the functional CD4 T cell subsets

Abstract Helper T cell choice of functional subset may depend on the mode and strength of T cell receptor interaction with peptide-MHC complex on antigen presenting cells. However, the extent, complexity, and homogeneity of this influence of TCR structure on the selection into a functional subset, as well as further stability of acquired clonal programs remain poorly studied. Here we present the first analysis of deep sequenced T-cell receptor repertoires of the eight effector CD4 T cell subsets (Th1, Th2, Th2a, Th17, Th1-17, Th22, Treg, and Tfh) from peripheral blood of 5 healthy donors, revealing unexpectedly prominent, multivariate subset-specific differences that are highly reproducible across unrelated donors. Similar analysis performed for the sorted naive CD4 T cell subsets showed that specific TCR features of Tregs (short and strongly interacting CDR3 region) are rooted in thymic selection, while differences between other subsets accumulate later upon T cell functional engagement. We next provide the first deep analysis of the plasticity of human effector CD4 subsets ex vivo. We demonstrate high plasticity within Th17 and Th22 functional subsets and prominent clonal exchange of both Th17 and Th22 with Th2. Th1-17 subset shares clones with Th1 but not with the Th17 subset. Finally, we investigate relative publicity of effector CD4 subset repertoires across donors. We reveal high publicity of Treg and Tfh, and high privacy of Th22 and Th2a repertoires, which corresponds to the number of added N-nucleotides for these subsets, suggesting their earlier fetal and evolutionary origin. Altogether, we provide the first detailed picture of distinct repertoire features, plasticity, and publicity of helper CD4 T cell subsets.

Regulation of thymic T-cell development by intestinal microbiota

Abstract Host commensal microbiota plays an important role in the development and functioning of a healthy immune system. Early life interference with commensal colonization is associated with immune disorders like inflammatory bowel disease (IBD), asthma, allergic rhinitis and atopic dermatitis. One important aspect of microbiota mediated immune regulation is its impact on T-cell differentiation and function. Although most studies show that microbial regulation of T-cells is limited to the periphery, some studies have suggested that the microbiota might impact antigen specific T-cell development in the thymus. However, there has been insufficient data. Interestingly, we are able to detect bacterial signatures by 16s sequencing within mouse and human thymus. Additionally, we observed development of antigen specific thymocytes in SFB colonized 7B8 TCR transgenic mice and in wildtype mice through SFB tetramer staining. We therefore, hypothesize that antigen-presenting cells (APCs) from the gut sample local microbial antigens and migrate to the thymus to present them to the developing thymocytes and influence selection. To test this hypothesis, we tracked the migration of intestinal APCs to the thymus using mice that express the photoconvertible Kikume protein. We performed targeted photo-conversion of the cecum through live surgery and discovered photoconverted APCs in the thymus 48 hours later. These observations open up the possibility that intestinal APCs migrate to the thymus early in life to shape the microbiota specific TCR repertoire.

Tumor-specific CD8 T cell activation in draining lymph nodes supports the anti-tumor CD8 T cell response

Abstract CD8 T cells are a critical part of the immune response to tumors, with CD8 T cell infiltration predicting disease progression in many types of cancer. Recent work in CD8 T cell immunology described how CD8 T cells respond to chronic diseases, finding two subsets of CD8 T cells within tumors. One is a stem-like CD8 T cell and the other is a terminally differentiated CD8 T cell with cytotoxic capabilities. Determining how tumor-specific CD8 T cells activate and differentiate is critical to understanding why some tumors are highly infiltrated. To study how tumor-specific CD8 T cells are activated, I have made a prostate cancer model that expresses the viral LCMV glycoprotein (GP) which acts as a tumor-specific antigen. We have used this model to study tumor-specific CD8 T cell activation by adoptively transferring LCMV GP specific TCR transgenic P14 CD8 T cells into TRAMPC1-LCMV-GP bearing mice. We have found when tumor-specific CD8 T cells are activated in the tumor-draining lymph node they acquire an undifferentiated but activated program, upregulating CD44, PD1 but retaining high TCF1 and CD62L expression. These undifferentiated activated CD8 T cells do not acquire a typical effector program that is seen in an acute viral infection such as LCMV Armstrong, yet they are able to migrate to the tumor to establish the anti-tumor response. In conclusion, tumor-specific CD8 T cells do not acquire an effector program after activation and instead gain an undifferentiated activated phenotype. These data suggest that tumor-specific CD8 T cells are activated in the TDLN and differentiate to become the stem-like CD8 T cells within the tumor, establishing the anti-tumor CD8 response.

Comprehensive profiling of TCR and BCR repertoire of infiltrated lymphocytes in the mouse injured skeletal muscle

Abstract Lymphocytes infiltrating to the injured skeletal muscle have been shown to play an important role in muscle regeneration. Site-specific infiltrating/expanding lymphocytes and its molecular features are potential resources for bioengineering such as a drug delivery system. Although some previous studies showed the accumulation of lymphocytes to the injured skeletal muscle, the actual frequency of distinct lymphocyte clones in situ is still unclear. Hence, a comprehensive picture of the lymphocyte repertoire has yet to be described in detail. To this end, we conducted a comprehensive profiling of TCR and BCR repertoire of infiltrating lymphocytes in the mouse injured skeletal muscle model by next-generation sequencing. In the TCR repertoire profiling, we found that the expression level of TCR transcripts contains unique CDR3 α and distinct population of CDR3 β were increased (7.6–17% and 6.7–15%) in infiltrating T cells in both of cardiotoxin- or cryo-injured skeletal muscle, whereas no clonal expansion of specific TCR α/β transcripts observed in control splenic T cells (<1% respectively). While in the BCR repertoire profiling, various transcripts of immunoglobulin kappa chains showed expansion in infiltrated fraction, despite the proportion of immunoglobulin heavy and lambda chain were remained similar to control splenic B cells. Together these data have allowed us to identify TCR/BCR candidates targeting injured skeletal muscles. Furthermore, we aim to utilize the T cells/B cells expressing high-affinity TCR/BCR for the drug delivery system, especially for large protein complexes.

Clinical and immune responses to anti-CD3 x anti-EGFR bispecific antibody armed activated T cells (EGFR BATs) in pancreatic cancer patients

ABSTRACT Purpose This was a phase I/II adoptive T cell trial in 7 locally advanced and metastatic pancreatic cancer patients using 3–8 infusions of anti-CD3 x anti-EGFR bispecific antibody armed activated T cells (BATs) to determine safety, the maximum tolerated dose (MTD), immune responses, time to progression (TTP), and overall survival (OS). Study Design: T cells obtained by apheresis were expanded and armed with EGFRBi, cryopreserved for infusions. In a phase I dose escalation, five patients received three weekly infusions of 10–40 × 109 BATs/infusion followed by a booster infusion 3 months later, and 2 patients received 8 infusions twice weekly for 4 weeks in a phase II. The trials were registered at http://www.clinicaltrials.gov, NCT01420874 and NCT02620865. Results: There were no dose-limiting toxicities (DLTs), and the targeted dose of 80 × 109 BATs was met. The median TTP is 7 months, and the median OS is 31 months. Two patients had stable disease for 6.5 and 25+ months, and two patients developed complete responses (CRs) after restarting chemotherapy. Infusions of BATs induced anti-pancreatic cancer cytotoxicity, innate immune responses, cytokine responses (IL-12, IP-10), and shifts in CD4 and CD8 Vβ repertoire with enhanced cytoplasmic IFN-γ staining in the Vβ repertoire of the CD8 subset that suggest specific clonal TCR responses. Conclusions: Infusions of BATs are safe, induce endogenous adaptive anti-tumor responses, and may have a potential to improve overall survival.

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.

Evaluation the potential of recombinant anti-CD3 nanobody on immunomodulatory function

T cells are the most predominant effector cells in immune-mediated elimination of cancer and circumventing tumor progression. Among various approaches, T cells activation by specific antibodies independently of their TCR specificity, is considered as an effective approach to circumvent tumor progression. The most common surface marker for all T cells which is crucial for T cell activation is regarded as CD3. Therefore, the goal of our study was to evaluate the preclinical efficacy of recombinant anti-CD3 nanobody. To this end, anti-CD3 sequence, was PCR amplified, following cloning and expression in E.coli and purification, the purified nanobody with a molecular weight of ∼17 kDa was confirmed by western blot. Furthermore, flow cytometry analysis demonstrated that purified nanobody could bind to CD3 on Jurkat cell line. Subsequently, results from inoculation of 3 μg/g of nanobody to tumor bearing balb/c mice indicate inhibition of tumor growth. Furthermore, circulating levels of tumoricidal cytokines such as IL-2 and IFNγ were raised whereas tolerogenic cytokines such as IL-4, 6 and 10 were decreased at the end of the treatment. Moreover, IHC analysis confirmed the presence and also the percentage of TILs in tumor sites in response to anti-CD3 therapy. Hence, our results suggest that the purified anti-CD3 nanobody may become a promising candidate for targeting and activating CTLs to induce anti-tumor responses and may provide groundwork for future studies involving other kind of cancers.

Novel Cellular Immunotherapy Using Allogeneic Vγ9/δ2-T Cells Gene-Modified to Express HTLV-1 P40Tax-Specific TCR for the Treatment of Adult T Cell Leukemia

Background: Adult T-cell leukemia/lymphoma (ATL) is a refractory peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection. Although only allogeneic hematopoietic stem cell transplantation (allo-HSCT) displaying the graft-vs. ATL (GvATL) can bring durable remission, allo-HSCT is largely ineligible for newly diagnosed ATL patients due to disease aggressiveness and advanced age-related conditions. Thus, a novel treatment with safety and efficacy instead of allo-HSCT still remains an unmet need, and a cellular immunotherapy using TCR or CAR gene-modified immune cells exerting GvATL could be such an option. However, to generate those effector cells from autologous T cells of heavily pre-treated ATL patients faces many obstacles. To circumvent those hurdles, an employment of unconventional allogeneic Vγ9/δ2-T cells which are potentially free from the risk of GVHD could provide greater treatment opportunity for ATL patients due to highly extended donor availability. Taking above, here, we have newly devised an adoptive immunotherapy using a novel HTLV-1 p40Tax-specific TCR gene-modified allogeneic Vγ9/δ2 T cells against ATL. Methods: After written informed conscent, we firstly established novel HLA-A24 restricted TCR-α/β genes from HTLV-1 P40Tax301-309 (SFHSLHLLF)/HLA-A24 tetramer-positive peripheral CD8+T-lymphocytes of ATL patinets in durable remission using a single cell cloning method. Then, we confirmed that T cells gene-modified with these TCR-α/β genes exerted the epitope-specific and HLA-A24-restricted responses. Next, in order to achieve highly stable expression of this TCR-α/β heterodimer on gene-modified Vγ9/δ2-T cells, we newly developed a retroviral vector co-expressing TCR-α/β and CD8 α/β genes using self-cleaving P2A and E2A peptides. Using this vector, allogeneic Vγ9/δ2-T cells from healthy donors numerously expanded with high purity in our novel culture system were subjected to gene-transfer to express relevant TCR α/β complex. Thereafter, we asssessed target-reactive cytokine production and cytocidal activity mediated by those gene-modified allogeneic Vγ9/δ2-T cells both in vitro and in vivo. Finally, we additionally assessed a potential risk of GVHD using intravenous administration of another TCR gene-modified Vγ9/δ2 T-cells in vivo. Results: To start with PBMCs from healthy donors, allogeneic Vγ9/δ2-T cells were stably multiplied greater than thousandfold with a quite high purity (≥95%) using our novel bisphosphonate derivative PTA (tetrakis-pivaloyloxymethyl2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate) combined with both 25 ng/ml of IL-7 and IL-15 in culture for 8 to 10 days. The stable expression of introduced TCR α/β heterodimer on Vγ9/δ2-T cells were successfully achieved by co-expression of CD8 α/β molecule. Those gene-modified Vγ9/δ2-T cells successfully recognized target peptide (SFHSLHLLF) in an HLA-A24 restricted fashion, and similarly demonstrated a cytocidal activity both in vitro and in vivo against HLA-A24 positive HTLV-1 infected cell lines (TL-Su and ILT#Hod), but not HLA-A24-negtive/Tax-positive cell line ILT#37 or HLA-A24-positve/Tax-negative cell line ATN-1. Furthermore, intravenously administered those TCR gene-modified Vγ9/δ2-T cells quickly and durably eradicated luciferase-gene modified TL-Su cells, but not ATN-1 cells in xenografted immunodeficient (NOG) mice, examined by in vivo imaging system. Finally, infused HLA-A2 restricted and NY-ESO-1 specific TCR (G50) gene-modified Vγ9/δ2-T cells exerted durable antitumor activity without causing GVHD using NOG mice xenografted with HLA-A2 positive melanoma cell line cells (NW-MEL-38). Conclusions: Our preclinical observations here obviously demonstrated the potential utility of TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells for the treatment of ATL without causing GVHD. Further studies regarding biological behaviors of HTLV-1 Tax specific TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells following target recognition in vivo are warranted, however, based on these lines of evidence and currently conducting assessments using clinical samples, we are planning to launch a novel clinical trial, particularly focusing on the applicability of HLA partially matched relative donors, as the source of gene-modified allogeneic Vγ9/δ2-T cells, which could highly extend the donor availability. Disclosures Fujiwara: BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPath Bio. Okumura:BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPth Bio.. Miyahara:BirghtPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Wan:BrightPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Tawara:Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Shiku:BrightPath Biotherapeutics, Co., Ltd.: Other: Chair of the department endowed by BrightPath Bio..

Single Cell RNA Sequencing Identifies Transcriptional Programs That Enhance Anti-Tumor Function of Transgenic CD4+ T Cells Redirected with TCR and CD8αβ

Introduction:Transgenic co-expression of a major histocompatibility complex class I restricted tumor associated antigen specific TCR and CD8αβ (TCR8) has been previously proposed as a strategy to redirect CD4+ T cells to tumors. However, it is unknown whether forced TCR8 expression induces more fundamental transcriptional consequences in both CD4+ and CD8+ T cells, and whether T cell lineage origin affects this outcome. Here we deeply interrogate the effects of transgenic TCR and TCR8 in human CD4+ and CD8+ T cells upon leukemia challenge by single cell RNA sequencing (scRNAseq) and investigate T cell function in vitro and in vivo. We identify profound changes of gene expression that have significant functional consequences. Methods:A previously characterized HLA-A*02:01 restricted survivin-specific TCR was used (Arber et al, JCI, 2015 Jan;125(1):157-68) and a new polycistronic vector with this TCR and CD8αβ was generated. CD4+ and CD8+ T cells were isolated and scRNAseq (25'474 cells in total) was performed on (1) freshly isolated cells, (2) retrovirally transduced (TCR or TCR8) expanded cells, and (3) TCR+CD8+, TCR8+CD8+ and TCR8+CD4+ T cells co-cultured with BV173 leukemia cells (HLA-A*02:01+survivin+). scRNAseq results were cross-validated in independent experiments with FACS analysis of selected markers, in vitro stress-killing assays, analysis of cytokine production, and assessment of anti-tumor function in vivo in xenograft mice. Results:CD4+ T cells only killed BV173 leukemia cells when redirected with TCR8 but not with TCR alone (p=0.0004, n=7), while killing by TCR+CD8+ and TCR8+CD8+ T was comparable (p=NS). To explore some of the possible underlying mechanisms, we used dimensionality reduction and unsupervised clustering of the scRNASeq data and identified 19 distinct cell clusters. CD4+ and CD8+ lineage origin clearly separated the samples, but separation by transgene type only became apparent upon co-culture. Analyzing differentially expressed genes, we found that co-cultured samples contained clusters with high expression of cytotoxic markers but with significant differences between CD4+ and CD8+ lineages (e.g. transcription of GZMB in CD4+ T cells and GNLY, NKG7, GZMK in CD8+ T cells). Next, we analyzed which genes were upregulated from the expanded to co-cultured states. Co-cultured TCR8+CD4+ T cells had more upregulated genes with a broader diversity compared to TCR+CD8+ or TCR8+CD8+ T cells. Among these upregulated pathways were cytotoxicity, co-stimulation, oxidative phosphorylation, NFkB regulation, cell growth and transcription factors. TCR8+CD4+ T cells also retained a less differentiated phenotype (e.g. high IL7R, SELL, CCR7, CXCR4) with preservation of replicative potential. Furthermore, co-cultured TCR8+CD4+ T cells expressed more co-stimulatory and less activation/ exhaustion markers. In addition, co-cultured TCR8+CD4+ T cells heavily relied on oxidative phosphorylation and had higher mitochondrial activity compared to co-cultured TCR+ or TCR8+ CD8+ T cells. In stress co-cultures with multiple rounds of tumor challenge, TCR8+CD4+ T cells outperformed TCR+CD8+ T cells (number of killings TCR8+CD4+ vs TCR+CD8+: 3.3±0.5 vs 1.3±1.1, p=0.01, n=7), but were comparable to TCR8+CD8+ T cells (TCR8+CD4+ vs TCR8+CD8+: 3.3±0.5 vs 2±1.4,p=NS, n=7). TCR8+CD4+ T cells expanded significantly better than TCR+CD8+ T cells (p=0.002) and TCR8+CD8+ T cells (p=0.015) and produced TH1 type cytokines. In the xenograft mouse model, we observed significant BV173 leukemia control in mice treated with TCR+CD8+ T cells compared to controls (NT), and further enhancement in mice treated with TCR8+CD8+ T cells (NT vs TCR: p=0.0002, NT vs TCR8: p<0.0001, TCR vs TCR8: p=0.01, n=5). TCR8+CD4+ T cells also significantly delayed leukemia progression compared to TCR+CD4+ or NT T cells (p=0.001, n=5). Conclusion:Transgenic TCR8 expression has previously been proposed as a strategy to enhance TCR-pMHC recognition. Here we identify profound transcriptional changes involving multiple pathways that are important for sustained anti-tumor function upon adoptive T cell transfer in vivo, such as cytotoxicity, co-stimulation, cell cycle and metabolism. Our results point towards previously unrecognized mechanisms by which TCR8 transgenes mediate their beneficial effect in both CD4+ and CD8+ T cells. Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees. Arber:Cell Medica: Patents & Royalties.