High-affinity TCRs Research Articles

Biology-based AI Predicts T-cell Receptor Antigen Binding Specificity

Adoptive cell transfer (ACT) using T cells modified by the T cell receptor (TCR) gene is an exciting and rapidly developing field. Numerous preclinical and clinical studies have shown varying feasibility, safety, and efficacy of using TCR-engineered T cells to treat cancer and viral infections. Although there is evidence that their use is effective, to what extent and how these therapies can be improved is still a question of research. Since TCR affinity has been generally accepted as the primary role in defining T cell specificity and sensitivity, selecting and generating high-affinity TCRS remains a fundamental approach to designing more effective T cells. However, the traditional approach of increasing affinity by random mutagenesis can cause adverse cross-reactions that result in on-target and off-target adverse events, produce depleted effectors through overstimulation, and ignore other kinetic and cellular parameters that have been shown to affect antigen specificity. In this paper, we review the preclinical and clinical potential of TCR-modified T cells, summarize contributions that challenge the role of TCR affinity in antigen recognition, and explore how structure-guided design can be used to manipulate antigen specificity and TCR cross-reactivity to improve the safety and efficacy of TCR-modified T cells for ACT.

Targeting the recurrent Rac1P29S neoepitope in melanoma with heterologous high-affinity T cell receptors.

Recurrent neoepitopes are cancer-specific antigens common among groups of patients and therefore ideal targets for adoptive T cell therapy. The neoepitope FSGEYIPTV carries the Rac1P29S amino acid change caused by a c.85C>T missense mutation, which is the third most common hotspot mutation in melanoma. Here, we isolated and characterized TCRs to target this HLA-A*02:01-binding neoepitope by adoptive T cell therapy. Peptide immunization elicited immune responses in transgenic mice expressing a diverse human TCR repertoire restricted to HLA-A*02:01, which enabled isolation of high-affinity TCRs. TCR-transduced T cells induced cytotoxicity against Rac1P29S expressing melanoma cells and we observed regression of Rac1P29S expressing tumors in vivo after adoptive T cell therapy (ATT). Here we found that a TCR raised against a heterologous mutation with higher peptide-MHC affinity (Rac2P29L) more efficiently targeted the common melanoma mutation Rac1P29S. Overall, our study provides evidence for the therapeutic potential of Rac1P29S-specific TCR-transduced T cells and reveal a novel strategy by generating more efficient TCRs by heterologous peptides.

γ9δ2 T-Cell Expansion and Phenotypic Profile Are Reflected in the CDR3δ Repertoire of Healthy Adults

γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire–phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics.

Abstract 3604: PSC-derived T cells as a novel source of tumor antigen-specific T cell receptors

Abstract T cell receptor (TCR)-engineered T cells are a promising approach to cell therapy for cancer. TCRs targeting public tumor associated antigens (TAAs) offer the widest potential coverage of patients within and across tumor types. TAA-specific TCRs, however, are rare in the blood and when detected are frequently of low affinity due to negative selection of high-affinity self-antigen reactive T cells in the thymus. Here, we present a TCR discovery platform based on in vitro generation of T cells from human pluripotent stem cells (PSCs) using the previously described artificial thymic organoid (ATO) system. We hypothesized that ATO-derived T cells offer several advantages for TCR discovery including a novel TCR repertoire and the absence of negative selection against TAAs during T cell development. Sequencing of the TCR repertoire of polyclonal CD8+ T cells generated from a HLA-A*02:01+ PSC line confirmed low junctional diversity at both the TCR beta and alpha loci, consistent with our previous finding of low TdT expression during T cell development from PSCs and an enrichment for germline-like TCRs. We then interrogated this novel TCR repertoire for reactivity against two well-described HLA-A2-restricted epitopes of NY-ESO-1 and WT1, reactivities to which are rare in the blood. Following co-culture with artificial APCs, a surprisingly high frequency of antigen-specific T cells was detected by MHC-tetramer staining. From these, high frequency TCR clonotypes were functionally validated by reconstitution in Jurkat TCR-reporter cells. Interestingly, from a single experimental run multiple antigen-specific TCRs were validated (5 out of 10 tested clonotypes for NY-ESO-1, and 2 out of 5 for WT1). This finding was reproducible across independent experimental runs, yielding further validated TCRs against these targets. To benchmark relative TCR affinities, we compared a panel of 5 ATO-derived NY-ESO-1 TCRs with 4 blood-derived TCRs with the same specificity, as well as the affinity-enhanced 1G4 TCR. ATO-derived TCRs showed not only superior performance to their blood derived counterparts in peptide dilution assays, but TCRs were identified with equivalent or superior cytotoxicity to 1G4, including against cell lines with endogenous NY-ESO-1 expression. Comparative in vivo anti-tumor efficacy studies are in process. A similarly high level of specificity and cytotoxicity was observed for ATO-derived WT1-specific TCRs. Finally, we demonstrate proof-of-concept identification of TCRs specific to a prostate-specific antigen for which high quality TCRs have remained elusive. In conclusion, the PSC ATO TCR-discovery platform efficiently identifies high-affinity TAA-specific TCRs, including against previously intractable targets. Considering the potential for MHC modularity of this in vitro system, this approach may theoretically enable rapid discovery of therapeutic TCRs based on any designated peptide/MHC-I combination. Citation Format: Chloe S. Wang, Suwen Li, Zhiyuan Mao, Olivia Zhou, Carlos Botero, William Satyadi, Patrick Chang, Sang Pil Yoo, Ho-Chung Chen, Shawn Lopez, Jami McLaughlin, Amélie Montel-Hagen, Owen N. Witte, Gay M. Crooks, Christopher S. Seet. PSC-derived T cells as a novel source of tumor antigen-specific T cell receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3604.

Breakdown of the blood brain barrier in Experimental Cerebral Malaria is caused by CD8+ T cells with low affinity TCRs

Abstract Malaria is a worldwide health concern killing over 400,000 people each year, many of them children under 5 years old. One severe outcome of Plasmodium infection is cerebral malaria caused by a dysregulation of the immune response in the central nervous system. Using an established mouse model of cerebral malaria called experimental cerebral malaria(ECM) using Plasmodium berghei ANKA (PbA) infections of C57BL/6J mice have shown that breakdown of the blood brain barrier is mediated by IFN-g producing CD8 T cells. Here, using novel techniques to determine the affinity of the MHC:peptide:TCR interaction we have discovered that although there are high affinity cells in the population trafficking to the CNS, surprisingly a large majority of these PbA-reactive pathogenic CD8 T cells have low affinity TCRs, and a large percentage of them cannot be detected by tetramer. Importantly we show that these low affinity cells are also producing IFN-g suggesting they are pathogenic. Through adoptive transfer experiments we show that high and low affinity T cells remain stable in their affinity profile. Furthermore we show here that low affinity cells are needed for breakdown of the blood brain barrier – CD8 T cells bearing high affinity TCRs for the GAP50 peptide of Plasmodium have an impaired ability to cause ECM compared to low affinity CD8 T cells. This work demonstrates that low affinity cells are the major component of the immunopathology causing ECM when infected with PbA and contrasts with current dogma which suggests that high affinity cells are the main cells responding in these types if infections. Supported by grants from NIH (R01 AI167422)

Engineering Cancer Antigen-Specific T Cells to Overcome the Immunosuppressive Effects of TGF-β.

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-β, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-β. Truncating the intracellular signaling domain from TGF-β receptor (TGFβR) II produces a dominant-negative receptor (dnTGFβRII) that dimerizes with endogenous TGFβRI to form a receptor that can bind TGF-β but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-β inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFβRII (e.g., GSK3845097). TGF-β isoforms and a panel of TGF-β-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-β-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFβRII may therefore improve the efficacy of TCR-transduced T cells.

Generation of a HuTCR mouse platform-derived MAGE-A1-directed high-affinity TCR with superior potency versus human-derived TCRs.

e14515 Background: As cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are suppressed via negative thymic selection. Therefore, patient- or donor-derived TCRs are typically of low affinity and result in a reduced antitumor effect. Using our proprietary HuTCR platform, which consists of mouse lines carrying the full human TCR α/β loci in combination with common human HLA alleles, we have isolated high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development. Furthermore, we validated MAGE-A1 as a potential cancer therapy target by using immunohistochemistry to evaluate expression in several major tumor types and healthy tissue. Methods: Using scRNAseq, TCRs were isolated from HuTCR mice. Human-derived MAGE-A1-specific TCR sequences were obtained from publicly available databases. All TCRs were expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional avidity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope peptides and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by co-culture with a panel of tumor cell lines varying in MAGE-A1 and/or MHC-class-I expression. MAGE-A1 expression on protein level was evaluated by immunohistochemistry. Results: Immunization of HuTCR mice with the antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR affinities ranging from 0.3 nM to 3 nM. By comparison, human-derived TCRs exhibited generally lower functional avidity from 3 nM to 60 nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cell lines expressing low MAGE-A1 and/or HLA-A2. Immunohistochemical analysis of MAGE-A1 expression in healthy tissues demonstrated highly selective expression of MAGE-A1 in testis, only. Screening for expression confirmed that a significant proportion of several major cancer types expresses MAGE-A1 as reported by various other groups [reviewed in Curr Opin Cell Biol. 2015 December; 37: 1–8]. Conclusions: The HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. In addition, it was confirmed that MAGE-A1 has a highly selective expression pattern in healthy tissues (testis, only), but shows distinct expression in several major human tumor types.

Enhanced target-specific delivery of docetaxel-loaded nanoparticles using engineered T cell receptors.

For effective targeted therapy of cancer with chemotherapy-loaded nanoparticles (NPs), antigens that are selective for cancer cells should be targeted to minimise off-tumour toxicity. Human leukocyte antigens (HLAs) are attractive cancer targets as they can present peptides from tumour-selective proteins on the cell surface, which can be recognised by T cells via T cell receptors (TCRs). In this study, docetaxel-loaded polymeric NPs were conjugated to recombinant affinity-enhanced TCRs to target breast cancer cells presenting a tumour-selective peptide-HLA complex. The TCR-conjugated nanoparticles enabled enhanced delivery of docetaxel and induced cell death through tumour-specific peptide-HLA targeting. These in vitro data demonstrate the potential of targeting tumour-restricted peptide-HLA epitopes using high affinity TCR-conjugated nanoparticles, representing a novel treatment strategy to deliver therapeutic drugs specifically to cancer cells.

TCR affinity influences helper T cell differentiation by biasing dendritic cell interactions

Abstract Naïve CD4+ T lymphocytes differentiate into various subsets when their TCRs detect microbial peptide-MHCII complexes presented by dendritic cells. However, the mechanism by which TCR signaling influences differentiation is unknown. We found that low affinity TCRs biased naïve T cells to become B cell helpers while high affinity TCRs promoted the phagocyte helper fate. We created software for high-throughput, multicolor, quantitative imaging called Chrysalis to determine if differentiation was influenced by the type of dendritic cell that the naïve T cell interacted with. Our results show that T cells with high affinity TCRs had prolonged interactions with XCR1+ dendritic cells and tended to become phagocyte helpers, while T cells with lower affinity TCRs preferentially interacted with SIRPα+ dendritic cells and tended to become B cell helpers. Thus, TCR affinity influences T cell differentiation by dictating interactions with distinct dendritic cell types.

Profiling TCR affinity distribution in human CD8+ T cell subsets responding to a cytomegalovirus epitope

Abstract CD8 T cell subsets have long been distinguished using phenotypic markers like CCR7, CD45RA, and IL-7Rα. These markers have allowed for the isolation of CD8 T cell populations with distinct functional and proliferative capacities as well as those that maintain better proliferative responses in vivo without antigenic stimulation thus contributing more to immunological memory. Although phenotypic analysis has allowed for the discerning of functional, proliferative, and homeostatic proliferative potential, there is still little known about the role TCR affinity plays in recruitment of various affinity TCRs into memory and effector phenotypes that generate long term immunological memory in human infection. Using the newly developed iTAST technique, CMV specific CD8 T cells were isolated from the blood and spleen of human patients seropositive for CMV. CMV specific T cells were split into memory and effector subsets using phenotypic markers CCR7, CD45RA, and IL-7Rα before isolation and quantification of 2D TCR affinity and sequence. A roughly 100-fold difference of 2D affinity was present in CMV specific cells within the immunological memory response; however, with an enrichment for high affinity TCRs across all phenotypes assessed. This is consistent with previous studies demonstrating high avidity TCR selection in CD8 T cell responses to CMV infection, coupled with a greater clonality observed in CMV specific TCRs within healthy older individuals infected with CMV. Overall the iTAST technique allows for quick and easy isolation of antigen specific populations, quantification of TCR affinity, and TCR sequence thus giving unique insight into immunological memory responses in individuals infected with human persistent viruses.

Comparison of T Cell Activities Mediated by Human TCRs and CARs That Use the Same Recognition Domains.

Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αβ heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at higher surface levels than TCRs, they were 10-100-fold less sensitive, even in the absence of the CD8 coreceptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high Ag density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high Ag density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for the next-generation design of receptors used in adoptive T cell therapies.

DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers.

Rare patients who spontaneously control HIV replication provide a useful model to inform HIV vaccine development. HIV controllers develop particularly efficient antiviral CD4+ T cell responses mediated by shared high-affinity TCRs. To determine whether the candidate DNA vaccine ADVAX could induce similar responses, we analyzed Gag-specific primary CD4+ T cells from healthy volunteers who received ADVAX DNA by electroporation. Vaccinated volunteers had an immunodominant response to the Gag293 epitope with a functional avidity intermediate between that of controllers and treated patients. The TCR repertoire of Gag293-specific CD4+ T cells proved highly biased, with a predominant usage of the TCRβ variable gene 2 (TRBV2) in vaccinees as well as controllers. TCRα variable gene (TRAV) gene usage was more diverse, with the dominance of TRAV29 over TRAV24 genes in vaccinees, whereas TRAV24 predominated in controllers. Sequence analysis revealed an unexpected degree of overlap between the specific repertoires of vaccinees and controllers, with the sharing of TRAV24 and TRBV2 public motifs (>30%) and of public clonotypes characteristic of high-affinity TCRs. MHC class II tetramer binding revealed a broad HLA-DR cross-restriction, explaining how Gag293-specific public clonotypes could be selected in individuals with diverse genetic backgrounds. TRAV29 clonotypes also proved cross-restricted, but conferred responses of lower functional avidity upon TCR transfer. In conclusion, DNA vaccination by electroporation primed for TCR clonotypes that were associated with HIV control, highlighting the potential of this vaccine delivery method. To our knowledge, this study provides the first proof-of-concept that clonotypic analysis may be used as a tool to monitor the quality of vaccine-induced responses and modulate these toward "controller-like" responses.

Dual Molecular Mechanisms Govern Escape at Immunodominant HLA A2-Restricted HIV Epitope.

Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted cytotoxic T lymphocyte epitope produce the SLFNTIAVL triple mutant “ultimate” escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent-exposed residues in the peptide destabilise the peptide–HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate re-evaluation of this exemplar model of HIV TCR escape.

The Clonal Invariant NKT Cell Repertoire in People with Type 1 Diabetes Is Characterized by a Loss of Clones Expressing High-Affinity TCRs.

Invariant NKT (iNKT) cells in healthy people express iNKT-TCRs with widely varying affinities for CD1d, suggesting different roles for high- and low-affinity iNKT clones in immune regulation. However, the functional implications of this heterogeneity have not yet been determined. Functionally aberrant iNKT responses have been previously demonstrated in different autoimmune diseases, including human type 1 diabetes, but their relationship to changes in the iNKT clonal repertoire have not been addressed. In this study, we directly compared the clonal iNKT repertoire of people with recent onset type 1 diabetes and age- and gender-matched healthy controls with regard to iNKT-TCR affinity and cytokine production. Our results demonstrate a selective loss of clones expressing high-affinity iNKT-TCRs from the iNKT repertoire of people with type 1 diabetes. Furthermore, this bias in the clonal iNKT repertoire in type 1 diabetes was associated with increased GM-CSF, IL-4, and IL-13 cytokine secretion among Ag-stimulated low-affinity iNKT clones. Thus, qualitative changes of the clonal iNKT repertoire with the potential to affect the regulatory function of this highly conserved T cell population are already established at the early stages in type 1 diabetes. These findings may inform future rationales for the development of iNKT-based therapies aiming to restore immune tolerance in type 1 diabetes.

Abstract IA18: Building better T cells for targeting and eliminating tumors

Abstract Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is not only safe but can infiltrate and retain function in the tumor microenvironment. We have used molecular expression profiling to detect antigens selectively or markedly over-expressed by tumors, and then used these antigens as stimuli to generate T cells from normal repertoires. We have developed a high throughput technology to identify those T cells that express high affinity TCRs, and to then isolate from these T cells the TCR genes, place them either directly or after affinity enhancement into shuttle vectors, and use these reagents to create recipient T cells with high avidity for tumor targets that can be administered in vivo. We have utilized in silico, in vitro, and preclinical mouse models to assess the safety and potential efficacy of T cells expressing such TCRs. We are currently pursuing targeting of 3 antigens that are expressed in both murine and human tumors and are pro-oncogenic, contributing to the malignant phenotype. This includes ongoing clinical trials that will be discussed in acute myelogenous leukemia and in non-small cell lung cancer or mesothelioma targeting WT1 with T cells transduced to express a high affinity TCR specific for WT1, as well as trials being designed to target Mesothelin (MSLN) with T cells transduced to express a high affinity TCR specific for MSLN in pancreatic and ovarian cancer that are anticipated to begin within 8-12 months. Our clinical results in treatment of AML and preclinical results in mouse models of pancreatic and ovarian cancer appear very promising. However, the composite clinical data, as well as the results in the preclinical mouse models that drive our clinical trials, demonstrate substantial obstacles to sustaining T cell function in vivo after transfer, particularly in the context of solid tumors. Engineered T cells with specificity for tumor antigens appear to have the capacity to infiltrate and accumulate in solid tumors, and to initially mediate anti-tumor activity, but frequently become dysfunctional in the tumor microenvironment. Studies are being pursued to identify the critical obstacles to maintaining T cell function and achieving more reproducible tumor eradication, including modulating the tumor microenvironment and engineering T cells to express immunomodulatory fusion proteins (IFP) that recognize ligands for inhibitory signals but deliver an activation/costimulatory signal. Our data suggest that engineering T cells to acquire novel properties not naturally found in unmanipulated T cells has the potential to create effective therapies for human cancers. Citation Format: Philip D. Greenberg, Aude Chapuis, Dan Egan, Ingunn Stromnes, Sunil Hingorani, Shannon Oda, Rachel Perret, Kristin Anderson, Tom Schmitt. Building better T cells for targeting and eliminating tumors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA18.

Abstract IA01: Engineering T cell responses to tumors: Taking the immune system where no responses have gone before

Abstract IA01: Engineering T cell responses to tumors: Taking the immune system where no responses have gone before

Structural and Functional Changes of the Invariant NKT Clonal Repertoire in Early Rheumatoid Arthritis.

Invariant NKT cells (iNKT) are potent immunoregulatory T cells that recognize CD1d via a semi-invariant TCR (iNKT-TCR). Despite the knowledge of a defective iNKT pool in several autoimmune conditions, including rheumatoid arthritis (RA), a clear understanding of the intrinsic mechanisms, including qualitative and structural changes of the human iNKT repertoire at the earlier stages of autoimmune disease, is lacking. In this study, we compared the structure and function of the iNKT repertoire in early RA patients with age- and gender-matched controls. We analyzed the phenotype and function of the ex vivo iNKT repertoire as well as CD1d Ag presentation, combined with analyses of a large panel of ex vivo sorted iNKT clones. We show that circulating iNKTs were reduced in early RA, and their frequency was inversely correlated to disease activity score 28. Proliferative iNKT responses were defective in early RA, independent of CD1d function. Functional iNKT alterations were associated with a skewed iNKT-TCR repertoire with a selective reduction of high-affinity iNKT clones in early RA. Furthermore, high-affinity iNKTs in early RA exhibited an altered functional Th profile with Th1- or Th2-like phenotype, in treatment-naive and treated patients, respectively, compared with Th0-like Th profiles exhibited by high-affinity iNKTs in controls. To our knowledge, this is the first study to provide a mechanism for the intrinsic qualitative defects of the circulating iNKT clonal repertoire in early RA, demonstrating defects of iNKTs bearing high-affinity TCRs. These defects may contribute to immune dysregulation, and our findings could be exploited for future therapeutic intervention.

Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies

Abstract SY31-03: Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies

TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential.

Autoreactive T cells infiltrating the target organ can possess a broad TCR affinity range. However, the extent to which such biophysical parameters contribute to T cell pathogenic potential remains unclear. In this study, we selected eight InsB9-23-specific TCRs cloned from CD4(+) islet-infiltrating T cells that possessed a relatively broad range of TCR affinity to generate NOD TCR retrogenic mice. These TCRs exhibited a range of two-dimensional affinities (∼ 10(-4)-10(-3) μm(4)) that correlated with functional readouts and responsiveness to activation in vivo. Surprisingly, both higher and lower affinity TCRs could mediate potent insulitis and autoimmune diabetes, suggesting that TCR affinity does not exclusively dictate or correlate with diabetogenic potential. Both central and peripheral tolerance mechanisms selectively impinge on the diabetogenic potential of high-affinity TCRs, mitigating their pathogenicity. Thus, TCR affinity and multiple tolerance mechanisms converge to shape and broaden the diabetogenic T cell repertoire, potentially complicating efforts to induce broad, long-term tolerance.

Transnuclear TRP1-specific CD8 T cells with high or low affinity TCRs show equivalent antitumor activity.

We have generated, via somatic cell nuclear transfer, two independent lines of transnuclear (TN) mice, using as nuclear donors CD8 T cells, sorted by tetramer staining, that recognize the endogenous melanoma antigen TRP1. These two lines of nominally identical specificity differ greatly in their affinity for antigen (TRP1(high) or TRP1(low)) as inferred from tetramer dissociation and peptide responsiveness. Ex vivo-activated CD8 T cells from either TRP1(high) or TRP1(low) mice show cytolytic activity in 3D tissue culture and in vivo, and slow the progression of subcutaneous B16 melanoma. Although naïve TRP1(low) CD8 T cells do not affect tumor growth, upon activation these cells function indistinguishably from TRP1(high) cells in vivo, limiting tumor cell growth and increasing mouse survival. The anti-tumor effect of both TRP1(high) and TRP1(low) CD8 T cells is enhanced in RAG-deficient hosts. However, tumor outgrowth eventually occurs, likely due to T cell exhaustion. The TRP1 TN mice are an excellent model for examining the functional attributes of T cells conferred by TCR affinity, and they may serve as a platform for screening immunomodulatory cancer therapies.