TCRβ Chain Research Articles

MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage.

Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.

Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains.

The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0060-1) contains supplementary material, which is available to authorized users.

Automation of immune repertoire amplification on the iC-Processor (TECH1P.851)

Abstract Immune repertoire amplification of T and B cell receptors coupled to Next Generation Sequencing (NGS) provides detailed sequence-level insight into the immune system. Previously, we developed a patented, nested multiplex PCR method (arm-PCR) to amplify an immune repertoire, inclusively and semi-quantitatively. Here, we describe the automation of this process in cassette format on the iC-system (iCubate, Inc.) and compare the sequencing results from repeat amplification experiments of the TCRβ and human Ig heavy chains from the same individual. The iC-system is an open, nested multiplex PCR diagnostic development platform, which allows for the development of automated diagnostic panels in a closed cassette format. In addition to the automation, the closed format helps to reduce contamination of the laboratory environment with multiplex primers. Template RNA is added to the cassette at the appropriate concentration with RT enzyme. After two sets of PCR, residual primer-dimers are removed by magnetic selection of the primary product in the cassette. The cassette is then removed from the iC-Processor, and the amplified library is eluted from the magnetic beads. The amplified immune library is directly ready for pooling, QC, and sequencing using the Illumina MiSeq NGS platform. Repeat amplification experiments from the same individual using this automated approach demonstrated high repeatability among shared CDR3 sequences for both the TCRβ and Ig heavy chains.

Autoimmune susceptibility imposed by public TCRβ chains (BA2P.125)

Abstract The MHC is the largest genetic risk factor for autoimmunity. It imposes risk in association with a complementary autoantigen specific TCR repertoire. How this occurs is unresolved. Each individual’s TCR repertoire is largely unique, and should therefore not contribute to population-wide disease risk. However, due to biases in TCR recombination, a subset of α and β sequences are shared by most individuals, or public. If these public α or β chains modulate a TCRαβ heterodimer’s likelihood of productively engaging autoantigen-MHC, they could influence autoimmune susceptibility. We demonstrate that disease-associated regulatory and effector TCRβ repertoires in autoimmune encephalomyelitis preferentially incorporate public TCR. Some disease-associated public TCRβ, when transgenically expressed, could further endow T cells with autoantigen specificity in the absence of priming, and demonstrate a subordinate role of the non-shared, endogenous TCRα chains in autoantigen specificity. Further, 2 of 7 Tg public and 0 of 7 Tg private TCRβ isolated from high frequency CNS-infiltrating T cells, provoked spontaneous, early-onset autoimmunity when associated with endogenous TCRα. These findings support a disproportionate contribution of public TCR to immunopathology and implicate them, in association with risk-associated MHC, in disease susceptibility. They further suggest that subsets of public sequences may serve as disease-specific biomarkers or therapeutic targets in autoimmunity.

T cell repertoire renewal following autologous stem cell transplantation for multiple sclerosis (THER3P.879)

Abstract Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies and also has been used in clinical trials for severe autoimmune diseases to “reset” the immune system and produce a new, hopefully non-autoimmune repertoire. While it has long been feasible to eliminate the vast majority of mature T cells, it has been very difficult to accurately and quantitatively determine the relationship of the regenerated repertoire to the baseline repertoire. Here, in a phase II study of HSCT for poor prognosis multiple sclerosis (MS) patients, we have used high throughput deep TCRβ chain sequencing to directly assess millions of individual TCRs per patient sample. We find that HSCT has distinctive, and separate, effects on the CD4 and CD8 repertoires. In the case of CD4 T cells, the dominant TCR clones present pre-treatment were not detectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8 clones were not effectively removed, and the reconstituted CD8 repertoire was created by clonal expansion of cells which were present pre-treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. This approach may enable monitoring of pathogenic or protective T cell clones following HSCT and or cellular therapies.

A119: Deep Sequencing Analysis of the T Regulatory and T Effector Repertoire in Juvenile Idiopathic Arthritis

Background/Purpose:T regulatory (Treg) function has been shown to be impaired in juvenile idiopathic arthritis (JIA) as synovial fluid (SF) Tregs are unable to suppress T effector cells (Teffs) found in the joint. The cause of this breach in immunologic tolerance is not fully understood, meriting the further study of Tregs and Teffs in JIA. The T cell receptor (TCR) endows T lymphocytes with antigen specificity and mediates interactions between T cells and their environment. Yet, little is known about the comparative specificities, clonality, and diversity of Tregs and Teffs in JIA. Further, the Treg repertoire has not been studied in adult or pediatric inflammatory arthritis. Therefore, we endeavored to characterize these TCR repertoires in JIA through deep sequencing technology.Methods:Mononuclear cells were isolated by Ficoll gradient centrifugation from paired peripheral blood (PB) and SF samples. Tregs (CD4+CD25+CD127low) and Teffs (CD4+CD25−) were sorted by flow cytometry. The TCRβ chain was amplified by multiplex PCR with genomic DNA serving as the template and then deep sequenced (ImmunoSEQ™). Sequences were aligned to the reference International ImMunoGeneTics information system. Further analysis utilizing Immunoglobulin Analysis Tool software was done to assess clonality, diversity, and variable (V), diversity (D), and joining (J) usage. Statistical analysis was completed with paired/unpaired Wilcoxon signed‐rank and Kruskal‐Wallis tests.Results:Three girls and 2 boys with oligoarticular (n = 2), extended oligoarticular (n = 2), and psoriatic (n = 1) arthritis were studied. Patients were similar in age (7–12 yrs) and ANA status (negative n = 4). Treatment consisted of NSAIDs (n = 2), methotrexate (n = 1), leflunomide (n = 1), and leflunomide with etanercept (n = 1). Tregs, measured as % of CD4+ cells, were enriched in the SF compared to PB (mean ± SEM: SF Tregs 13.2 ± 1.8; PB Tregs 5.1 ± 0.6). SF Teff repertoire was less diverse (p = 0.008) and more clonal (p = 0.008) than PB Teffs. In SF Teffs, a single complementarity determining region 3 (CDR3) was shared across all patients, though different V and J genes were used to create this CDR3, suggesting antigen selection. SF Tregs had the largest clonal expansions compared to PB Tregs (p = 0.016), PB Teffs (p = 0.008) and SF Teffs (p = 0.032). The degree of SF Treg clonality correlated with the active joint count. V gene usage, particularly Vβ29, in unique clonotypes was skewed in SF Tregs compared to PB Teffs, PB Tregs, and SF Teffs (p = 0.022); Vβ29 gene overuse was most prominent in patients with more active joints.Conclusion:We report on the first deep sequencing analysis of T cells in JIA. An antigen‐driven process was suggested by the clonality and shared CDR3 clonotypes in SF Teffs. This study is the first to demonstrate abnormalities in the Treg repertoire in inflammatory arthritis. The SF Treg repertoire had the most pronounced clonality and skewing of V gene usage, and these abnormalities correlated with disease severity. These results suggest that Treg insufficiency in JIA may be partially due to an aberrant Treg repertoire in the arthritic joint. Studies of additional patients with functional correlates are needed to augment these findings.

S77. Proffered paper: In vitro induced response patterns of antileukemic T-cells – characterization by combination of functional assays, spectratyping and next generation sequencing

Meeting abstracts T-cell receptor (TCR) diversity is characterised by somatic alterations in the complementary determining region 3 (CDR3) of the human TCR β-chain. Complemented with the TCR alpha-chain, TCR diversity can hypothetically result in up to 1018 different TCR molecules. Myeloid

T cell repertoire following autologous stem cell transplantation for multiple sclerosis

Autologous hematopoietic stem cell transplantation (HSCT) is commonly employed for hematologic and non-hematologic malignancies. In clinical trials, HSCT has been evaluated for severe autoimmunity as a method to "reset" the immune system and produce a new, non-autoimmune repertoire. While the feasibility of eliminating the vast majority of mature T cells is well established, accurate and quantitative determination of the relationship of regenerated T cells to the baseline repertoire has been difficult to assess. Here, in a phase II study of HSCT for poor-prognosis multiple sclerosis, we used high-throughput deep TCRβ chain sequencing to assess millions of individual TCRs per patient sample. We found that HSCT has distinctive effects on CD4+ and CD8+ T cell repertoires. In CD4+ T cells, dominant TCR clones present before treatment were undetectable following reconstitution, and patients largely developed a new repertoire. In contrast, dominant CD8+ clones were not effectively removed, and the reconstituted CD8+ T cell repertoire was created by clonal expansion of cells present before treatment. Importantly, patients who failed to respond to treatment had less diversity in their T cell repertoire early during the reconstitution process. These results demonstrate that TCR characterization during immunomodulatory treatment is both feasible and informative, and may enable monitoring of pathogenic or protective T cell clones following HSCT and cellular therapies.

Visualization and quantification of monoallelic TCRα gene rearrangement in αβ T cells

T-cell receptor α (TCRα) chain rearrangement is not constrained by allelic exclusion and thus αβ T cells frequently have rearranged both alleles of this locus. Thereby, stepwise secondary rearrangements of both TCRα loci further increase the odds for generation of an α-chain that can be positively selected in combination with a pre-existing TCRβ chain. Previous studies estimated that approximately 2-12% of murine and human αβ T cells still carry one TCRα locus in germline configuration, which must comprise a partially or even fully rearranged TCRδ locus. However, these estimates are based on a relatively small amount of individual αβ T-cell clones and αβ T-cell hybridomas analyzed to date. To address this issue more accurately, we made use of a mouse model, in which a fluorescent reporter protein is introduced into the constant region of the TCRδ locus. In this TcrdH2BeGFP system, fluorescence emanating from retained TCRδ loci enabled us to quantify monoallelically rearranged αβ T cells on a single-cell basis. Via fluorescence-activated cell sorting analysis, we determined the frequency of monoallelic TCRα rearrangements to be 1.7% in both peripheral CD4(+) and CD8(+) αβ T cells. Furthermore, we found a skewed 5' Jα gene utilization of the rearranged TCRα allele in T cells with monoallelic TCRα rearrangements. This is in line with previous descriptions of a tight interallelic positional coincidence of Jα gene segments used on both TCRα alleles. Finally, analysis of T cells from transgenic mice harboring only one functional TCRα locus implied the existence of very rare unusual translocation or episomal reintegration events of formerly excised TCRδ loci.

Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells

Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies. Here, we show that pre-TCRα, a TCRα surrogate that pairs with TCRβ chains to signal proper TCRβ folding during T-cell development, can be expressed in TCRα knockout mature T-cells to support CD3 expression at the cell surface. Cells expressing pre-TCR/CD3 complexes can be activated and expanded using standard CD3/CD28 T-cell activation protocols. Thus, heterologous expression of pre-TCRα represents a promising technology for use in the manufacturing of TCR-deficient T-cells for adoptive immunotherapy applications.

Immune selection of tumor cells in TCR β-chain transgenic mice

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single β-chain transgenic B10.D2(R101) (KdIdDb) mice to allogeneic EL4 (H-2b) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2Kb molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8+ T-lymphocytes with endogenous β-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic β-chains became CD8+CD44+CD62L− effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40–60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2Kb molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

Gene Therapy With a High-Affinity Single-Chain p53-Specific TCR Mediates Potent Anti-Tumor Response Without Inducing Gvhd In Vivo

The adoptive transfer of tumor-reactive cells is a promising approach in the treatment of human malignancies, but the challenge of isolating T cells with high-avidity for tumor antigens in each patient has limited its widespread application. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264-272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. However, a safety concern of TCR gene transfer is the risk of pairing between introduced and the naturally expressed endogenous TCR chains, resulting in the generation of self-reactive T cells (off-target autoimmunity). We first genetically modified p53TCR constructs to minimize mispairing and improve correct pairing of the introduced TCR. We and others have shown that, cysteine modifications are able to increase the expression of the introduced TCR but fail to prevent mispairing formation in mouse and human T cells. To further enhance preferential TCR pairing, cell surface expression and TCR function, we introduced additional cysteine residues into the TCR α and β chain constant domains along with codon-optimization of the TCR sequences and cloning of the TCR constructs into one single 2A-based retroviral vector. To overcome TCR mispairing formation, we designed a single chain (sc) TCR by connecting the variable TCRa domain to the TCRb chain via a short peptide linker co-expressed with a truncated constant TCR a domain. Beside off-target toxicity, adoptive transfer of high-avidity T cells may potentially cause severe on-target toxicity for normal cells expressing low level of antigens. In this respect, pre-clinical in vivo studies are still missing.Here, we evaluated the safety issues raised by the risk of p53TCR gene transfer-associated on/off-target toxicities in relevant mouse models of adoptive transfer.In vitro studies showed that, scTCR-modified CD4+ and CD8+ T cells displayed similar high-avidity compared to the full-length TCR, as determined by peptide titration in cytotoxicity assays and were able to mediate specific lysis of p53 mutant A2.1+ tumor cells. Though, genetic modifications preserved the antigen specificity of these TCRs, the full-length version of the TCR could not prevent mispairing-mediated lethal off-target autoimmunity in vivo. In sharp contrast, T cells engrafted with the modified scTCR did not induce graft-versus-host disease (GVHD) following adoptive transfer. We next assessed the potential of scTCR-modified T cells to cause on-target autoimmunity in vivo, using mice which express the human wild type p53 and A2.1Kb (Hupki-A2.1Kb). We found that lymphodepleting preconditioning regimens plus vaccination-induced expansion of transferred TCR-specific T cells did not result in a depletion of hematopoietic cells, as mice recovered normal white blood cell counts, including lymphocytes and monocytes and survived without any sign of toxicity. Importantly, our study demonstrated that high-avidity scTCR-engineered human T cells were able to eradicate established tumors and persist for more than 6 months after infusion without inducing xeno-GVHD in NodScid IL-2R gamma chain-null mice.Taken together, our study provided evidence that an optimized high-affinity scTCR-specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53+A2.1+ tumor cells in vivo without inducing off-target or self-directed toxicities in humanized mouse models of adoptive T-cell transfer. These data strongly support the improved safety and therapeutic efficacy of high-affinity scp53TCR for TCR-based immunotherapy of p53-associated malignancies. Disclosures:No relevant conflicts of interest to declare.

Biased usage and preferred pairing of α- and β-chains of TCRs specific for an immunodominant gluten epitope in coeliac disease

CD4⁺ T cells that recognize dietary gluten antigens presented by the disease-associated HLA-DQ2 or DQ8 molecules are central players in coeliac disease. Unbiased sequencing of the human TCRα variable (TRAV) and humanTCRβ variable (TRBV) genes of 68 HLA-DQ2.5-glia-α2-specific T cells from coeliac disease patients confirmed previous reports of over-usage of the TRBV7-2 gene segment, a conserved Arg residue in the complementarity-determining region (CDR) 3β loop and prevalent usage of the canonical ASSxRxTDTQY CDR3β loop among T cells with this specificity. In 30 clones that had the canonical TCRβ chain, we found a strict usage of the TRAV26-1 gene segment in the TCRα chain. There was variable usage of the TRAJ genes and diverse CDR3α sequences with no apparent conserved motifs. This study extends previous reports on biased TCR usage in both HLA-DQ2.5- and DQ8-restricted gluten-specific TCRs and provides data for further studies on TRAV and TRBV pairing.

Development of transgenic mice expressing a coronavirus-specific public CD4 T cell receptor

Mice that are transgenic (Tg) for T cell receptor (TCR) expression are used extensively to analyze longitudinal T cell responses during effector and memory phases of the T cell response. Generation of TCR Tg mice generally requires T cell stimulation and cloning in vitro prior to amplification, processes which introduce biases into selection of the TCR that is ultimately chosen for TCR Tg mouse generation. Here we describe an alternative approach that involves no T cell stimulation or propagation in vitro. We generated mice that were transgenic for a TCR responding to a CD4 T cell epitope (epitope M133) that is immunodominant in mice infected with a neurotropic coronavirus, the JHM strain of mouse hepatitis virus. The CD4 T cell response to epitope M133 is of particular interest because it may be pathogenic, protective or regulatory, depending upon the physiological setting. We applied an iterative process in which we identified a TCR-β chain expressed by all mice that were examined (‘public sequence’). This TCR-β chain was introduced into bone marrow cells with a lentivirus vector, generating TCR-β retrogenic mice. A TCR-α chain that paired with this TCR-β was then identified and used to generate a second set of TCR (α/β) retrogenic mice. After demonstrating that these cells were functional and responded to epitope M133, these TCR chains were used to generate an epitope M133-specific TCR Tg mouse. This method should be generally useful for engineering TCR Tg mice without introduction of bias caused by in vitro manipulation and propagation.

Immune surveillance by CD8αα+ skin-resident T cells in human herpes virus infection

SummaryMost herpes simplex virus 2 (HSV-2) reactivations in humans are subclinical and associated with rapid expansion and containment of virus. Previous studies have shown CD8+ T cells persist in genital skin and mucosa at the dermal-epidermal junction (DEJ), the portal of neuronal release of reactivating virus, for prolonged time periods after lesions are cleared1,2. The phenotype and function of this persistent CD8+ T-cell population remain unknown. Here, using cell type-specific laser capture microdissection, transcriptional profiling and T-cell receptor beta (TCRβ) genotyping on sequential genital skin biopsies, we show CD8αα+ T cells are the dominant resident population of DEJ CD8+ T cells that persist at the site of prior HSV-2 reactivation. CD8αα+ T cells located at the DEJ lack chemokine receptor expression required for lymphocyte egress and recirculation, express gene signatures of T-cell activation and antiviral activity, and produce cytolytic granules during clinical and virological quiescent time periods. Sequencing of the TCRβ chain repertoire revealed that the DEJ CD8αα+ T cells are oligoclonal with diverse usage of TCR VB genes, which differ from those commonly described for MAIT and NKT cells. Dominant clonotypes overlapped among multiple recurrence episodes over a period of two and a half years. Episodes of rapid asymptomatic HSV-2 containment were also associated with a high CD8 effector-to-target ratio and focal enrichment of CD8αα+ T cells. These studies indicate DEJ CD8αα+ T cells are tissue resident cells that appear to play a fundamental role in immune surveillance and in initial containment of HSV-2 reactivation in human peripheral tissue. Elicitation of CD8αα+ T cells might be a critical component for developing effective vaccines against skin and mucosal infections.

Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression

Current tumor immunotherapy approaches include the genetic modification of peripheral T cells to express tumor antigen-specific T-cell receptors (TCRs). The approach, tested in melanoma, has led to some limited success of tumor regression in patients. Yet, the introduction of exogenous TCRs into mature T cells entails an underlying risk; the generation of autoreactive clones due to potential TCR mispairing, and the lack of effective negative selection, as these peripheral cells do not undergo thymic selection following introduction of the exogenous TCR. We have successfully generated MART-1-specific CD8 T cells from genetically modified human hematopoietic stem cells (hHSC) in a humanized mouse model. The advantages of this approach include a long-term source of antigen specific T cells and proper T-cell selection due to thymopoiesis following expression of the TCR. In this report, we examine the molecular processes occurring on endogenous TCR expression and demonstrate that this approach results in exclusive cell surface expression of the newly introduced TCR, and the exclusion of endogenous TCR cell surface expression. This suggests that this stem cell based approach can provide a potentially safer approach for anticancer immunotherapy due to the involvement of thymic selection.

The role of pTα/pre-TCR in susceptibility to autoimmunity (P1464)

Abstract Autoimmune diseases result from a breakdown in self-tolerance, with a major contribution of pathogenic self-reactive T cells. Individuals with a particular autoimmune disease are often at risk of developing additional autoimmunity directed towards other target tissues. These observations suggest that there might be a mechanism leading to a general defect in self-tolerance, resulting in increased susceptibility to autoimmune disease. Tolerance towards self is initially established in the thymus during the process of thymic selection. Developing thymocytes are first immature CD4 and CD8 double negative thymocytes. Cells with a productive TCRβ gene rearrangement will express a pre-TCR/CD3 complex on the cell surface by combining a TCRβ chain with the pTα chain. Signaling through the pre-TCR, β-selection, plays a crucial role in the generation αβT cells, by enabling further maturation to the CD4 and CD8 double positive stage. The crucial role of pTα at β-selection together with the genetic localization of the ptcra gene and the fact that variations in pTα will affect all developing T cells, suggests that pTα could be a common factor leading to a general defect in self-tolerance. We have discovered differences in the isoform expression pattern and amino acid sequence of pTa between NOD and B6 mice. These findings are consistent with the hypothesis that alterations in the pre-TCR might affect selection, giving a modified TCR repertoire and increased susceptibility to autoimmunity.

A role for CD3 conformation in the preTCR/CD3 complex (P4428)

Abstract Mature T cells recognize and respond to antigens through the T cell antigen receptor (TCR). We have established that upon TCR antigenic engagement, the TCR/CD3 complex undergoes a conformational change (CD3dc) of its CD3 epsilon tails to expose a proline-rich sequence and allow downstream signaling to occur. CD3dc has been shown to be required for full TCR/CD3 signaling and function of mature T cells. Pre-TCR assembly with the CD3 complex serves as a developmental checkpoint that ensures the expression of a function variant TCRβ chain in thymocytes. Interestingly, stimulation of the preTCR complex has been found to be ligand-independent; assembly is sufficient. Knowing this, we first determined the stoichiometry of the preTCR/CD3 complex and asked the question, how does assembly impact its ability to undergo conformational change? To answer this question, we employed various novel biochemical methods developed in our laboratory including a flow-cytommetry based immunoprecipitation (IP-FCM) as well as a CD3-pull down assay (CD3-PD). We aim to study the conformation of CD3 in the preTCR/CD3 complex and establish whether or not the assembly of this receptor is enough to induce the active CD3 conformation that supports functional CD3 signaling in mature cells. With our data, we are further able to characterize previously unstudied requirements for CD3 conformational change in the preTCR/CD3 complex with the potential of using this knowledge to manipulate CD3dc in the future.

RasGRP1, but Not RasGRP3, Is Required for Efficient Thymic β-Selection and ERK Activation Downstream of CXCR4

T cell development is a highly dynamic process that is driven by interactions between developing thymocytes and the thymic microenvironment. Upon entering the thymus, the earliest thymic progenitors, called CD4−CD8− ‘double negative’ (DN) thymocytes, pass through a checkpoint termed “β-selection” before maturing into CD4+CD8+ ‘double positive’ (DP) thymocytes. β-selection is an important developmental checkpoint during thymopoiesis where developing DN thymocytes that successfully express the pre-T cell receptor (TCR) undergo extensive proliferation and differentiation towards the DP stage. Signals transduced through the pre-TCR, chemokine receptor CXCR4 and Notch are thought to drive β-selection. Additionally, it has long been known that ERK is activated during β-selection; however the pathways regulating ERK activation remain unknown. Here, we performed a detailed analysis of the β-selection events in mice lacking RasGRP1, RasGRP3 and RasGRP1 and 3. We report that RasGRP1 KO and RasGRP1/3 DKO deficient thymi show a partial developmental block at the early DN3 stage of development. Furthermore, DN3 thymocytes from RasGRP1 and RasGRP1/3 double knock-out thymi show significantly reduced proliferation, despite expression of the TCRβ chain. As a result of impaired β-selection, the pool of TCRβ+ DN4 is significantly diminished, resulting in inefficient DN to DP development. Also, we report that RasGRP1 is required for ERK activation downstream of CXCR4 signaling, which we hypothesize represents a potential mechanism of RasGRP1 regulation of β-selection. Our results demonstrate that RasGRP1 is an important regulator of proliferation and differentiation at the β-selection checkpoint and functions downstream of CXCR4 to activate the Ras/MAPK pathway.

Abstract PR12: Early TCRα expression during T cell development for the generation of enhanced-affinity TCRs for TCR gene therapy.

Abstract Thymic selection eliminates T cells expressing TCRs with a high affinity for self-antigen. However, many of the most promising tumor antigens for T cell-based cancer immunotherapies are unmodified self-antigens. Therefore, tumor-specific T cells often have a low avidity for tumor antigens, thus limiting the efficacy of this approach. TCR gene therapy using TCR genes that have been modified for increased affinity can overcome this limitation. The generation of enhanced affinity TCRs generally involves saturation mutagenesis targeting the complementarity determining regions of the TCR genes to be modified. However, this approach requires considerable trial and error, and is still not always successful at generating a higher affinity TCR without promiscuous off-target activity. We have developed a system for selecting high affinity tumor antigen-specific TCRs from a population of T cells selected by agonist signaling during T cell development in vitro. The αβ/γδ lineage choice is in part dictated by the strength of TCR/pre-TCR signaling during early T cell development, such that the pre-TCR provides a weak signal that drives αβ T cell development while the mature γδ TCR provides a stronger signal that drives γδ T cell development. In TCR transgenic mice, early expression of the transgenic TCRα chain prior to β-selection can also provide a stronger γδ-like signal through the mature αβ TCR, leading to the development of lineage redirected TCRαβ+ CD4/CD8 double negative (DN) γδ wanna-be” cells. The development of these DN TCRαβ+ cells is enhanced in the presence of cognate antigen or TCR crosslinking; suggesting that early expression of an antigen-specific TCRα chain before β-selection could promote the generation of DN TCRαβ+ cells in the presence of antigen, through a process in which the introduced TCRα chain pairs with an endogenous TCRβ chain to produce an αβ TCR with high affinity for the target antigen. To test this hypothesis, the TCRα chain from a tumor antigen-specific TCR was transduced into hematopoietic progenitor cells and a library of TCRβ chains was isolated from the lineage redirected DN TCRαβ+ T cells that developed. Several TCRβchains of varying CDR3 lengths were isolated that confer enhanced affinity for the target antigen when paired with the introduced TCRα chain. This abstract is also presented as Poster A13. Citation Format: Thomas M. Schmitt, Philip D. Greenberg, David H. Aggen, David Kranz, Sebastian Ochsenreither. Early TCRα expression during T cell development for the generation of enhanced-affinity TCRs for TCR gene therapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR12.