Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis
Ravi SharmaSanjay V. BoddulNiyaz YoosufSara TurcinovAnatoly DubnovitskyGenadiy KozhukhFredrik WermelingWilliam W. KwokLars KlareskogVivianne Malmström
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Abstract Objectives To search for common features in the autoreactive T cell receptor (TCR)-repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Methods Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Results Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the citTNC22-specific TCRs was evident in blood and synovial fluid of different patients. Conclusion The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients.Summary: T‐cell activation requires contact between T cells and antigen‐presenting cells (APCs) to bring T‐cell receptors (TCRs) and major histocompatibility complex peptide (MHCp) together to the same complex. These complexes rearrange to form a concentric circular structure, the immunological synapse (IS). After the discovery of the IS, dynamic imaging technologies have revealed the details of the IS and provided important insights for T‐cell activation. We have redefined a minimal unit of T‐cell activation, the ‘TCR microcluster’, which recognizes MHCp, triggers an assembly of assorted molecules downstream of the TCR, and induces effective signaling from TCRs. The relationship between TCR signaling and costimulatory signaling was analyzed in terms of the TCR microcluster. CD28, the most valuable costimulatory receptor, forms TCR–CD28 microclusters in cooperation with TCRs, associates with protein kinase C θ, and effectively induces initial T‐cell activation. After mature IS formation, CD28 microclusters accumulate at a particular subregion of the IS, where they continuously assemble with the kinases and not TCRs, and generate sustained T‐cell signaling. We propose here a ‘TCR–CD28 microcluster’ model in which TCR and costimulatory microclusters are spatiotemporally formed at the IS and exhibit fine‐tuning of T‐cell responses by assembling with specific players downstream of the TCR and CD28.
Immunological synapse
Jurkat cells
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Abstract T-cell involvement may be central to the pathogenesis of systemic sclerosis (SSc). Like αβ TCR+ T-cells, γδ TCR+ T-cell diversity during T-cell development results in a repertoire of different T-cells each with a unique TCR. Therefore the probability of finding identical TCR on defined T-cell populations is negligible, except in the context of an antigen-driven T-cell response. To determine whether γδ TCR+ T-cells are clonally expanded in skin biopsies and/or peripheral blood of patients with SSc (n=7), γ-chain (VγI & Vγ9) and δ-chain (Vδ1 & Vδ2) TCR transcripts were amplified by gene-specific PCR, followed by cloning and sequencing. We report the presence of substantial proportions of identical VγI-chain transcripts (14.3%-37.2%; p<0.05), and Vγ9 transcripts (24%-83.3%; p<0.05) in skin biopsies and/or PBMC of patients with SSc. Likewise, there were multiple identical Vδ1-chain transcripts (25%-50%; p<0.05), and Vδ2-chain transcripts (18.1%-80%; p<0.05) in the samples analyzed. Extensive clonal expansions of γ- and δ-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of γδ TCR+ T-cells in these patients. These γδ TCR+ T-cells may have undergone activation and clonal expansion in vivo in response to as yet unidentified antigens. Future studies to identify the antigens recognized by these clonally expanded γδ TCRs will facilitate better understanding of SSc pathogenesis.
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TCR‐microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation independently of lipid rafts. TCR‐MCs contain receptors, kinases and adaptors, and function as the signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported to the center to form cSMAC. The co‐stimulation receptor CD28 joins the signaling region of cSMAC and recruits PKCθ and Carma1. CTLA‐4 accumulates in the same region and competes with CD28 for negative regulation of T cell activation. T cell activation is therefore mediated by two spatially distinct signaling compartments: TCR signaling by the peripheral TCR‐MC and co‐stimulation signal by the central signaling cSMAC.
Immunological synapse
Co-stimulation
Jurkat cells
Lipid raft
Cell Signaling
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Summary T‐cell activation results from engagement of the T‐cell receptor (TCR) by cognate peptide–major histocompatibility complex (pMHC) complexes on the surface of antigen‐presenting cells (APC). Previous studies have provided evidence supporting the notion that the half‐life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters that can influence T‐cell activation. However, whether the half‐life of the TCR/pMHC interaction can modulate T‐cell activation in response to a pathogen challenge remains unknown. To approach this question, we generated strains of bacteria expressing variants of the ovalbumin (OVA) antigen, carrying point mutations in the SIINFEKL sequence. When bound to H‐2K b , this peptide is the cognate ligand for the OT‐I TCR. Variants of the H‐2K b /SIINFEKL bind to the OT‐I TCR with distinct half‐lives. Here we show that dendritic cells (DCs) infected with bacteria expressing OVA variants were incapable of activating OT‐I T cells when the half‐life of the TCR/H‐2K b /OVA interaction was excessively short. Consistent with these data, T‐cell activation was only observed in mice infected with bacteria expressing OVA variants that bound to OT‐I with a half‐life above a certain threshold. Considered together, our data suggest that the half‐life of TCR/pMHC interaction can significantly modulate T‐cell activation in vivo , as well as influence recognition of antigens expressed by bacteria. These observations underscore the importance of the TCR/pMHC half‐life on the clearance of pathogens.
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Abstract TCR interactions with cognate peptide-MHC (pepMHC) ligands are generally low affinity. This feature, together with the requirement for CD8/CD4 participation, has made it difficult to dissect relationships between TCR-binding parameters and T cell activation. Interpretations are further complicated when comparing different pepMHC, because these can vary greatly in stability. To examine the relationships between TCR-binding properties and T cell responses, in this study we characterized the interactions and activities mediated by a panel of TCRs that differed widely in their binding to the same pepMHC. Monovalent binding of soluble TCR was characterized by surface plasmon resonance, and T cell hybridomas that expressed these TCR, with or without CD8 coexpression, were tested for their binding of monomeric and oligomeric forms of the pepMHC and for subsequent responses (IL-2 release). The binding threshold for eliciting this response in the absence of CD8 (KD = 600 nM) exhibited a relatively sharp cutoff between full activity and no activity, consistent with a switchlike response to pepMHC on APCs. However, when the pepMHC was immobilized (plate bound), T cells with the lowest affinity TCRs (e.g., KD = 30 μM) responded, even in the absence of CD8, indicating that these TCR are signaling competent. Surprisingly, even cells that expressed high-affinity (KD = 16 nM) TCRs along with CD8 were unresponsive to oligomers in solution. The findings suggest that to drive downstream T cell responses, pepMHC must be presented in a form that supports formation of appropriate supramolecular clusters.
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