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Objectives The aim of this study was to combine deep T cell phenotyping with assessment of citrulline-reactive CD4+T cells in the pre-rheumatoid arthritis (RA) phase.Methods 20 anti-CCP2 positive individuals (HLA-DRB1*04:01) presenting musculoskeletal complaints without clinical or ultrasound signs of synovitis; 10 arthritis progressors and 10 matched non-arthritis progressors were included. Longitudinal samples (1–3 time points) of peripheral blood mononuclear cells were assessed using HLA-class II tetramers with 12 different citrullinated candidate autoantigens combined in a >20-colour spectral flow cytometry panel.Results The baseline CD4+T cell phenotype was similar between individuals who progressed to arthritis (ie, in the pre-RA phase) and the non-progressors, when studying markers associated with Th1, Th17, T-peripheral and T-regulatory cells as well as with T-cell activation. Citrulline-reactive CD4+T cells were present in both groups but at significantly lower frequency in the progressor group. CD4+T cells specific for citrullinated tenascin-C were the most frequently observed among the progressors, and their frequencies diminished during follow-up that is, closer to arthritis onset. Notably, PD-1 and CD95 expression on the memory cit-tenascin-C-specific T cells in this group indicated repeated antigen exposure.Conclusions Our data lend support to citrullinated tenascin-C as an interesting T cell antigen in RA. Moreover, lower frequency of circulating citrulline-specific cells in arthritis progressing individuals suggest an initiated homing of these cells to the joints and/or their associated lymph nodes in the pre-RA phase and a possible window of opportunity for therapeutic preventive interventions.

Abstract We aimed 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). 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. 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 cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. 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.

Abstract Background HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. Results Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-β, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28

Tenascin-C (TNC), an extracellular matrix protein that has proinflammatory properties, is a recently described antibody target in rheumatoid arthritis (RA). In this study, we utilized a systematic discovery process and identified 5 potentially novel citrullinated TNC (cit-TNC) T cell epitopes. CD4+ T cells specific for these epitopes were elevated in the peripheral blood of subjects with RA and showed signs of activation. Cit-TNC–specific T cells were also present among synovial fluid T cells and secreted IFN-γ. Two of these cit-TNC T cell epitopes were also recognized by antibodies within the serum and synovial fluid of individuals with RA. Detectable serum levels of cit-TNC–reactive antibodies were prevalent among subjects with RA and positively associated with cyclic citrullinated peptide (CCP) reactivity and the HLA shared epitope. Furthermore, cit-TNC–reactive antibodies were correlated with rheumatoid factor and elevated in subjects with a history of smoking. This work confirms cit-TNC as an autoantigen that is targeted by autoreactive CD4+ T cells and autoantibodies in patients with RA. Furthermore, our findings raise the possibility that coinciding epitopes recognized by both CD4+ T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of RA.