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Objectives Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation–associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein. Methods We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n  = 8) and serum (n  = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains. Results All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein. Conclusions Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade. [ABSTRACT FROM AUTHOR]

Mutations of the gene encoding four-and-a-half LIM domain 1 (FHL1) are the causative factor of several X-linked hereditary myopathies that are collectively termed [FHL1.sup.-]related myopathies. These disorders are characterized by severe muscle dysfunction and damage. Here, we have shown that patients with idiopathic inflammatory myopathies (IIMs) develop autoimmunity to FHL1, which is a muscle-specific protein. Anti-FHL1 autoantibodies were detected in 25% of IIM patients, while patients with other autoimmune diseases or muscular dystrophies were largely anti-FHL1 negative. Anti-FHL1 reactivity was predictive for muscle atrophy, dysphagia, pronounced muscle fiber damage, and vasculitis. FHL1 showed an altered expression pattern, with focal accumulation in the muscle fibers of autoantibody-positive patients compared with a homogeneous expression in anti-[FHL1.sup.-]negative patients and healthy controls. We determined that FHL1 is a target of the cytotoxic protease granzyme B, indicating that the generation of FHL1 fragments may initiate FHL1 autoimmunity. Moreover, immunization of myositis-prone mice with FHL1 aggravated muscle weakness and increased mortality, suggesting a direct link between anti-FHL1 responses and muscle damage. Together, our findings provide evidence that FHL1 may be involved in the pathogenesis not only of genetic [FHL1.sup.-]related myopathies but also of autoimmune IIM. Importantly, these results indicate that anti-FHL1 autoantibodies in peripheral blood have promising potential as a biomarker to identify a subset of severe IIM., Introduction Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare systemic autoimmune diseases collectively called myositis, which causes progressive muscle weakness. Several forms ofthe disease, including polymyositis (PM), dermatomyositis [...]

Objective: CD4+ T cells are implicated in rheumatoid arthritis (RA) pathology from the strong association between RA and certain HLA class II gene variants. This study was undertaken to examine the synovial T cell receptor (TCR) repertoire, T cell phenotypes, and T cell specificities in small joints of RA patients at time of diagnosis before therapeutic intervention. Methods: Sixteen patients, of whom 11 patients were anti–citrullinated protein antibody (ACPA)–positive and 5 patients were ACPA–, underwent ultrasound‐guided synovial biopsy of a small joint (n = 13) or arthroscopic synovial biopsy of a large joint (n = 3), followed by direct sorting of single T cells for paired sequencing of the αβ TCR together with flow cytometry analysis. TCRs from expanded CD4+ T cell clones of 4 patients carrying an HLA–DRB1*04:01 allele were artificially reexpressed to study antigen specificity. Results: T cell analysis demonstrated CD4+ dominance and the presence of peripheral helper T–like cells in both patient groups. We identified >4,000 unique TCR sequences, as well as 225 clonal expansions. Additionally, T cells with double α‐chains were a recurring feature. We identified a biased gene usage of the Vβ chain segment TRBV20‐1 in CD4+ cells from ACPA+ patients. In vitro stimulation of T cell lines expressing selected TCRs with an extensive panel of citrullinated and viral peptides identified several different virus‐specific TCRs (e.g., human cytomegalovirus and human herpesvirus 2). Still, the majority of clones remained orphans with unknown specificity. Conclusion: Minimally invasive biopsies of the RA synovium allow for single‐cell TCR sequencing and phenotyping. Clonally expanded, viral‐reactive T cells account for part of the diverse CD4+ T cell repertoire. TRBV20‐1 bias in ACPA+ patients suggests recognition of common antigens. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease affecting synovial joints where different CD4+ T cell subsets may contribute to pathology. Here, we perform single cell sequencing on synovial CD4+ T cells from anti-citrullinated protein antibodies (ACPA)+ and ACPA- RA patients and identify two peripheral helper T cell (TPH) states and a cytotoxic CD4+ T cell subset. We show that the adhesion G-protein coupled receptor 56 (GPR56) delineates synovial CXCL13high TPH CD4+ T cells expressing LAG-3 and the tissue-resident memory receptors CXCR6 and CD69. In ACPA- SF, TPH cells display lower levels of GPR56 and LAG-3. Further, most expanded T cell clones in the joint are within CXCL13high TPH CD4+ T cells. Finally, RNA-velocity analyses suggest a common differentiation pathway between the two TPH clusters and effector CD4+ T cells. Our study provides comprehensive immunoprofiling of the synovial CD4+ T cell subsets in ACPA+ and ACPA- RA. Antibodies against citrullinated proteins (ACPA) can divide rheumatoid arthritis patients but the difference in immune phenotype is not clear. Here the authors characterise T peripheral helper cells in arthritic joints comparing ACPA+ and ACPA- patients showing changes in immune cell phenotype and surface molecules. [ABSTRACT FROM AUTHOR]

The pre-T ten receptor (TCR) functions as a critical checkpoint during [alpha][beta] T cell development. Signaling through the pre-TCR controls the differentiation of immature CD[4.sup.-]CD[8.sup.-]CD[25.sup.+]CD[44.sup.-] [double-negative (DN)3] thymocytes into CD[4.sup.+]CD[8.sup.+] double-positive (DP) cells through the CD[4.sup.-]CD[8.sup.-]CD[25.sup.-]CD[44.sup.-](DN4) stage. In addition, pre-TCR activity triggers expansion and survival of thymocytes and inhibits TCR[beta] gene rearrangement through a process referred to as allelic exclusion. Whereas many proteins involved in the pre-TCR transduction cascade have been identified, little is known about the nuclear factors associated with receptor function. Here, we use gene targeting to inactivate the Ets-1 transcription factor in mice and analyze pre-TCR function in developing Ets-1deficient (Ets-[1.sup.-/-]) thymocytes. We find that inactivation of Ets-1 impairs the development of DN3 into DP thymocytes and induces an elevated rate of cell death in the DN4 subset. This defect appears specific to the [alpha][beta] lineage because [gamma][delta] T cells maturate efficiently. Finally, the percentage of thymocytes coexpressing two different TCR[beta] chains is increased in the Ets-[1.sup.-/-] background and, in contrast with wild type, forced activation of pre-TCR signaling does not block endogenous TCR[beta] gene rearrangement. These data identify Ets-1 as a critical transcription factor for pre-TCR functioning and for allelic exclusion at the TCR[beta] locus.