Background/Purpose: Idiopathic inflammatory myopathies (IIM) also known as myositis, are rare chronic autoimmune disorders represented by lesions in muscle, skin and lung. One of the most common autoantibodies in myositis, with a prevalence of 25-35%, is the anti Jo-1 autoantibodies, targeting the histidyl-transfer RNA synthetase (HisRS). The presence of autoantibodies in patients along with strong associations with the HLA-DRB1*03:01 genotype, which is responsible to present peptides to activate CD4+ T-cells, suggest the existence of autoantigens being recognized by autoreactive CD4+T-cells. Moreover, we have previously shown that upon stimulation of both peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid cells (BALF) with HisRS protein and peptides, CD4+ T-cells were activated and produced inflammatory cytokines. Thus, presence of antigen specific autoreactive CD4+T-cells has not been established in myositis yet but there are clear indications about their existence.The main aim of this study is to investigate the HisRS specific CD4+T-cell population using HLA Class II tetramers. HLA Class II tetramer is an important tool for the characterization of specific CD4+T-cells and is being widely used in a large variety of diseases and vaccine studies. These cells are of specific interest to understand autoimmunity and to develop new therapies in autoimmune diseases.

Methods: HLA-DRB1*03/*01 monomers with selected tetanus and HisRS peptides were produced in-house in E.coli system. The peptides of interest were attached to the N-terminus of the HLA β-chain via a flexible peptide linker. HLA-tetramers were assembled using a commercial fluorescently labeled streptavidin. The efficacy of the peptide-HLA tetramers was validated by stimulating PBMC from HLA-matched healthy controls with tetanus peptide. The frequency of tetanus specific CD4+ T-cells were detected at different time points (6,13 and 21 days) from the cultures using tetanus peptide bound HLA-DRB1*03/*01 tetramers. The presence of tetanus specific T-cells was confirmed by the secretion of significantly higher IFNg levels upon re-stimulation of cells with tetanus peptide. HisRS specific CD4+ T-cells were investigated from PBMC of anti-Jo1+ and HLA-DRB1*03/*01 patients using tetramers and surface marker expressions.

Results: Our findings demonstrate the presence of HisRS+CD4+ T-cells in PBMC of Jo-1+ patients (n=4) using two HisRS tetramers with different fluorochromes and activation markers following stimulation with the respective peptide. We are now including more patient samples to confirm our findings, and further support our findings with functionality assays by flow cytometry and ELISA/fluorospot assays.

Conclusion: Myositis is a rare and chronic autoimmune disorder, with no currently available cure. Previous studies indicate the importance of T cells in this disease. However, the phenotype, functionality and role of these cells in the disease pathogenesis has not been fully established. Characterization of this autoreactive T-cell population will help us enhance our understanding of the disease pathogenesis and thus to develop better treatment options.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/investigation-of-antigen-specific-cd4-t-cells-in-patients-with-idiopathic-inflammatory-myopathies/