Pos0606 detection of hisrs specific autoreactive cd4+ t cells in patients with idiopathic inflammatory myopathies

Background Idiopathic inflammatory myopathies (IIM) or myositis are a group of chronic autoimmune disorders represented by lesions in muscle, skin, joints and lung. One of the most common autoantibodies with a prevalence of 25-35 % is anti-Jo1, targeting histidyl-transfer RNA synthetase (HisRS). Importance of T-cells in disease is established by their presence at sites of inflammation such as the muscle. We have shown that upon stimulation of PBMC with HisRS, CD4 + T-cells are activated and produce inflammatory cytokines. Genetic association of HLA-DRB1*03:01 further implicates the recognition of autoantigens by CD4 + T-cells. However, the presence of antigen specific CD4 + T-cells has not yet been shown in patients with IIM. Objectives The aim of this project is to identify HisRS CD4 + T-cells using HLA Class-II tetramers (Tmr) and single T cell receptor (TCR) sequencing. Methods HLA-DRB1*03:01 monomers with selected HisRS- and tetanus peptides as controls were produced in E.coli. The peptides were covalently linked to the HLA b-chain via a flexible peptide linker. HLA-tetramers were assembled using a commercial APC or PE labeled streptavidin. The efficacy of Tmrs was validated upon stimulation of PBMCs from HLA-matched healthy controls with tetanus peptide. In parallel, upregulation of T cell activation markers such as PD-1, CD69, CD137 was also investigated. Next, we stimulated PBMCs from anti-Jo1 positive patients with IIM that were HLA-DRB1*03 (n=10) with both tetanus and HisRS peptides and cultured the cells for 6 days followed by tetramer staining. Fluorospot was performed for the detection of IFNg and IL2 secreting cells and culture supernatants were collected for cytokine analysis using cytokine bead arrays. Summary of the workflow is presented in Figure 1. Results We detected HisRS+CD4 + T cells from 5 out of 10 patients using tetramers upon stimulation with HisRS peptide. We detected increased IFNg levels in the supernatants where Tmr+ cells were detected further supporting the activation of T cells. HisRS + CD4 + T cells from patients (n=5) were single cell sorted and TCR a/b chains were sequenced (n=538). A bioinformatics pipeline for the alignment and assembly of the TCR sequences, was developed and the clonality analysis revealed presence of expanded T cell clones in 4 out of 5 patients. Expanded T cells had high mean fluorescent intensity values (MFI) for HisRS tetramers, suggesting the responsiveness of autoreactive T-cells to HisRS. Conclusion IIM are rare, chronic autoimmune disorders with no available cure. Previous studies indicate the importance of T cells in this disease. However, the phenotype and role of these cells in the disease pathogenesis has not been fully established. Our results indicate the presence of HisRS+CD4 + T-cells in IIM which will introduce the possibility of new targeted treatment approaches. Figure 1. Summary of experimental setup starting from the stimulation of PBMCs and tetramer staining (Step 1), sorting of tetramer+ cells (Step 2), preparation of cells for TCR sequencing (Step 3) and analysis of TCRs (Step 4). Acknowledgements This project is supported by the Myositis Association, Lung and Heart Foundation, Swedish Research Council (VR), Professor Nana Svartz Foundation and Karolinska Insitutet Foundations. Disclosure of Interests Begum Horuluoglu: None declared, Angeles Shunashy Galindo-Feria: None declared, Ravi Kumar: None declared, Genadiy Kozhukh: None declared, Juan Sebastian Díaz-Boada: None declared, Daniel Ramsköld: None declared, Karine Chemin: None declared, Vivianne Malmström: None declared, Ingrid E. Lundberg Shareholder of: Roche and Novartis, Consultant of: Corbus Pharmaceuticals, Grant/research support from: Astra Zeneca, Employee of: Advisory board for Astra Zeneca, Bristol Myers Squibb, EMD Serono Research & Development Institute, Argenx, Octapharma, Kezaar, Orphazyme, Pfizer and Janssen.