Background:Idiopathic inflammatory myopathies (IIM) also known as myositis, are rare chronic autoimmune disorders which are represented by muscle weakness and extra-muscular features such as skin rash, interstitial lung disease (ILD) and arthritis. 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). Although the exact mechanism of how these antibodies are developed is unknown, we have previously shown that upon stimulation of both peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid cells (BALF) with HisRS protein, CD4+ T cells were activated and produced inflammatory cytokines. Hitherto the presence of antigen specific autoreactive T cells has not been established in myositis, however previous studies by our group showed a strong indication of their presence with a reactivity to a specific HisRS peptide.Objectives:The main aim of this project is to detect and characterize HisRS specific CD4+T cells using HLA Class II tetramers. HLA Class II tetramers allow the detection of rare antigen specific CD4+ T cells and are widely used in studies of immunity, vaccine development, allergy monitoring and in autoimmunity. These cells are of specific interest to understand autoimmunity and to develop new therapies in autoimmune diseasesMethods:HLA-DRB1*03:01 monomers with selected tetanus and HisRS peptides were in-house in E.coli system. The peptides of interest were attached to the N-terminus of the HLA b-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 PBMCs from HLA-matched healthy controls with tetanus peptide. The drequency of tetanus specific CD4+ T cells were detected at different time points (6,13 and 21 days) from the cultures using tetanus peptides bound HLA-DRB1*0301 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. The same protocol is applied for the HisRS-peptide tetramers. Peripheral blood cells are analysed from anti-Jo1+ and HLA-DRB1*0301 positive patients with IIM.Results:Applying this method, our preliminary findings demonstrate the presence of HisRS+CD4+ T cells in peripheral blood from Jo-1+ patients (n=3) using HisRS tetramers following stimulation with the respective peptide. We are now including more patient samples to confirm our findings, and further characterize their phenotype and functionalities by flow cytometry and ELISA/fluorospot assaysConclusion: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.Acknowledgements:This work has been supported by grants from Karolinska Instiutet Resarch Foundation, Professor Nanna Svartz Stiftelse, Hjärt-Lung Fonden and Vetenskapsrådet in Sweden.Disclosure of Interests:Begum Horuluoglu: None declared, Angeles Shunashy Galindo-Feria: None declared, Karine Chemin: None declared, Genadiy Kozhukh: None declared, Anatoly Dubnovitsky: None declared, Vivianne Malmström: None declared, Ingrid E. Lundberg Consultant of: Consulting fees from Corbus Pharmaceuticals, Ind, Grant/research support from: Research grants from Bristol Myers Squibb and Astra Zeneca.
Characterization of Intracellular Cytokine Profile of CD4(+) T Cells in Peripheral Blood and Tumor-draining Lymph Nodes of Patients with Gastrointestinal Cancer