Op0154 Tlr2 Increasing Cd8+t Cell Cytotoxicity by Activating Mitophagy in Primary Sjogren’s Syndrome

Background: Primary Sjogren's syndrome is one of the systemic autoimmune diseases. In recent years, studies have suggested that CD8+T cells also play an important role in the pathogenesis of pSS, but the specific molecular mechanism by which CD8+T cells cause exocrine gland damage is still unclear1. Therefore, in-depth study of the molecular regulatory mechanism of CD8+T will help us clarify the specific role of CD8+T cell subsets in the pathogenesis of pSS and find potential new targets for the treatment of pSS. Objectives: The aim of this study is to: 1) detect the frequency of CD8+T cells in peripheral blood, and screen and validate CD8+T cell genes through transcriptome sequencing analysis; 2) Exploring the mechanism of CD8+T cell cytotoxicity and mitochondrial dynamics disorders. Methods: We obtained 11 human SG biopsy samples from pSS patients and 5 SG samples from non-SS control individuals who had sicca symptom but not meeting any of the classification criteria of pSS.The peripheral blood samples from 5 patients with pSS and 3 healthy controls were also collected and digested into single-cell suspensions, and subjected to scRNA-seq.Phenotypical analysis of CD8+T cell in PBMCs from 37 pSS patients and 40 healthy donors (HD) was performed by flow cytometry.Isolating CD8+T cells from PBMCs and observing their morphology under transmission electron microscopy.Transcriptional analysis of sorted CD8+T cell from 6 pSS patients and 5 HD was performed by RNA-seq. Differential gene expression was validated using RT-PCR and WB. Additionally, primary CD8+T cells cultured in vitro were used to detect their functional effects under activation or inhibition of TLR2.Simultaneously detect the morphology and function of mitochondria in primary CD8+T cells after cultivation. Results: Here, We obtained a total of 79,596 immune cell transcriptomes by the integration of PBMCs and SG scRNA-seq data. Among these, cytotoxic CD8+T cells significantly expand in peripheral blood of pSS. And we observed an increase in the proportion of CD8+T cells in the blood of pSS patients, exhibiting cytotoxic effects (p+T cells of pSS patients by TEM. The transcriptome sequencing results identified 9 key genes through the bioinformatics algorithm, among which TLR2 was found to have the most significant expression difference (p+T cells, activation of TLR2 expression significantly promoted the cytotoxicity and inflammatory response of CD8+T cells (p+T cell. JC-1 results showed impaired mitochondrial membrane potential, and WB detected a significant increase in the expression of mitochondrial autophagy proteins PINK1 and Parkin.These findings suggest that mitophagy in CD8+T cells is associated with their cytotoxic effects. Conclusion: In summary, our research results demonstrate the TLR2 promotes cytotoxicity and inflammatory response of CD8+T cells by activating mitophagy, which further leads to the pathogenic role of CD8+T cells in pSS.Our research provides new potential therapeutic targets for pSS. REFERENCES: 1 Gao CY, Yao Y, Li L, et al. Tissue-Resident Memory CD8+T Cells Acting as Mediators of Salivary Gland Damage in a Murine Model of Sjögren's Syndrome. Arthritis Rheumatol. 2019;71(1):121-132. Acknowledgements: NIL. Disclosure of Interests: None declared.