OP0154 TLR2 INCREASING CD8+T CELL CYTOTOXICITY BY ACTIVATING MITOPHAGY IN PRIMARY SJOGREN’S SYNDROME
Authors: Z. Zhou, Z. Tan, N. Xiang, L. Wang, X. Li
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Primary Sjogren's syndrome is one of the systemic autoimmune diseases. In recent years, studies have suggested that CD8<sup>+</sup>T cells also play an important role in the pathogenesis of pSS, but the specific molecular mechanism by which CD8<sup>+</sup>T cells cause exocrine gland damage is still unclear[1]. Therefore, in-depth study of the molecular regulatory mechanism of CD8<sup>+</sup>T will help us clarify the specific role of CD8<sup>+</sup>T cell subsets in the pathogenesis of pSS and find potential new targets for the treatment of pSS. <h3>Objectives:</h3> The aim of this study is to: 1) detect the frequency of CD8<sup>+</sup>T cells in peripheral blood, and screen and validate CD8<sup>+</sup>T cell genes through transcriptome sequencing analysis; 2) Exploring the mechanism of CD8<sup>+</sup>T cell cytotoxicity and mitochondrial dynamics disorders. <h3>Methods:</h3> 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<sup>+</sup>T cell in PBMCs from 37 pSS patients and 40 healthy donors (HD) was performed by flow cytometry.Isolating CD8<sup>+</sup>T cells from PBMCs and observing their morphology under transmission electron microscopy.Transcriptional analysis of sorted CD8<sup>+</sup>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<sup>+</sup>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. <h3>Results:</h3> 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<sup>+</sup>T cells significantly expand in peripheral blood of pSS. And we observed an increase in the proportion of CD8<sup>+</sup>T cells in the blood of pSS patients, exhibiting cytotoxic effects (p&lt;0.01) and enhanced inflammatory response(p&lt;0.05). Besides the organelle damage was also observed in CD8<sup>+</sup>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&lt;0.001) by RT-PCR. During in vitro culture of CD8<sup>+</sup>T cells, activation of TLR2 expression significantly promoted the cytotoxicity and inflammatory response of CD8<sup>+</sup>T cells (p&lt;0.05). Specially mitochondria observed with partial or complete loss of cristae and increase significantly in autophagosomes was observed under transmission electron microscopy (p&lt;0.01).The seahorse experiment confirmed that TLR2 affects both basal and maximal oxidative mitochondrial oxidative phosphorylation in CD8<sup>+</sup>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<sup>+</sup>T cells is associated with their cytotoxic effects. <h3>Conclusion:</h3> In summary, our research results demonstrate the TLR2 promotes cytotoxicity and inflammatory response of CD8<sup>+</sup>T cells by activating mitophagy, which further leads to the pathogenic role of CD8<sup>+</sup>T cells in pSS.Our research provides new potential therapeutic targets for pSS. <h3>REFERENCES:</h3> [1] Gao CY, Yao Y, Li L, et al. Tissue-Resident Memory CD8<sup>+</sup>T Cells Acting as Mediators of Salivary Gland Damage in a Murine Model of Sjögren's Syndrome. Arthritis Rheumatol. 2019;71(1):121-132. <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>