POS0102 GLOBAL CHARACTERIZATION OF SALIVARY GLANDS IMMUNE MICROENVIRONMENT IN PRIMARY SJÖGREN’S SYNDROME BY SINGLE-CELL SEQUENCING

Abstract

BackgroundPrimary Sjögren’s syndrome (pSS) is a heterogeneous, chronic, complex systemic autoimmune disease. The hallmark symptom of the disease is exocrinopathy, chiefly salivary and lachrymal glands, which often results in dryness of the mouth and eyes. As of today, although a lot of genetic and epigenetic studies have reveal the complexity of pSS to a certain extent, but the knowledge of existing pSS disease heterogeneity is still limited and the immune mechanisms of salivary glands (SG) injury have been challenging to clarify.ObjectivesSingle-cell RNA sequencing (scRNA-seq) is a powerful tool capable of defining cell types and states on the basis of their individual transcriptome in a given sample from health and disease. To characterize the salivary glands immune microenvironment of patients with pSS, we performed droplet-based single cell mRNA sequencing (scRNA-seq) (10X Genomics) to provide a deeper insight into the cellular and molecular characteristics of salivary glands from pSS patients.Methods11 patients and 5 non-pSS controls were recruited from the The First Affiliated Hospital of USTC. The non-pSS were subjects who had experienced subjective symptoms of dryness, but no not meet any of the classification criteria of pSS. The clinical characteristics and laboratory findings of enrolled patients were also collected. After resection, salivary glands tissue samples were obtained after labial gland biopsy, rapidly digested to a single-cell suspension and subjected to scRNA-seq using the 10X platform. After rigorous quality control (QC) definition, low-quality cells were filtered. Following gene expression normalization for read depth and mitochondrial read count, we applied principle component analysis on genes variably expressed across all 72,853 cell.ResultsA total of 72,853 cells were obtained from all salivary glands samples. Our results revealed 12 major unique cell populations of salivary glands cell, including T cells, B cells, plasma cells, epithelial cells, myoepithelial cells, endothelial cells, myofibroblast, pericytes, melanocytes, fibroblast, myeloid cells and a cluster of unknown cells. As expected, lymphocytes (T and B cell populations) were significant increase in the salivary glands of patients with pSS. For further subsets analysis, we identify 41 subsets, including novel subpopulations in cell types hitherto considered to be homogeneous, as well as transcription factors underlying their heterogeneity. Strikingly, we found that differentially expressed genes (DEGs) that myoepithelial cells uniquely downregulated in pSS patients were involved in regeneration, stem cell population maintenance, cell division, and epithelial cell proliferation. This indicated an impaired stem cell property and regeneration capacity of myoepithelial cells in the SG of pSS patients which may result in the reduction of normal epithelial cells differentiation and proliferation. Our results identified three distinct endothelial subtypes according to the differentially expressed cell markers. ACKR1+ endothelial cells were expanded in the SG of pSS patients which may enhance Leukocyte transendothelial migration. A clear interferon response was observed in most celltypes. We also found a significantly expand PD-1hiCXCR5–CD4+T peripheral helper (Tph), GZMK+CD8+ T cells and a patient-specific fibroblasts in pSS patients. Cellular interaction analysis of SG revealed a strong interaction between epithelial cells and immune cells from pSS patients through CD74-MIF, MIF-TNFRSF14 and HLA-C-FAM3C receptor/ ligand pairs. Chemokine receptors CXCR4 were broadly expressed in SG immune cells implying a potentially central role in cell trafficking.ConclusionThis resource provides deeper insights into pSS salivary glands immune microenvironment that will be helpful in understanding of the disease heterogeneity and advancing pSS therapy.Disclosure of InterestsNone declared