Application of imaging mass cytometry for spatially profiling the microenvironment of salivary glands in primary Sjögren's syndrome.

Primary Sjogren's syndrome (pSS) is a slowly progressive, systemic autoimmune disorder characterized by gradual lymphocytic infiltration of exocrine glands. However, the spatially profiling the immune microenvironment in pSS is largely unclear, limiting the understanding of the complex interplay among cells within the microenvironment. Based on imaging mass cytometry (IMC) analysis of clinical pSS samples, we first revealed that labial salivary gland (LSG) comprised of epithelial, immune cells and stromal cells, and epithelial was the main cell type in LSG. Eight immune cells populations were identified, including CD8(+) T, CD4(+) T, Treg, B, NK cells, neutrophils, resident macrophages and a mixed immune cell cluster. We found that CD8(+) T cells, but not CD4(+) T cells, were the most prominent T cells in immune infiltrates of pSS LSG. With the increase of pSS disease activity and severity, the infiltration abundance of CD8(+) T cells gradually increased and was accompanied by the activation of inflammatory response. sc-RNA-seq analysis based on the GSE272409 dataset confirmed that CD8(+) T cells were the main immune cells, and dominated the most intercellular ligand-receptor interactions. CD8(+) T cells were further clustered into five cell subsets, of which CD160(+)CD8(+) T cells subset appeared to present only in pSS patients. Further experiments demonstrated that CD160 expression on CD8(+) T cells was associated with an enhanced expression of proinflammatory and cytotoxic cytokines IFN-γ, GZMB and TNF-α, and the injury of salivary gland epithelial cells. Besides, proportion of GZMK(+)CD8(+) T cells subset was increased in pSS patients. Trajectory analysis confirmed an enhanced frequency of CD8(+) T cell differentiation and activation during the progression of pSS. This study provided single cell profile with spatial information for analyzing the LSG immune microenvironment in pSS, which could not be achieved by conventional immunofluorescence and immunohistochemistry assays.