Characterization of Peripheral Blood Immune Cell Features in SSA/SSB Double‑Positive Primary Sjögren’s Disease Based on Single‑Cell RNA Sequencing

Abstract

Objective Primary Sjögren’s disease (pSjD) is a chronic systemic autoimmune disease characterized by lymphocyte infiltration of the exocrine glands. Anti-SSA (Ro) and anti-SSB (La) antibodies are diagnostic serological markers of pSjD. However, whether the coexistence of anti-SSA and anti-SSB antibodies defines an immunologically distinct subtype at the single-cell level remains unclear. Methods From our established single-cell peripheral blood mononuclear cells (PBMCs) RNA sequencing database for pSjD, 3 healthy controls, 5 SSA+/SSB− pSjD patients, and 5 SSA+/SSB+ pSjD patients were selected. After quality control and removal of doublets using DoubletFinder, batch effects were corrected with Harmony. Unsupervised clustering was performed to identify major cell lineages. Subsequently, subpopulation clustering, differential gene expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis, pseudotime trajectory analysis, and cell-cell communication analysis were carried out. Flow cytometry was further used to validate the findings from the single-cell sequencing. Results Seven major immune cell populations were identified, including T cells, B cells, monocytes/macrophages, NK cells, plasmacytoid dendritic cells (pDCs), mast cells, and platelet-megakaryocytes. Further subpopulation analysis revealed a more pronounced immune transcriptional reprogramming in the SSA+/SSB+ group compared to the SSA+/SSB− group. Specifically, cytotoxicity-related genes were upregulated in CD8+ T cells. Mucosal-associated invariant T (MAIT) cells exhibited interferon-related transcriptional program remodeling accompanied by a tissue-homing tendency. Plasma cells showed altered expression of immunoglobulin variable region genes. Monocytes/macrophages displayed changes in metabolic-related transcriptional signatures. GO/KEGG enrichment analysis, pseudotime trajectory analysis, and CellChat analysis further supported that the SSA+/SSB+ subtype possesses stronger systemic immune activation characteristics. Flow cytometry further supported altered peripheral T-cell subset distribution in SSA+/SSB+ patients, characterized by an increased proportion of CD8+ T cells and decreased proportions of CD45RA+ naïve-like T cells and MAIT cells compared with SSA+/SSB− patients. Conclusion SSA+/SSB+ pSjD may represent a subtype with stronger systemic immune activation, which partly explains why these patients exhibit higher disease activity in clinical practice.