Immunological profiling of rheumatoid factor-positive primary Sjögren's syndrome by single-cell RNA sequencing

Primary Sjögren's syndrome (pSS) is a highly heterogeneous autoimmune disease. Previous studies have suggested that rheumatoid factor (RF)-positive patients appear to have a higher incidence of systemic involvement. However, the underlying mechanisms remain unclear. This study aims to elucidate the potential biological mechanisms through single-cell RNA sequencing (scRNA-seq).

Peripheral blood mononuclear cells from 3 healthy controls, 6 RF-negative (RF^-) pSS patients, and 6 RF-positive (RF⁺) pSS patients were subjected to scRNA-seq. Subsequent analyses included pathway enrichment analysis, trajectory analysis, cell-cell communication analysis, and B cell receptor (BCR) and T cell receptor (TCR) repertoire profiling. Potential differences were further validated by flow cytometry.

Analysis of B cell subsets revealed a significantly increased proportion of plasma cells in RF⁺ patients, and differential gene expression analysis indicated enhanced pathways related to antibody production, clonal expansion, and type I interferon activation. In T-cell subsets, the RF⁺ group showed a notably higher proportion of CD8⁺ T cells, which exhibited stronger cytotoxicity and activation of interferon signaling. In contrast, mucosal-associated invariant T (MAIT) cells demonstrated activated interferon signaling but were present at a lower proportion compared to the RF^- group. Cell-cell communication analysis revealed strengthened crosstalk between B cells and T cells in the RF⁺ group. Pseudo-time trajectory analysis further indicated that in RF⁺ patients, both plasma cells and CD8⁺ T cells exhibited a developmental skew toward a terminally differentiated state. Additionally, both BCR and TCR repertoire analyses indicated RF⁺ and RF^- patients exhibiting distinct patterns in clonal distribution and frequency. Flow cytometry further validated the increased proportion of CD8⁺ T cells and the decreased proportion of MAIT cells in the peripheral blood of RF⁺ pSS patients.

The RF status in pSS patients is closely associated with specific immune cell profiles. RF⁺ patients exhibit more active plasma cell responses, enhanced CD8⁺ T-cell cytotoxicity and interferon responses, as well as heightened interferon signaling yet reduced proportion of MAIT cells. RF status serves as a key stratification marker for immune heterogeneity in pSS patients, providing new insights for understanding disease mechanisms and developing targeted therapeutic strategies.