Peripheral CD4 + CD8 + double-positive (DP) T cells have been implicated in the pathogenesis of autoimmune diseases; however, the precise pathogenic mechanisms of circulating DP T cells remain unclear. This study aims to identify the key pathogenic DP T cell subset and develop corresponding targeted therapeutic strategies for autoimmune diseases, including rheumatoid arthritis (RA). We utilized single-cell RNA sequencing (scRNA-seq) to profile DP T cells from peripheral blood of RA patients and healthy controls (HC), with variations in key subset frequencies validated by flow cytometry. Integrated analysis of differentially expressed genes, pseudotime trajectory, and cell-cell communication identified the IL-27/ANXA1 signaling axis. For in vitro validation, DP T cells from RA patients were treated with recombinant IL-27 (rIL-27) or anti-IL-27 neutralizing antibody (anti-IL-27), and the frequency of ANXA1 + GZMB + cells was assessed by flow cytometry. In a collagen-induced arthritis (CIA) mouse model, systemic administration of rIL-27 or anti-IL-27 was performed; splenic DP T cells and the GZMB + subset were analyzed by flow cytometry, ANXA1 expression in sorted GZMB + DP T cells was quantified by RT-qPCR, and joint inflammation was evaluated clinically and histopathologically. scRNA-seq revealed a distinct GZMB + DP T cell subset in RA patients, characterized by significant downregulation of IL27RA and ANXA1-mediated aberrant communication with monocytes. Flow cytometry confirmed the expansion of GZMB + DP T cells in peripheral blood of RA patients compared with HC. In vitro, IL-27 suppressed the proportion of ANXA1 + cells among DP T cells, whereas IL-27 blockade reversed this effect. In CIA mice, IL-27 treatment reduced the splenic GZMB + DP T frequency and alleviated joint swelling, while IL-27 inhibition exacerbated arthritis. Mechanistically, IL-27 constrains DP T cell-mediated pathogenic responses via an ANXA1-dependent pathway. GZMB + CD4 + CD8 + T cells represent a potential pathogenic subset within DP T cells that actively contributes to RA progression. Importantly, IL-27 appears to exert therapeutic effects by modulating DP T cell differentiation—particularly the GZMB + DP T cell subset—and suppressing ANXA1-mediated intercellular communication between GZMB + CD4 + CD8 + T cells and monocytes, thereby offering a potential treatment strategy for RA.
Xue et al. (2026) studied this question.