Objective 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). Methods 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. Results 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. Conclusion 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.

GZMB⁺CD4⁺CD8⁺ T cells in rheumatoid arthritis: a potential therapeutic target for IL-27 intervention

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2026年6月2日

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Jiawei Xue,
Suna Jiang,
Haonan Jia,
Weiyan Wang,
Wenjing Li,
Yanli Wang,
Jianyang Zhuang,
Xuemin Wei,
Hongying Li,
Maolin Chu,
Liang Zhang,
Juan Zhang

摘要

Objective

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).

Methods

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.

Results

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.

Conclusion

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.

GZMB+CD4+CD8+ T cells in rheumatoid arthritis: a potential therapeutic target for IL-27 intervention

摘要

GZMB⁺CD4⁺CD8⁺ T cells in rheumatoid arthritis: a potential therapeutic target for IL-27 intervention