Single cell sequencing reveals expanded cytotoxic CD4+ T cells and two clusters of peripheral helper T cells in synovial fluid of ACPA+ RA patients

Rheumatoid arthritis is an autoimmune disease affecting the synovial joints where CD4+ T cells play a pathogenic role. However, a deeper analysis of cytotoxic CD4+ T cells is still lacking, particularly in the context of the recently described peripheral helper T-cell subset (TPH). Here, we show, using 10X-single-cell sequencing and multi-parameter flow cytometry that cytotoxic CD4+ T cells are enriched in synovial fluid (SF) of anti-citrullinated peptides antibody (ACPA)-positive RA patients. We also identify two distinct TPH clusters differentially characterized by the expression of CXCL13 and PRDM1, respectively. Our data reveal that the adhesion G-Protein Coupled Receptor 56 (GPR56), a marker of circulating cytotoxic cells, delineates the TPH CD4+ T-cell subset in SF. At the site of inflammation, GPR56+ CD4+ T cells expressed the tissue-resident memory markers LAG-3, CXCR6, and CD69. Further, TCR clonality analysis revealed that most of the expanded clones in PB and SF are contained within the cytotoxic CD4+ T-cell population. Finally, the detection of common TCRs between the two TPH and cytotoxic CD4+ T cell clusters suggests a common differentiation pathway between these three subsets. Our study provides a better overview of the different pathogenic T-cell subsets at the site of inflammation in ACPA+ RA and suggests GPR56 as a therapeutic target to modulate TPH cells and cytotoxic CD4+ T cell function.EGA study EGAS00001005241