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

Rheumatoid arthritis is an autoimmune disease affecting the synovial joints where different subsets of CD4 + T cells are suspected to play a pathogenic role. So far, our understanding of the contribution of cytotoxic CD4 + T cells is incomplete, particularly in the context of the recently described peripheral helper T-cell subset (T PH ). Here, using single cell sequencing and multi-parameter flow cytometry, we show that cytotoxic CD4 + T cells are enriched in synovial fluid of anti-citrullinated peptides antibody (ACPA)-positive RA patients. We identify two distinct T PH states 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 synovial T PH CD4 + T-cell subset. 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 expanded clones in SF are contained within the cytotoxic and the CXCL13 + T PH CD4 + T-cell populations. Finally, the detection of common TCRs between the two T PH and cytotoxic CD4 + T-cell clusters suggest a shared differentiation. Our study provides comprehensive immunoprofiling of the heterogenous T-cell subsets at the site of inflammation in ACPA+ RA and suggests GPR56 as a therapeutic target to modulate T PH cells and cytotoxic CD4 + T cell function.