Single cell sequencing reveals clonally expanded cytotoxic cd4+ t cells in the joints of acpa+ ra patients

Argyriou, A.; Wadsworth, M. H.; Lendvai, A.; Christensen, S.; Hensvold, A.; Gerstner, C.; Kravarik, K.; Winkler, A.; Malmström, V.; Chemin, K.

Background: CD4+ T cells with cytotoxic functions (CD4+ CTL) have gained attention in recent years. Accumulating evidence supports their importance in defense against human viral infections such as CMV1, EBV2, dengue3, HIV4, 5 and SARSCoV-26. Moreover, expansion of so called CD28null cytotoxic CD4+ T cells have been reported in the blood of patients with rheumatic diseases such as rheumatoid arthritis (RA)7, myositis8 and vasculitis9 as well as in cardiovascular diseases10. Objectives: Here, we aimed to investigate the presence and clonal expansion of CD4+ CTL in the peripheral blood (PB) and synovial fluid (SF) of RA patients using single cell technologies. Methods: We assessed the expression of cytotoxic effector molecules and transcription factors in CD4+ T cells in synovial fluid (n=21) and paired peripheral blood (n=16) from ACPA-and APCA+ RA patients by multi-parameter flow cytometry. We performed single cell sequencing, in combination with 5 TCRab sequencing, on purified CD4+ T cells from the peripheral blood (PB) and synovial fluid (SF) of ACPA+ RA patients (n=7). Results: Flow cytometry experiments show that Granzyme-B+ Perforin-1+ CD4+ CTL are significantly increased in the SF of ACPA+ RA patients as compared to ACPA-RA patients (p=0.0072). The presence of CD4+ CTL could be confirmed by single cell sequencing in SF of each ACPA+ RA patient tested (n=7). Moreover, we found that the adhesion G-protein coupled receptor GPR56 is selectively expressed on the recently described peripheral helper (TPH) T-cell subset11 and associates with the expression of tissue resident memory markers LAG-3, CXCR6 and CD69. In blood, we confirmed a previous report12 showing that GPR56 delineates cytotoxic CD4+ T cells. Finally, expanded TCR clones expressing cytotoxic effector molecules were identified in synovial fluid of ACPA+ RA patients and, for some patients, in their corresponding peripheral blood. Conclusion: We identified GPR56 as a marker of TPH cells in SF of ACPA+ RA patients that associates with tissue residency receptors. The combination of single cell sequencing and multi-parameter flow cytometry highlights the importance of CD4+ CTL in ACPA+ RA and suggests a potential therapeutic target (Figure 1).