OP0072 SINGLE CELL SEQUENCING REVEALS CLONALLY EXPANDED CYTOTOXIC CD4+ T CELLS IN THE JOINTS OF ACPA+ RA PATIENTS
Authors: A. Argyriou, M. H. Wadsworth, A. Lendvai, S. Christensen, A. Hensvold, C. Gerstner, K. Kravarik, A. Winkler, V. Malmström, K. Chemin
Journal: Annals of the Rheumatic Diseases (2021)

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 CMV 1 , EBV 2 , dengue 3 , HIV 4, 5 and SARS-CoV-2 6 . 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 , myositis 8 and vasculitis 9 as well as in cardiovascular diseases 10 . 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 (T PH ) T-cell subset 11 and associates with the expression of tissue resident memory markers LAG-3, CXCR6 and CD69. In blood, we confirmed a previous report 12 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 T PH 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). References: [1]Casazza J. P. et al ., J Exp Med 2006, 203 (13), 2865-77. [2]Landais E. et al., Blood 2004, 103 (4), 1408-16. [3]Kurane I. et al . J Exp Med 1989, 170 (3), 763-75. [4]Appay V. et al . J Immunol 2002, 168 (11), 5954-8. [5]Juno J. A. et al . Front Immunol 2017, 8 , 19. [6]Meckiff B. J. et al . Cell 2020, 183 (5), 1340-1353 e16. [7]Schmidt D. et al . J Clin Invest 1996, 97 (9), 2027-37. [8]Fasth A. E. et al . J Immunol 2009, 183 (7), 4792-9. [9]Moosig F. et al . Clin Exp Immunol 1998, 114 (1), 113-8. [10]Sato K. et al . J Exp Med 2006, 203 (1), 239-50. [11]Rao D. A., et al . Nature 2017, 542 (7639), 110-114. [12]Peng Y. M. et al . J Leukoc Biol 2011, 90 (4), 735-40. Acknowledgements: We thank the patients who donated samples and the medical staff at the Rheumatology Clinic of Karolinska University Hospital. Julia Boström, Gloria Rostvall, and Susana Hernandez Machado are acknowledged for organizing the sampling, storage, and administration of biomaterial. This study is supported by grants from Dr. Margaretha Nilssons, the Nanna Svartz, the Ulla and Gustaf af Ugglas foundations and the Swedish association against rheumatism. Disclosure of Interests: Alexandra Argyriou: None declared, Marc H Wadsworth II Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Adrian Lendvai: None declared, Stephen Christensen Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Aase Hensvold: None declared, Christina Gerstner: None declared, Kellie Kravarik Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Aaron Winkler Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Vivianne Malmström: None declared, Karine Chemin: None declared