Multi-HLA Class II Tetramer Analyses of Citrulline-Reactive T Cells and Early Treatment Response in Rheumatoid Arthritis

Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterization of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described. Results: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-b, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n=7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n=14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n=5) and early RA patients (n=5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n=10) and found that the group of patients achieving minimum disease activity (DAS28 <2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells. Conclusions: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterization of antigen-specific T cells in ex vivo patient samples including RA ‘at risk’ subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.