Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

Abstract

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.

Biased TCR gene usage in citrullinated Tenascin C specific T-cells in rheumatoid arthritis

“…Still, the scarcity of autoreactive T cells continues to make studies challenging. Our method of in-vitro stimulating patient-derived primary cells with proposed autoantigens in presence of recombinant human IL-2 and IL-7 allows the propagation and subsequent capture of these rare cells for downstream studies of the T cell receptor repertoire 16 , where IL-7 was especially important for the synovial cultures.…”

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis

“…So far, we lack the knowledge on well-defined PTA presented by human LNSCs and the availability of corresponding autoreactive human T cells. However, recently a multi-tetramer assay has been developed to detect citrulline-specific T cells in both healthy individuals and RA patients [ 54 ]. These tetramers might allow us to study tolerance capacity of LNSCs in controlling citrulline-specific T cells using our in vitro model containing expanded human LNSCs.…”

The shared susceptibility epitope of HLA-DR4 binds citrullinated self-antigens and the TCR

“…Multiple citrullinated peptides have been shown to be B cell antigens in patients with RA, as is evidenced by the high level of ACPA positivity in patients with RA (13)(14)(15)(16). Numerous citrullinated peptides have also been shown to be T cell antigens in RA including -enolase (24)(25)(26), vimentin (24,25), cartilage intermediate layer protein (24,25), and fibrinogen (24,25). Consistent with their role in disease, CD4 + T cells produce proinflammatory cytokines in response to citrullinated self-epitopes (27), citrulline-reactive CD4 + T cells are present in HLA-DR4 transgenic mice immunized with citrullinated selfantigens (28) and in the RA joint (26), and citrulline-reactive T helper 1 cells are elevated in patients with HLA-DR4 + RA (11,24).…”