The T cell phenotype and receptor repertoire in different stages of rheumatoid arthritis : an immunological timeline

Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by symmetrical inflammation in hands and feet. Antibodies recognizing citrullinated protein (ACPA) and/or rheumatoid factor (RF) are found in most patients and associates with a worse prognosis. Genetic predisposition (i.e., specific HLA-DRB1 alleles) combined with environmental threats (e.g., smoking) increase the risk to develop seropositive RA and is thought to evoke a break in tolerance toward citrullinated autoantigens. Whereas it is well known that autoantibodies are present in the circulation long before disease onset, this is an unexplored area for autoreactive T cells recognizing citrullinated antigens. Additionally, little is known about such autoreactive T cells as the disease progress. The aim of this thesis was to explore the occurrence, phenotype, and T cell receptor repertoire of autoreactive CD4+ T cells during different stages of disease. We have studied several cohorts including individuals at-risk of developing disease, patients with recent onset RA and patients with longstanding disease. Samples from peripheral blood, lymph nodes, synovial fluid, and synovial tissue have been assessed at the single cell level primarily using HLA-class II tetramers (£12 citrullinated peptides), surface phenotyping, paired ab-T cell receptor (TCR) sequencing or FluoroSpot. In addition, we have re-expressed TCRs into artificial cell lines to test antigen specificity. With the exception of TCR-repertoire analysis of synovial tissue, all individuals were HLA-DRB1*04:01 carriers. Autoreactive CD4+ T cells recognizing citrullinated peptides were rare, but detectable already prior to disease onset in individuals at-risk of developing RA. Notably, the frequencies were significantly lower in those who later progress to arthritis. We found citrullinated peptides from tenascin-C (a large extracellular matrix protein) to be a novel T cell autoantigen and notably, when assessed, this was the most frequent specificity during the different stages of disease, both in blood and synovial fluid. Citrullinated a-enolase, cartilage intermediate layer protein (CILP), fibrinogen-b and to a less degree vimentin peptides was also recognized by autoreactive CD4+ T cells during the course of RA. Intriguingly, lymph node CD4+ T cells primarily recognized citrullinated vimentin both in the at-risk phase and early stage of RA. The T cell receptor repertoire in the synovial tissue was diverse already at time of diagnosis, whereas both CD4+ and CD8+ cells contributed to clonal expansions. Viral reactive T cells constituted a small fraction of the clonally expanded CD4+ T cells, whereas the majority were orphans in terms of antigen specificity. However, a bias in gene usage of CD4+ T cell (TRBV20-1) was seen amongst ACPA+ patients and interestingly such bias was also noted in CD4+ T cells specific to citrullinated tenascin-C peptides derived from both synovial fluid and blood in longstanding RA. To conclude, autoreactive CD4+ T cells constitute a small, but detectable part of the T cell compartment during the different stages of rheumatoid arthritis. The T cell receptor repertoire is diverse early in disease – whereas genetic bias might imply recognition of a common antigen. The knowledge of such cells and their specificity, of which tenascin-C was most frequent in our studies can direct future efforts in tolerizing therapies to cure or ultimately prevent disease

Shared recognition of citrullinated tenascin-C peptides by T and B cells in rheumatoid arthritis

Tenascin-C (TNC), an extracellular matrix protein that has proinflammatory properties, is a recently described antibody target in rheumatoid arthritis (RA). In this study, we utilized a systematic discovery process and identified 5 potentially novel citrullinated TNC (cit-TNC) T cell epitopes. CD4+ T cells specific for these epitopes were elevated in the peripheral blood of subjects with RA and showed signs of activation. Cit-TNC–specific T cells were also present among synovial fluid T cells and secreted IFN-γ. Two of these cit-TNC T cell epitopes were also recognized by antibodies within the serum and synovial fluid of individuals with RA. Detectable serum levels of cit-TNC–reactive antibodies were prevalent among subjects with RA and positively associated with cyclic citrullinated peptide (CCP) reactivity and the HLA shared epitope. Furthermore, cit-TNC–reactive antibodies were correlated with rheumatoid factor and elevated in subjects with a history of smoking. This work confirms cit-TNC as an autoantigen that is targeted by autoreactive CD4+ T cells and autoantibodies in patients with RA. Furthermore, our findings raise the possibility that coinciding epitopes recognized by both CD4+ T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of RA

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 characterisation 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 alpha-enolase, fibrinogen-beta, 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 characterisation 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