Background/Purpose: Although autoreactive CD4+ T cell and antibody responses against citrullinated antigens are known to contribute to loss of immune tolerance in rheumatoid arthritis (RA), identifying the immunodominant antigens remains an important priority.  In this study, we focused on tenascin-C, an extracellular matrix protein with proinflammatory properties, based on our previous studies reporting the presence of autoantibodies targeting a citrullinated epitope in the fibrinogen-like globe of tenascin-C in patients with established RA and patients with early synovitis. Here, we extend these findings utilizing a systematic discovery approach to identify novel citrullinated tenascin C epitopes that elicit T and B cell responses in patients with RA.

Methods: An algorithm was used to predict HLA-DRB1*0401 restricted epitopes along the entire length of the tenascin-C monomer. Predicted epitopes were then tested for their ability to bind HLA-DRB1*0401 and to elicit a T cell response in vitro. Multiplex HLA class II tetramer staining combined with cell surface marker antibodies was used to assess the frequency and phenotype of T cells specific for the immunogenic citrullinated-tenascin-C peptides in peripheral blood. Fluorospot assays were used to measure cytokines produced by synovial fluid mononuclear cells in response to citrullinated-tenascin-C peptides. Antibodies to citrullinated-tenascin-C peptides were detected in sera and synovial fluid using ELISAs and an extracellular matrix peptide microarray. The relationship between antibody seropositivity and clinical variables was modeled using logistic regression models with age and sex as co-variates. All RA subjects met the 2010 American College of Rheumatology criteria, and ACPA positivity was determined based on clinical testing for CCP.

Results: We identified five novel citrullinated-tenascin-C epitopes that were restricted by HLA-DRB1*0401. Notably, CD4 T cells specific for these epitopes were increased in the peripheral blood of patients with RA compared to healthy control subjects, and were present in synovial fluid. Furthermore, two of these citrullinated tenascin-C epitopes were also B cell epitopes that were recognized by antibodies in sera and synovial fluid from RA subjects. Strikingly, antibodies were present in 75% of the patients for one of these epitopes. Serum levels of these citrullinated-tenascin-C reactive antibodies were associated with rheumatoid factor, CCP seropositivity, the shared epitope, and smoking.

Conclusion: Taken together this work demonstrates the importance of citrullinated-tenascin C as an autoantigen in RA. Furthermore, our findings suggest that a unique set of epitopes recognized by both CD4 T cells and B cells have the potential to amplify autoimmunity and promote the development and progression of RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/t-and-b-cell-responses-to-common-tenascin-c-peptides-in-ra/