Characterization of T-Cell Responses to Homocitrulline in a Mouse Model of Rheumatoid Arthritis
Abstract
Rheumatoid Arthritis (RA) is an incurable autoimmune disease that affects 1% of the global population. Patients with RA have autoantibodies to homocitrullinated proteins, indicative of more severe disease. However, little is known about T helper (Th)-cell responses. This study aims to characterize Th-cell responses to homocitrulline in HLA-DRB1*04:01 transgenic (DR4tg) mice, expressing the strongest RA genetic risk factor. Male and female DR4tg mice were immunized with a synthetic homocitrullinated peptide (HomoCitJED). Spleens and dLNs were collected on days 10, 30, and 100 post-immunization, and Th cells were analyzed by flow cytometry. Furthermore, a tetramer assay was developed to quantify homocitrulline-specific Th cells. On day 10, the proportion of T-bet+ Th1 cells was 16-fold higher in HomoCitJED-immunized mice compared to PBS control. By day 100, all HomoCitJED-immunized mice developed ankle swelling indicative of arthritis. These mice had significantly higher RORγt+FOXP3+ Th17/Treg cells than negative controls. We also observed a significant increase in activated T-bet+RORγt+ Th1/Th17 cells post-immunization with HomoCitJED. At day 100, frequencies of exhausted Th cells were 3.5-fold higher in HomoCitJED immunized mice than in controls, which positively correlated with ankle swelling. There was also a significant shift towards effector memory phenotype in HomoCitJED immunized mice, contributing to chronic disease. Lastly, using the tetramer assay, we captured tetramer-positive Th cells; however, quantification and phenotyping of those cells still require optimization. Overall, homocitrullinated antigens appear to induce pro-inflammatory T-cell responses and lead to exhaustion. We believe this research will bring us closer to identifying novel targets for RA therapies.