Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Microbiology and Immunology

Supervisor

Barra, Lillian

2nd Supervisor

Cairns, Ewa

Co-Supervisor

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.

Summary for Lay Audience

Rheumatoid Arthritis (RA) is an incurable autoimmune disease characterized by joint inflammation, which causes pain and severely impairs quality of life. Two main types of immune cells involved in RA progression are B and T cells. In RA, these cells get activated in response to molecules containing homocitrulline. Although B-cell activation to homocitrulline has been studied before, less is known about T cells. In this study, we investigated T-cell responses to homocitrulline in a mouse model of RA. Male and female mice were injected with homocitrulline or negative control. Since lymph nodes and spleens have a lot of T-cells, they were collected. We studied the following time points: 10, 30, and 100 days after injection. We also used a unique method to capture T cells specific to homocitrulline.

Our results showed an increase in inflammatory T cells 10 days after injection with homocitrulline. By day 100, all the homocitrulline-injected mice developed ankle swelling, suggestive of arthritis. At the same time, we observed a significant increase in activated T cells, primarily of the inflammatory subtype. Additionally, some T cells became more dysfunctional in homocitrulline-injected mice at day 100 and correlated positively with higher ankle swelling. In contrast, other T cells became memory cells that can circulate in the bloodstream and cause chronic inflammation. Lastly, using our unique method, we captured T cells activated explicitly in response to homocitrulline.

Overall, homocitrulline mainly caused pro-inflammatory responses and increased proportions of dysfunctional T cells. Studying T cells in RA will help us understand immune responses and guide the development of RA medications to help patients live pain-free and high-quality lives.

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Available for download on Saturday, May 31, 2025

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