Investigating Homocitrulline-Specific T-Cell Responses in Rheumatoid Arthritis

Objectives Rheumatoid Arthritis (RA) is an incurable autoimmune disease affecting 1 in every 100 Canadians. Both B cells and T cells are involved in the pathogenesis of RA. These cells respond to peptides containing modified amino acids, including citrulline and homocitrulline. Although B-cell responses to homocitrullinated peptides (HomoCitP), also referred to as anti-carbamylated protein (anti-CarP) antibodies, have been studied, less is known about T-cell responses to HomoCitP. This study aims to investigate T cells that respond to HomoCitP in RA patients and in a humanized mouse model expressing the most significant RA genetic risk factor: HLA-DR4. Methods Male and female HLA-DR4-transgenic mice (n=6-9) were injected subcutaneously with HomoCitP or phosphate-buffered saline (PBS) as a control. Draining lymph nodes were collected on day 10 post-immunization for flow cytometry analysis. For human studies, blood samples were obtained from RA patients and age- and sex-matched healthy controls. CD4+ T cells were isolated using magnetic negative selection beads and stained with HomoCitP-loaded HLA-DR4-encoded MHC II tetramers to identify and phenotype HomoCitP-specific T cells. Peripheral blood mononuclear cells (PBMCs) were analyzed for cytokine production, and serum was collected for antibody assays. Results Mice immunized with HomoCitP, compared to PBS, had a 25-fold increase in the frequency of pro-inflammatory T helper 1 (Th1) cells (Table 1). Additionally, we detected a 1.8-fold increase in total activated cells with pro-inflammatory Th17 cells being the most abundant (30.5%). Furthermore, mice injected with HomoCitP had elevated levels of terminally exhausted and central memory T cells, which may contribute to chronic disease. When immune cells from these mice were exposed to HomoCitP in vitro, production of the cytokines TNF-α and IFN-γ was triggered, corroborating the inflammatory nature of the response to HomoCitP. Similar preliminary results were observed in RA patients, where PBMCs showed increased production of TNF-α, IFN-γ, IL-17A, and IL-6 cytokines in response to HomoCitP in vitro (Table 1). Using a tetramer assay, we detected 3-times more HomoCitP-specific T cells in RA patients compared to healthy controls. The majority of these cells were pro-inflammatory Th1 (51%) and Th17 (39%). Lastly, 8/10 RA patients tested positive for HomoCitP antibodies, compared to 1/6 healthy controls. Table 1. Summary of results for mouse and human cell experiments . Values represent the mean percentages of cells or the mean number of cells per 1 million cells. For human HomoCitP antibody levels, an optical density less than 0.1 was considered negative, 0.1–0.5 was considered low, 0.5–1.0 was considered medium, and greater than 1.0 was considered high. HomoCitP antibody concentration was significantly higher in RA patients compared to healthy controls (p = 0.0180). The median age of RA patients was 71 years, while the median age of healthy controls was 61 years (p = 0.0909). The percentage of female RA patients was 80%, compared to 83% in healthy controls. Conclusion This study identified HomoCitP-specific T-cell responses and cytokine production in both mice and RA patients. The observed pro-inflammatory responses provide valuable insights into RA pathogenesis and suggest potential new therapeutic targets for better control of disease activity.

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