Homocitrullinated Peptides Drive Pro‐Inflammatory T Cell Responses in a Humanized Mouse Model of Rheumatoid Arthritis

OBJECTIVE: Anti-homocitrullinated protein/peptide antibodies are specific to rheumatoid arthritis (RA) and predictive of worse prognosis, suggesting a pathogenic role for autoreactivity to homocitrullinated peptides (HomoCitP). However, T cell responses to HomoCitP remain largely unexplored. We investigated these responses in a humanized HLA-DRB1*04-transgenic (DR4tg) mouse model of RA, which expresses the strongest genetic risk factor for this disease. METHODS: DR4tg mice (n = 6-15) were injected subcutaneously with a HomoCitP called HomoCitJED, whereas control mice received phosphate-buffered saline. After 10 days, T cells were analyzed for their phenotypic characteristics, cytokine production, and proliferative capacities in draining lymph nodes (dLNs) and spleens by flow cytometry, enzyme-linked immunosorbent assays, and ProQuantum immunoassays. RESULTS: HomoCitJED immunization, compared with controls, drove robust expansion of T helper subsets in the dLNs, including more than a seven-fold increase in Th1, Th17, and hybrid Th1/Th17 cells (P < 0.0087). Cytokine staining confirmed effector activity, with higher frequencies of IL-17A+, TNF-α+IL-17A+, and IFN-γ+IL-17A+ cells (P < 0.0207). Exhaustion-associated markers were also increased, including lymphocyte activation gene-3 (LAG-3)+ and T cell Ig and mucin-domain-containing protein 3 (Tim-3)+ CD4+ T cells (P < 0.0411). CD8+ T cells mirrored these findings, with a six-fold increase in CD25 and doubled killer cell lectin-like receptor G1 (KLRG1) expression, accompanied by augmented IL-17A and IFN-γ/IL-17A production and antigen-specific proliferation (P < 0.0379). Expression of exhaustion-associated markers Tim-3+ and LAG-3+ on CD8+ T cells was likewise elevated (P < 0.0205). CONCLUSION: HomoCitP elicited Th1/Th17 and Tc1/Tc17 responses marked by concurrent expression of activation and exhaustion-associated markers, consistent with a dysregulated state. These findings position homocitrulline-driven CD4+ and CD8+ T cell imbalance as a potential mechanistic contributor to RA pathogenesis and a target for future immunomodulatory therapies.