Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell–mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibβ-74cit_69-81 peptide led to a population of HLA-DR4^{Fibβ-74cit69-81} tetramer⁺ T cells that exhibited biased T cell receptor (TCR) β chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibβ-72,74cit_69-81) altered the responding HLA-DR4 tetramer⁺ T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR β chain usage toward the Fibβ-74cit_69-81 peptide was observed in healthy HLA-DR4⁺ individuals and patients with HLA-DR4⁺ RA, thereby suggesting a link to human RA.

Purpose: To study the role of regulatory T cells (Tregs) in patients with tubercular uveitis. Methods: Frequencies of peripheral Tregs, Th1, Th17 cells, and intracellular cytokines were determined in 17 tubercular uveitis patients and 18 disease controls. Function of Tregs, Th1, and Th17 cells was assessed in vitro. Simultaneously, ocular levels of IFN-γ, IL-17A, IL-4, and IL-10 were also measured. Results: Frequencies of peripheral Tregs in tubercular uveitis subjects were significantly lower compared with disease controls. Furthermore, expression of TGF-β and IL-2Rα, but not CTLA4, was reduced in Tregs of the tubercular uveitis group. The tubercular uveitis group demonstrated heightened Th1, Th17 responses following in vitro stimulation with phorbol myristate acetate (PMA)/ionomycin. Interestingly, Treg suppression assay did not show a significant difference between the two groups. Ocular levels of IFN-γ, IL-17A, and IL-10 were also elevated in tubercular uveitis group. Conclusions: Low Treg frequency and hyporesponsive function contribute to proinflammatory responses manifesting at ocular level in tubercular uveitis.

Purpose: To assess cellular composition and local cytokine response in vitreous humor of tubercular uveitis. Methods: Cells were collected from vitreous cassettes and peripheral blood of 8 tubercular uveitis and 5 control subjects, undergoing vitrectomy and analyzed by flow cytometry for cellular composition, activation status, proinflammatory cytokine expression, and uptake of TLR9 ligand, CpG ODN 2216. Results: CD3 + T cells with equal proportion of CD4+ and CD8 + T cells formed major fraction of infiltrating cells. The vitreous humor showed higher expression of recent activation marker, CD69, and proinflammatory cytokines, IFN-γ and IL-17A, in CD4 + T cells as compared to peripheral blood. Lastly, intraocular CD4 + T cells showed reduced uptake of ODN 2216 than peripheral blood. Conclusions: Our results indicate that local antigenic stimuli trigger T cell infiltration and activation of CD4 + T cells that are hyporesponsive to TLR9 stimulation. These infiltrating T cells might be responsible in further aggravating ocular inflammation.

Objectives: The aim of this study was to combine deep T cell phenotyping with assessment of citrulline-reactive CD4+T cells in the pre-rheumatoid arthritis (RA) phase. Methods: 20 anti-CCP2 positive individuals (HLA-DRB1*04:01) presenting musculoskeletal complaints without clinical or ultrasound signs of synovitis; 10 arthritis progressors and 10 matched non-arthritis progressors were included. Longitudinal samples (1–3 time points) of peripheral blood mononuclear cells were assessed using HLA-class II tetramers with 12 different citrullinated candidate autoantigens combined in a >20-colour spectral flow cytometry panel. Results: The baseline CD4+T cell phenotype was similar between individuals who progressed to arthritis (ie, in the pre-RA phase) and the non-progressors, when studying markers associated with Th1, Th17, T-peripheral and T-regulatory cells as well as with T-cell activation. Citrulline-reactive CD4+T cells were present in both groups but at significantly lower frequency in the progressor group. CD4+T cells specific for citrullinated tenascin-C were the most frequently observed among the progressors, and their frequencies diminished during follow-up that is, closer to arthritis onset. Notably, PD-1 and CD95 expression on the memory cit-tenascin-C-specific T cells in this group indicated repeated antigen exposure. Conclusions: Our data lend support to citrullinated tenascin-C as an interesting T cell antigen in RA. Moreover, lower frequency of circulating citrulline-specific cells in arthritis progressing individuals suggest an initiated homing of these cells to the joints and/or their associated lymph nodes in the pre-RA phase and a possible window of opportunity for therapeutic preventive interventions.

Anti‐cyclic citrullinated peptide2 (CCP2) antibody positivity in rheumatoid arthritis (RA) and in the predisease phase, together with the success of B‐cell depletion, support a crucial role for B cells in RA pathogenesis. Yet, knowledge of B cells in the transition from autoimmunity to RA is limited, and therefore we here investigated B‐cell changes during the risk‐RA phase. B‐cell phenotypes in 18 CCP2‐positive risk‐RA individuals with musculoskeletal complaints were studied, parallel with ten CCP2‐positive RA patients and nine healthy controls. Nine of the risk‐RA individuals progressed to RA. B‐cell phenotypes were investigated using spectral flow cytometry. The results demonstrate that unswitched and switched memory B‐cell frequencies in the risk‐RA cohort were more similar to controls than RA patients. Yet, risk‐RA progressors displayed an early activation profile amongst naïve B cells. Deeper characterization of the memory compartment revealed expansion of CD27‐negative IgG+ B cells both in RA compared with controls (p = 0.0172) and in risk‐RA progressors versus non‐progressors (p = 0.0295). Overall, we demonstrate that the phenotypic distribution of B cells is altered in the risk‐RA phase. This includes changes in CD27‐negative class‐switched B cells, which have been attributed to autoreactive and anergic features implicating a possible contribution to RA development.