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Objective. Functionally impaired Treg cells expressing abnormally low levels of CTLA-4 have been well documented in rheumatoid arthritis (RA). However, the molecular defect underlying this reduced expression is unknown. The aims of this study were to assess the role of DNA methylation in regulating CTLA-4 expression in Treg cells isolated from RA patients and to elucidate the mechanism of their reduced suppressor function. Conclusion. We show for the first time that epigenetic modifications contribute to defective Treg cell function in RA through an inability to activate the IDO pathway. Therefore, this study sets a precedent for investigating potential therapeutic strategies aimed at reinforcing the IDO pathway in RA patients. Methods. CTLA-4 expression in

Objective We have previously shown, in a cohort of untreated rheumatoid arthritis (RA) patients, that the suppressive function of Treg cells is defective. However, other studies in cohorts of patients with established RA have shown that Treg cell function is normal. We hypothesized that treatment may restore Treg cell function and lead to reduced disease activity. The aim of this study was to investigate whether treatment with methotrexate (MTX) can result in epigenetic changes that lead to restoration of the Treg cell suppressive function in RA. Methods Peripheral blood samples from RA patients were assessed using 3H‐thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme‐linked immunosorbent assay to determine Treg cell suppression of interferon‐γ production. CTLA‐4 and FoxP3 expression was measured by flow cytometry and quantitative polymerase chain reaction (qPCR) in Treg cells from healthy individuals and RA patients. CD4+ T cells isolated from healthy individuals were cultured with interleukin‐2 (IL‐2), IL‐6, and tumor necrosis factor α in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the FOXP3 upstream enhancer was analyzed by bisulfite sequencing PCR. Results Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX‐exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA‐4 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the FOXP3 upstream enhancer. Conclusion This study identifies a novel mechanism of action of MTX, in which treatment of RA patients with MTX restores defective Treg cell function through demethylation of the FOXP3 locus, leading to a subsequent increase in FoxP3 and CTLA‐4 expression.

Objective. To assess the role of the activating receptor NKG2D in arthritis.Methods. Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, realtime polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days.Results. We demonstrated expression of NKG2D Natural killer (NK) cells are lymphocytes of the innate immune system with both cytotoxic and cytokineproducing effector functions, regulated by a repertoire of cell receptors, including activating receptors NKG2D, CD16 (Fc␥ receptor III), NKp30, NKp44, and NKp46; killer cell immunoglobulin-like receptor; and the inhibitory receptors CD94/NKG2A and LY49 (mouse) (for review, see ref. 1). The role of NK cells in the defense against infections and cancer has been studied extensively (1), but their role in autoimmunity and chronic

Background Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.