{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,25]],"date-time":"2026-10-25T19:16:25Z","timestamp":1792955785991,"version":"4.5.0"},"reference-count":44,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2015,4,27]],"date-time":"2015-04-27T00:00:00Z","timestamp":1430092800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"Kennedy Trust for Rheumatology Research"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Arthritis &amp; Rheumatology"],"published-print":{"date-parts":[[2015,5]]},"abstract":"<jats:sec><jats:title>Objective<\/jats:title><jats:p>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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Peripheral blood samples from RA patients were assessed using <jats:sup>3<\/jats:sup>H\u2010thymidine incorporation to measure Treg cell suppression of T cell proliferation, and by enzyme\u2010linked immunosorbent assay to determine Treg cell suppression of interferon\u2010\u03b3 production. CTLA\u20104 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\u20102 (IL\u20102), IL\u20106, and tumor necrosis factor \u03b1 in the presence or absence of MTX, and FoxP3 expression was determined using qPCR and flow cytometry. Methylation of the <jats:italic>FOXP3<\/jats:italic> upstream enhancer was analyzed by bisulfite sequencing PCR.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Defective Treg cell function was observed only in RA patients who had not been treated with MTX, whereas Treg cells from MTX\u2010exposed RA patients had restored suppressive function. This restored suppression was associated with increased expression of FoxP3 and CTLA\u20104 in Treg cells. Bisulfite sequencing PCR of Treg cells cultured in MTX revealed a significant reduction in methylation of the <jats:italic>FOXP3<\/jats:italic> upstream enhancer.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>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 <jats:italic>FOXP3<\/jats:italic> locus, leading to a subsequent increase in FoxP3 and CTLA\u20104 expression.<\/jats:p><\/jats:sec>","DOI":"10.1002\/art.39031","type":"journal-article","created":{"date-parts":[[2015,1,20]],"date-time":"2015-01-20T13:03:21Z","timestamp":1421759001000},"page":"1182-1192","source":"Crossref","is-referenced-by-count":156,"title":["Methotrexate Restores Regulatory T Cell Function Through Demethylation of the FoxP3 Upstream Enhancer in Patients With Rheumatoid Arthritis"],"prefix":"10.1002","volume":"67","author":[{"given":"Adam P.","family":"Cribbs","sequence":"first","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alan","family":"Kennedy","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henry","family":"Penn","sequence":"additional","affiliation":[{"name":"Northwick Park Hospital Harrow UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Parisa","family":"Amjadi","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Green","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jordan E.","family":"Read","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fionula","family":"Brennan","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernard","family":"Gregory","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard O.","family":"Williams","sequence":"additional","affiliation":[{"name":"Kennedy Institute of Rheumatology and University of Oxford Oxford UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2015,4,27]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2011.05.025"},{"key":"e_1_2_7_3_1","first-page":"899","article-title":"Occurrence of rheumatoid arthritis in a nationwide series of twins","volume":"13","author":"Aho K","year":"1986","journal-title":"J Rheumatol"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/hdy.2010.54"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg2540"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.187.4173.226"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/eji.201444453"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/eji.200838105"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20040165"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0806855105"},{"key":"e_1_2_7_11_1","first-page":"2344","article-title":"Treg cell function in rheumatoid arthritis is compromised by CTLA\u20104 promoter methylation resulting in a failure to activate the indoleamine 2,3\u2010dioxygenase pathway. 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