{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T16:52:51Z","timestamp":1786121571794,"version":"build-2736575974"},"reference-count":33,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"\n Objective<\/jats:title>\n Short-term methotrexate (MTX) discontinuation restores vaccine responses in rheumatoid arthritis (RA). We investigated how the timing of MTX exposure relative to lymphocyte activation influences cellular pharmacokinetics and vaccine immunogenicity.<\/jats:p>\n <\/jats:sec>\n \n Methods<\/jats:title>\n \n T and B cells from healthy donors (n = 4) were stimulated and treated with MTX either continuously or as a 24-hour pulse at defined activation time points. Activation, proliferation, and apoptosis were assessed by flow cytometry. Intracellular MTX polyglutamate (MTX-PG) levels were quantified using liquid chromatography mass spectrometry. Expression of reduced folate carrier 1 (RFC1), folylpolyglutamate synthase (FPGS), and \u03b3-glutamyl hydrolase (GGH) was quantified. A\n post-hoc<\/jats:italic>\n analysis of two clinical trials including 318 RA patients evaluated the effects of MTX timing on influenza vaccine responses.\n <\/jats:p>\n <\/jats:sec>\n \n Results<\/jats:title>\n Continuous MTX suppressed activation and proliferation and induced apoptosis in activated, but not resting T and B cells from healthy controls. A 24-hour MTX pulse on days 2 and 3 of activation inhibited cell activation and proliferation. Activated cells accumulated rapidly intracellular MTX-PG, peaking at 24 hours of MTX exposure, followed by near-complete clearance by 48 hours after MTX withdrawal. Resting cells showed minimal MTX-PG accumulation. Protein expression of RFC1 and FPGS peaked at 48 hours post-activation and declined thereafter. In RA patients, MTX administration on days 3\u20134 post-vaccination impaired vaccine responses, whereas delaying MTX for one week post-vaccination had minimal impact.<\/jats:p>\n <\/jats:sec>\n \n Conclusions<\/jats:title>\n MTX has a rapid intracellular turnover and exerts activation-phase-dependent effects on lymphocytes, mediated in part by dynamic expression of RFC1 and FPGS. These might explain the rapid recovery of vaccine responses after brief MTX interruption.<\/jats:p>\n <\/jats:sec>","DOI":"10.3389\/fimmu.2026.1898581","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T05:27:50Z","timestamp":1786080470000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Rapid on\u2013off effects of methotrexate on lymphocytes may facilitate recovery of suppressed vaccine immunogenicity after its short-term methotrexate discontinuation in rheumatoid arthritis"],"prefix":"10.3389","volume":"17","author":[{"given":"Heejin","family":"Jeong","sequence":"first","affiliation":[{"name":"Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seojin","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun Won","family":"Park","sequence":"additional","affiliation":[{"name":"Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Winthrop","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Oregon Health and Science University","place":["Portland, OR, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun Bong","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University","place":["Seoul, Republic of Korea"]},{"name":"Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin Kyun","family":"Park","sequence":"additional","affiliation":[{"name":"Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine","place":["Seoul, Republic of Korea"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1136\/annrheumdis-2021-221244","article-title":"Impact of disease-modifying antirheumatic drugs on vaccine immunogenicity in 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