Rapid on–off effects of methotrexate on lymphocytes may facilitate recovery of suppressed vaccine immunogenicity after its short-term methotrexate discontinuation in rheumatoid arthritis

Objective 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. Methods 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 γ-glutamyl hydrolase (GGH) was quantified. A post-hoc analysis of two clinical trials including 318 RA patients evaluated the effects of MTX timing on influenza vaccine responses. Results 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 3–4 post-vaccination impaired vaccine responses, whereas delaying MTX for one week post-vaccination had minimal impact. Conclusions 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.