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.