Time-resolved immune dynamics in rheumatoid arthritis under methotrexate therapy

OBJECTIVES: Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA), yet its immunological mechanisms remain incompletely understood. This study aimed to define the time-resolved cellular and molecular changes induced by MTX. METHODS: In a prospective longitudinal study, newly diagnosed patients with RA initiating MTX were systematically profiled over time. We applied an integrated single-cell multiomics approach combining high-parameter immunophenotyping and single-cell RNA sequencing of PBMCs (peripheral blood mononuclear cells) obtained from 6 patients at baseline and at sequential time points during therapy. Functional in vitro assays were conducted to evaluate direct effects of MTX. Key findings were validated in an independent cohort of 22 patients with RA, stratified by clinical response, and integrated with external transcriptomic datasets. RESULTS: MTX-induced rapid immune remodelling that was detectable as early as 3 weeks after treatment initiation and preceded clinical improvement. The earliest and most pronounced alterations occurred in T follicular helper (Tfh) cells and plasmablasts, which declined selectively in clinical responders but remained largely unchanged in nonresponders across independent cohorts and external datasets, underscoring their potential as early biomarkers of treatment response. Functional plasmablast differentiation assays further linked MTX treatment to impaired development. Transcriptomic network analyses identified novel regulatory hubs associated with MTX exposure, including LMNA, SCO2, GVQW3, and members of the GIMAP family. To promote data accessibility, we developed an interactive single-cell browser for community-driven exploration. CONCLUSIONS: Together, these findings reveal an unexpectedly rapid and coordinated immune reconfiguration after MTX initiation and position early alterations in Tfh cells and plasmablasts as biomarkers of treatment response.