Background: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by immune dysregulation, involving altered peripheral blood mononuclear cell (PBMC) populations and dysregulated cytokine signaling. Methotrexate (MTX) remains the preferred first-line treatment for RA, yet its precise mechanisms of immune modulation are not fully understood. A deeper understanding of MTX’s effects at the cellular and molecular levels could enhance therapeutic strategies for RA management.

Objectives: This study aimed to elucidate the mechanisms by which MTX modulates immune dynamics in RA by identifying early cellular and molecular changes following MTX therapy, exploring key signaling pathways affected by treatment, and aim to uncover potential biomarkers for therapeutic response.

Methods: A multi-omics approach was employed, combining single-cell RNA sequencing (scRNA-seq) and flow cytometry-based immunophenotyping. RA patients initiating MTX therapy were followed for 12 weeks, with immune cell profiles assessed at baseline, 3 weeks, and 12 weeks post-treatment. Differential gene expression and network analyses were conducted to identify key pathways and regulatory nodes modulated by MTX.

Results: Methotrexate (MTX) treatment led to significant immune modulation, with effects becoming detectable as early as three weeks after the initiation of therapy. One of the primary outcomes was the restoration of naïve T and B cell populations. Plasmablast levels emerged as a potential biomarker of early therapeutic response. Transcriptional analysis demonstrated the modulation of key signaling pathways, including TNF-α signaling, B cell receptor signaling, and T cell receptor-mediated apoptosis.Additionally, network analysis identified critical regulatory hub genes, such as EGR1, JAK2, and SOCS1, predominantly within monocytes and CD4 memory T cells.

Conclusion: In conclusion, these findings advance our understanding of MTX’s effects on immune cell dynamics at different stages of treatment, showing for the first time the early cellular changes leading to immune modulation in RA.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: Lisa Göschl: None declared. Teresa Preglej: None declared. Anela Tosevska: None declared. Marie Brinkmann: None declared. Daniela Sieghart: None declared. Philipp Schatzlmaier: None declared. Elisabeth Simader: None declared. Thomas Krausgruber: None declared. Lina Dobinkar: None declared. Christoph Bock: None declared. Hannes Stockinger: None declared. Daniel Aletaha Daniel Aletaha received grants and consulting fees from AbbVie, Amgen,Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, Daniel Aletaha received grants and consulting fees from AbbVie, Amgen,Lilly, Merck, Novartis, Pfizer, Roche and Sandoz, Michael Bonelli Michael Bonelli received grants from AlphaSigma.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.