(Vienna, 30 July 2026) Methotrexate (MTX) has been the mainstay of treatment for rheumatoid arthritis for decades. However, how the drug alters the immune system during the first few weeks of treatment has not been fully understood until now. Researchers at the Medical University of Vienna have now shown that MTX triggers coordinated changes in the immune system within just a few weeks. A team from the Department of Medicine III identified early cellular and molecular signatures that could indicate a good or poor response to treatment. The study, published in the journal *Annals of the Rheumatic Diseases*, combined single-cell RNA sequencing with immunophenotyping.
The study combines state-of-the-art single-cell RNA sequencing with advanced immunophenotyping to track changes in immune cell populations and molecular signalling pathways in patients from the start of MTX treatment onwards.
The immune response to methotrexate over time
The study was led by Teresa Preglej and Anela Tosevska from the Division of Rheumatology at the Department of Medicine III, Medical University of Vienna, and was carried out in close collaboration with rheumatologists, immunologists, computational biologists and international partners from Sweden and Germany. "Methotrexate has been used successfully for decades, yet many aspects of its mechanism of action have, until now, been surprisingly poorly understood," explains lead author Teresa Preglej. "By monitoring patients right from the start of treatment, we were able to observe changes in the immune system that occur long before any clinically measurable improvement. "
"Our findings provide a detailed picture of how patients’ immune systems respond to methotrexate," adds lead author Anela Tosevska. "A better understanding of these early changes could help in the future to identify those patients who are likely to benefit most from th e therapy."
Early changes distinguish responders from non-responders
The researchers monitored patients with RA who had never received treatment before during the first twelve weeks of MTX therapy. Significant changes in the immune system were detected as early as three weeks into treatment – at a time when clinical improvements were barely discernible. Among the most striking observations were a decrease in T follicular helper cells (Tfh cells) and in plasmablasts, specialised antibody-producing B cells. These changes occurred primarily in those patients who subsequently responded well to the treatment, whilst non-responders showed only minor comparable immune changes.
"One of the most exciting observations was that key immune cell populations change within just a few weeks," says Michael Bonelli, co-senior author of the study. "Such early signatures could help to predict treatment success much earlier in future than is currently possible," adds Lisa Göschl, also a co-senior author of the study (both from the Division of Rheumatology at MedUni Vienna).
New insights into the mechanism of action of an established standard therapy
In addition to changes in immune cell populations, the study identified extensive molecular reprogramming of the immune system and described previously unrecognised signalling pathways associated with MTX therapy. Supplementary laboratory experiments also demonstrated direct effects of MTX on the development of plasmablasts. The study thus provides one of the most comprehensive analyses to date of the biological mechanisms of action of methotrexate and lays a valuable foundation for future research.
Prospects for personalised therapy
With its high-temporal-resolution depiction of the immune response to methotrexate, the study provides new insights into one of the most important therapies in rheumatology and opens up new prospects for personalised treatment strategies. Although methotrexate remains the standard first-line treatment for rheumatoid arthritis, a significant proportion of patients do not respond adequately to the treatment. Early biological markers of treatment success could, in future, help to tailor treatment more precisely and enable those affected to access alternative treatment options more quickly.
Publication: Annals of the Rheumatic Diseases
Time-resolved immune dynamics in rheumatoid arthritis under methotrexate therapy
Teresa Preglej*, Anela Tosevska* Marie Brinkmann, Philipp Schatzlmaier, Franziska Schmidt, Jakov Korzhenevich, Elisabeth Simader, Daniela Sieghart, Philipp Hofer, Thomas Krausgruber, Lina Dobnikar, Christoph Bock, Thomas Karonitsch, Renate Kain, Hannes Stockinger, Josef S. Smolen, Wilfried Ellmeier, Leonid Padyukov, Marta Rizzi, Daniel Aletaha, Lisa Göschl#, Michael Bonelli#