Methotrexate does not exert immunomodulatory effects on regulatory T cells but on effector T cells

Abstract

BACKGROUND: Methotrexate (MTX) is the most commonly used anti-rheumatic drug in juvenile idiopathic arthritis (JIA). MTX induces nullremission on medicationnull in 70% of patients and continuous nullremission off medicationnull in up to 50% of patients (1). MTX is unique in the realm of anti-rheumatic drugs since it is currently the only drug that appears capable of establishing immune tolerance in JIA. In spite of this, it is unclear which immunomodulatory mechanisms enable MTX to achieve sustained remission. We thus examined the immunomodulatory effects that MTX exerts on two major players in immune tolerance - regulatory (Tregs) and effector (Teffs) T cells. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from 20 extended oligoarticular and polyarticular JIA patients were isolated before MTX start, 3 and 6 months after MTX start. We analyzed frequency and phenotype of Tregs by flow cytometry. The function of Tregs was evaluated in CFSE-sup-pression assays. Proliferation of Teffs was examined in proliferation assays in the presence of anti-CD3. Teff cytokine production was measured ex vivo with flow cytometry upon PMA-ionomycin stimulation and in 4-5 day-culture supernatants with the luminex technique. RESULTS: The frequency of FoxP3+ Tregs and the FoxP3 content per cell did not change upon MTX start. Suppressive capacity of Tregs appeared to be lower 6 months after MTX start compared to 3 months after MTX and prior to MTX start. In cross-over experiments, however, suppressive capacity of Tregs from all time points was equal. Teffs after MTX start proliferated significantly more in comparison to Teffs prior to MTX upon stimulation with anti-CD3. We observed an increase in IFN(gamma) production by flow cytometry and luminex and an increase in TNFa and IL-10 production by luminex only. IL-17 production decreased in supernatants of Teff cultures. CONCLUSIONS: MTX does not exert its immunomodulatory actions on Tregs. Instead, we observe changes in Teffs upon the start of therapy. Strikingly, we show that Teffs after MTX start demonstrate increased proliferation upon general stimulation. We will further explore the effect of MTX on Teff proliferation upon antigen-specific stimulation with HSP60 self-antigens as well as cytokine production of Teffs in cultures upon general and antigen-specific stimulation.