Background/Purpose: Rheumatoid Arthritis (RA) is a chronic immune-mediated heterogenous disease characterized by a defect in the compartment of regulatory CD4+Foxp3+ T cells (Treg) which are essential for maintenance of self-tolerance and exert a suppressive effect. The first-line anchor drug for RA is low-dose methotrexate (MTX). However, 46% of RA patients started on MTX therapy discontinue treatment by 3 years and our ability to predict who will experience a good response versus non response remains very limited. Accordingly, the aim of the present study was to evaluate the role of (CD4+ CD25+ FOXP3) regulatory T cells in MTX unresponsiveness in naïve RA patients and to examine their correlation with the traditional clinical and serological markers of disease activity.

Methods: Fifty random early untreated RA patients diagnosed according to the ACR/EULAR 2010 criteria of RA, naïve to disease-modifying anti-rheumatic drugs (DMARDS) were enrolled. Information on disease duration, disease activity, presence and duration of morning stiffness, articular and extra articular complaints, smoking habits and comorbidities was recorded. The British version of the Health Assessment Questionnaire (HAQ) was performed to assess functional status. Disease activity with DAS-28 activity score was calculated for all patients. Peripheral blood (CD4+ CD25+ FOXP3) Tregs were determined in all RA patients before and after MTX treatment using multi-color flow cytometry. All patients received MTX monotherapy at a dose of 15 to 20 mg/week, maintained for at least four weeks before peripheral blood collection. Patients were stratified according to their response to MTX therapy into two groups: Unresponsive: (UR-MTX), treated with MTX by a dose of ≥ 15mg/week for at least 6 months and still presented active disease (DAS28 >4.0) and Responsive: (R-MTX), treated with MTX by a dose of ≥ 15mg/week for at least 6 months and presented remission (DAS28 < 3.0) using the EULAR response criteria.

Results: Six months after MTX monotherapy, 30 patients were classified as responders and 20 as non-responders. The mean value of CD4+ CD25+ FOXP3 Treg cells % was significantly higher in the (R-MTX) group; 0.63 ± 0.57, than in the (UR-MTX) group; 0.18 ± 0.14, p< 0.001. In the (R-MTX) group, the absolute change of (CD4+ CD25+ FOXP3) Tregs % was inversely correlated to DAS-28 score (r_s= -0.652, p< 0.001), ESR (r_s= -0.654, p< 0.001), CRP (r_s= -0.840, p< 0.001), VAS (r_s= -0.550, p=0.002), and HAQ score. (r_s= -0.548, p=0.002), but directly correlated to serum Hb levels (r_s = 0.535, p=0.002).

Conclusion: MTX responsiveness is directly related to the expansion of (CD4+ CD25+ FOXP3) Tregs % after 6 months of MTX monotherapy, likely via the ADO/A2aR pathway. The absolute change of Tregs % correlates inversely with disease activity in responsive patients, which suggests an important role of (CD4+ CD25+ FOXP3) Treg cells in the pathogenesis of disease activity and flares. Our data also suggest that, low expression of (CD4+ CD25+ FOXP3) Treg cells after 6 months of MTX monotherapy, could be a biomarker for predicting unresponsiveness to MTX in RA patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-regulatory-t-cells-cd4cd25foxp3-in-methotrexate-unresponsiveness-in-a-cohort-of-naive-rheumatoid-arthritis-patients/