P176 Anti-IL6R treatment acts on CD4+ FoxP3+ regulatory T cells in a model of rheumatoid arthritis

Abstract

Introduction Studies have demonstrated the clinical efficacy of tocilizumab, a humanized anti-IL-6 receptor (R) antibody (Ab), in patients with rheumatoid arthritis (RA). The rational for blocking IL-6 in this disease mainly lays on the pro-inflammatory role of this cytokine in the disease. However, only few works have studied the consequences of anti-IL-6R treatment on Tregs cells and mainly focuses on their frequency.In this study, we hypothesized that IL-6 inhibition leads to Tregs modifications in RA models given that (i) anti-IL6R is therapeutic in RA and its models by inhibiting Th17 cells, and (ii) IL-6 plays a determining role in inducing Th17 when present, or Tregs when it is absent. Our objective was thus to elucidate anti-IL-6R mode of action in RA models by studying the consequences of this treatment on Tregs phenotype and biological activity. Methods Mice with collagen-induced arthritis were treated at day 0 by MR16-1 (a rat anti-mouse IL-6 receptor monoclonal Ab provided by Chugai Pharmaceutical Co. LTD, Japan) and the evolution of Tregs (defined as CD4+ FoxP3+) during arthritis course was assessed at key time points (day 9-17-28 and 43 after CIA induction) by studying their number, frequency and phenotype (expression of GITR, ICOS, Helios, CD62L, CTLA-4 and CD39) in lymph nodes (LN), thymus, spleen by flow cytometry. Finally, the immunosuppressive activity of the Treg cells on CFSE-labeled CD4+ CD25-T effector (Teff) cells proliferation was also studied. Numerical analysis of Th17 and Th1 cells was also performed at the same times by flow cytometry. Results Clinical and histological evaluation of arthritides in mice treated with anti-mouse IL-6R mAb showed, as expected, a less severe disease as compared to control Ig treated mice. A decreased Teff/Tregs ratio, together with an increased Treg/Th17 overtime were observed in the LN of MR16–1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4+ CD8-FoxP3+. Tregs phenotype was modified in treated mice, with a decreased frequency of Tregs expressing Helios (spleen) but an increased frequency of CD39+ Tregs (LN and spleen). Lastly, the immunosuppressive activity of the Treg cells on Teff cells proliferation was not modified, suggesting that Tregs immunosuppression activity was probably due to another mechanism. Conclusion Tregs are modified by anti-IL-6R treatment in CIA, that could result in an enhanced central and peripheral generation of Tregs. The increased CD39 expression may reflect the generation of a more suppressive Tregs population, acting through ATP hydrolysis. This hypothesis should be verified in the future. All together, this study shows that inhibition of IL-6 in CIA also acts by enhancing Tregs/Th17 balance and by Tregs phenotype.