Background/Purpose: 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. Our objective was thus to elucidate anti-IL-6R mode of action, by studying the consequences of this treatment on Tregs phenotype and biological activity. in RA patients treated with tocilizumab and in a RA model, namely collagen-induced arthritis (CIA).

Methods: Mice with CIA 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 8-18-28 and 42 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 and spleen by flow cytometry. 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.

Twenty patients with severe and active RA, refractory to methotrexate or anti-TNF therapies were recruited and treated with 8mg/kg of tocilizumab monthly. Peripheral blood was recovered for each patient at day 0, as well as 1 and 3 month, and Th17and Tregs were analyzed by flow cytometry.

Results:

In mice, 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. Th17 frequency was unchanged, but Tregs frequency was enhanced in the LN of MR16-1 treated mice. In the thymus, we observed an enhanced frequency of Tregs CD4⁺CD8^–FoxP3⁺. Tregs phenotype was also modified in treated mice, with an increased frequency of CD39⁺ Tregs (LN and spleen), suggesting an enhanced ATP hydrolysis immunosuppressive activity of Tregs. In contrast, the immunosuppressive activity of the Treg cells on Teff cells proliferation was not modified.

In RA patients, we also shown that tocilizumab did not changed Th17 frequency but rather acted on Tregs. Indeed, in responder patients to tocilizumab, CD4⁺CD25⁺FoxP3⁺ staining was confined to CD127^low, suggesting the induction of induced Tregs following treatment.

Conclusion: Tregs, but not Th17, are modified by anti-IL-6R treatment in RA, that could result in an enhanced central and peripheral generation of Tregs. These results highlight the benefit of therapeutic approaches based on Treg promotion in RA, and stress the relevance of the monitoring of cell populations in arthritis following cytokine blockade.