Fig. 7.

Anti-IL-6R reduces the expression of IFN-γ in activated Xiap^−/− Treg cells. a, b Anti-IL-6R decreases IFN-γ expression in re-stimulated Xiap^−/− Treg cells. WT and Xiap^−/− iTreg cells were stimulated with anti-CD3/CD28 and IL-2, or with additional IL-12 as indicated, in the absence or presence of anti-IL-6R (50 μg ml⁻¹) for 4 days. iTreg cells were re-stimulated with TPA/A23187 for 24 h and IFN-γ production was determined by ELISA (a), or reactivated with TPA/A23187 for 5 h and the expressions of Foxp3 and IFN-γ were determined by intracellular staining (b). c, d Anti-IL-6R inhibits IFN-γ expression in human Treg cells. Control and human XIAP-knockdown iTreg cells were stimulated as in (a, b), with additional IL-6 as indicated, and secretion (c) or intracellular expression (d) of IFN-γ was determined. e Inability of anti-TNF or anti-IL-1R to inhibit the production of IFN-γ in activated Xiap^−/− Treg cells. WT and Xiap^−/− iTreg cells were stimulated, as described in (a), in the presence or absence of anti-TNF or anti-IL-1R (50 μg ml⁻¹ each) for 4 days. Treg cells were re-stimulated with TPA/A23187 for 24 h and the secreted IFN-γ was determined by ELISA. f Anti-IL-6R rescues the impaired suppressive activity of Xiap^−/− tTreg cells in vivo. CD45.2⁺ WT or Xiap^−/− tTreg cells were co-transferred with CD45.1⁺ CD4⁺CD25^- effector T cells into male CD45.1⁺ RagI^−/− mice. Anti-IL-6R antibody (500 μg per mouse) was intraperitoneally administrated at day 0, followed by weekly dosing of 500 μg. The body weights of mice were monitored at the indicated time-points. ***P < 0.001 by two-way ANOVA. g, h Anti-IL-6R restores Foxp3 stability and reduces IFN-γ expression in Xiap^−/− tTreg cells transferred in vivo. Lymphocytes were isolated from spleen and mesenteric lymph nodes of mice in (f) and reactivated with TPA/A23187. The expressions of Foxp3 (g) and IFN-γ (h) in CD45.2⁺ T cells were determined by intracellular staining. Values are mean ± SD of triplicate samples in an experiment. *P < 0.05, **P < 0.01, ***P < 0.001 for unpaired t-test (a, c, g, h). Experiments (a–e) were independently repeated three times with similar results