FIGURE 6.

The TNF‐α/TNFR2 signaling pathway promotes the proliferation and enhances the immunosuppressive phenotype and function of Tregs. (A) The proportions of Foxp3⁺ Tregs in CD4⁺ T cells and the expression levels of TNFR2, CTLA‐4 and ICOS in control Tregs and activated Tregs were compared by flow cytometry analysis. Three biological duplicates of independent experiments were carried out. (B) Tregs were stimulated by either TNF‐α or TNF‐α plus Mab726 for 72 hours in the absence of anti‐CD3/CD28 beads. The proportion of Foxp3⁺ Tregs in CD4⁺CD25⁺ T cells was determined by flow cytometry. Representatives of three independent experiments with biological duplicates are shown. (C) Tregs were stimulated with either TNF‐α or TNF‐α plus Mab726 for 72 hours in the absence of anti‐CD3/CD28 beads. The level of LAP expression in activated Tregs was determined by flow cytometry. (D) Proliferation suppression assay. CFSE‐labeled CD8⁺ T cells were cultured alone or coincubated with activated Tregs and stimulated with either TNF‐α or TNF‐α plus Mab726 for 72 hours to quantify the proliferation percentage by flow cytometry. (E) Purified CD8⁺ T cells were cultured alone or coincubated with activated Tregs and stimulated by either TNF‐α or TNF‐α plus Mab726 for 72 hours. The IFN‐γ production in CD8⁺ T cells was detected by ELISA. Statistical analyses were performed using GraphPad Prism 8.0.1. The statistical tests used are indicated in the figure legends: *P < .05; **P < .01; ***P < .001; ****P < .0001 [Color figure can be viewed at wileyonlinelibrary.com]