Fig. 3

TRAPs promote CD4⁺ T cell expression of IL-6 in an NF-κB/p38/Akt-dependent manner and induce IL-10 and IL-21 via IL-6–STAT3 signaling. a Western blot analyses of the phosphorylation of JNK, ERK, p38, Akt, IKKα/β, IκBα, p65 and STAT3 in CD4⁺ T cells treated with TRAPs (3 μg/ml) for the indicated time. b CD4⁺ T cells pre-treated with the indicated inhibitors for 1 h and then co-cultured with TRAPs (3 μg/ml) for 72 H. IL-6, IL-10 and IL-21 levels in the supernatants were determined by ELISA. c Western blot analyses of the phosphorylation of STAT3 in CD4⁺ T cells pre-treated with the STAT3 inhibitor Stattic at the indicated concentrations (0.5, 1 or 2 μM) for 1 h, and then co-cultured with TRAPs (3 μg/ml) for 2 h. ELISA of IL-10 and IL-21 secretion by CD4⁺ T cells treated as above for 72 h. d Western blot analyses of the phosphorylation of STAT3 in CD4⁺ T cells treated with anti-IL-6 neutralizing antibody (1 μg/ml) and TRAPs (3 μg/ml) for 2 h. ELISA of IL-10 and IL-21 secretion by CD4⁺ T cells treated as above for 72 h. e Western blot analyses of the phosphorylation of STAT3 in WT or Il6^−/− CD4⁺ T cells treated with TRAPs (3 μg/ml) for 2 h and ELISA of IL-10 and IL-21 secretion by WT or Il6^−/− CD4⁺ T cells for 72 h. f, g Flow cytometric and statistical analyses of the percentage of IL-21⁺ CD4⁺ T cells (f) or IL-10⁺ CD4⁺ T cells (g) in the iLN and spleens of WT or Il6^−/− C57BL/6 mice (n = 6) 7 days after i.v. administration of normal saline (NS) or TRAPs (30 μg/mouse) every other day for 3 times. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by one-way ANOVA with the Tukey-Kramer multiple test, 2-tailed unpaired t-test or Mann-Whitney U test