Figure 6

Prevention of CIA by in vivo ergotype peptide–induced Treg. (A) Total CD4 + T cells, CD4 + CD25-T cells or CD4 + CD25 + T cells derived from CIA or peptide-treated CIA mice were transferred into naive DBA/1 through intravenous injection. On the same day, naïve mice were induced with CII. Severity of arthritis was assessed by scoring clinical features 2 wks after the adoptive transfer. Values are the mean ± SD of 10 mice per group. *: P < 0.05, **: P < 0.01 versus C-P group; #: P < 0.05, ##: P < 0.0, ###: P < 0.001 versus internal CD4 + CD25–; &: P < 0.05 versus C-P group. (B) The levels of CII-specific IgG, IgG1 and IgG2a in sera collected on d 14 post adoptive transfer were determined by ELISA. Data are represented as mean ± SD. *: P < 0.05 versus C-P group. (C) Mononuclear cells from the spleens of transferred mice were isolated and stimulated with CII at 20 μg/mL. H3–TdR (1μCi/well) was added at 48 h and cell proliferation was assessed at 72 h. The stimulation index normalized to unstimulated controls is displayed. Data are represented as mean ± SD. **: P < 0.01 versus C-P group. (D) The expressions of Th1, Th17 and Treg related cytokines IFN-γ, IL-17, TGF-β and IL-10 were measured by ELISA in sera and in supernatants by splenic mononuclear cells with CII stimulation (20 μg/mL) for 48 h. Values are the mean ± SD of 10 mice per group. *: P < 0.05, **: P < 0.01, ***: P < 0.001 versus C-P group. (E) mRNA levels of IFN-g, T-bet, IL-17, STAT3, RORg t and FOXP3 in splenic mononuclear cells stimulated with CII (20μg/mL) for 24 h were measured by real-time qPCR. Values are represented as mean ± SD. *: P < 0.05, **: P < 0.01, ***: P < 0.001 versus C-P group. Data are representative of three separate experiments with similar results.