Figure 5

Treg function affected by ergotope peptide treatment. Flow cytometric analysis of CTLA-4 extracellular (A) and intracellular (B) expressions in CD4 + CD25 + (upper panel) and CD4 + CD25- (lower panel) splenocytes from untreated, C-P, P5-6 and P14-16 treated groups are shown. Statistical analysis is shown on the right. Values are the mean ± SD. *: P < 0.05 versus C-P group. (C) Inhibition rate was evaluated by H³-TdR incorporation assay using the formula described in the Materials and Methods section. CD4 + CD25-T cells were isolated from mice with CIA and then cocultured with increasing numbers of CD4 + CD25 + regulatory T cells from untreated, C-P, P5-6 and P14-16 treated CIA mice on d 35 post first immunization for 3 d. Ratio indicates CD4 + CD25-T cells:regulatory T cells. Values are the mean ± SD of five mice per group from three independent experiments (each performed in triplicate). (D) Splenocytes were isolated from untreated, C-P, P5-6 and P14-16 treated mice with CIA on d 35 post first immunization; CD4 + CD25- effect T cells (Teff) and CD4 + CD25 + regulatory T cells (Treg) were sorted by MACS. H³-TdR incorporation assay was used to test the cell proliferation of Treg, Teff, Teff coculture with Treg at a ratio of 5:1. Anti-IL-10, anti-CTLA-4 and anti-IL-10 + anti-CTLA-4 blocking antibodies were added to the culture and cells were stimulated with anti-CD3 and anti-CD28 for 72 h. H³-TdR (1 µCi/well) was added at the last 16 h. Data are the mean ± SD of five mice per group from three independent experiments (each performed in triplicate). **: P < 0.01, ***: P < 0.001 versus C-P group; #: P < 0.05, ##: P < 0.01, ###: P < 0.001 versus Teff:Treg = 5:1 of internal group.