Fig. 2

Expression of Foxp3 in sorted or expanded cells and that of CTLA-4 and GITR on CD4+CD25+Foxp3+ Tregs. (A) Representative gating strategy of Foxp3, CTLA-4, and GITR expression. Cells were identified using their forward- (FSC) and side-scatter (SSC) properties. Live cells were gated using Zombie Aqua™ Fixable Viability Kit staining and then examined for the coexpression of CD3 and CD4. Foxp3 expression in CD4+CD25+ T cells is plotted on the basis of Foxp3 fluorescence minus one (FMO). CTLA-4 and GITR expression (red histogram) on CD4+CD25+Foxp3+ Tregs were identified via the corresponding FMO (blue histogram). (B) Percentage of CD4+CD25+ Foxp3+ Tregs expanded for 0, 18, and 42 days (n = 3, each group). (C) Correlation between the percentage of CD4+CD25+CD127− Tregs and CD4+CD25+ Foxp3+ Tregs (n = 9). (D) Percentage of CTLA-4+ cells in CD4+CD25+Foxp3+ Tregs expanded for 0, 18, and 42 days (n = 3, each group). (E) Percentage of GITR+ cells in CD4+CD25+ Foxp3+ Tregs expanded for 0, 18, and 42 days (n = 3, each group). P-values for differences between groups were determined using one-way ANOVA, followed by Tukey’s multiple comparisons test in (B), (D), and (E). Pearson correlation analysis was used in (C). *p < 0.05, **p < 0.01, ns indicates no significant difference