Figure 6.

IL-2/IL-2Ab enhances the protective effect of Tregs. Tregs were isolated from donor C57BL/6 mice that were pretreated with IL-2/IL-2Ab (IL-2/IL-2Ab Tregs) or IsoAb (IsoAb Tregs) for 3 d. A total of 1 × 10⁶ IsoAb Tregs or IL-2/IL-2Ab Tregs was transferred into recipient C57BL/6 mice through tail vein 2 h after tMCAO. Control mice received the same volume of PBS. Animals were killed 3 d after tMCAO. A, Schematic images for experimental design. B, Representative images of MAP2 staining. Scale bar, 1 mm. C, Quantification of infarct volume. n = 9/group. **p < 0.01 IL-2/IL-2Ab Treg versus PBS. #p < 0.05 IL-2/IL-2Ab Tregs versus IsoAb Tregs. D, Suppressive function of IL-2/IL-2Ab or IsoAb-treated Tregs. Teffs were labeled with CFSE (1 μm, 37°C, 10 min). Tregs prepared from IL-2/IL-2Ab or IsoAb-treated mice were added at a ratio of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, or 1:64 to the number of Teffs. Cells were incubated for 3 d. Suppression of Teff proliferation was determined by CFSE dilution on a flow cytometer. Top, Representative plots of suppression assay using CFSE-labeled Teffs incubated with Tregs at various ratios. Bottom, Bar graph indicating CFSE dilution in CD4⁺CD25⁻ gated Teffs. The percentage of suppression was calculated as 100 − (% divided with Tregs/% divided without Tregs) × 100. Data are shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 versus IsoAb, unpaired t test; #p < 0.05, ###p < 0.001 versus Teff alone, one-way ANOVA followed by Bonferroni post hoc.