Figure 3. Enhanced peripheral population and suppressive activity of S1P₁-KO T_reg cells.

(a) Flow cytometry of gated CD4 T cells from the spleen and peripheral lymph nodes (PLN) of WT and S1P₁-KO mice. The panel on the right shows the proportions of Foxp3⁺ T_reg cells among total CD4⁺ T cell population, with the mean (+s.d.) calculated from 4 mice of each genotype. (b) Flow cytometry analysis of T_reg markers (Foxp3, CD25, GITR and CTLA4) in PLN of WT and S1P₁-KO mice. Data are representative of 2 independent experiments. Similar findings were observed in other peripheral lymphoid organs (not shown). (c) In vitro T-cell suppression assays using Foxp3⁺ CD4SP cells from WT and S1P₁-KO mice. The left panel shows a representative proliferative assay of 4 independent experiments, the middle panel is the percentage of suppression with the mean (±s.d.) calculated from 4 experiments, and the right panel shows a representative of 2 independent experiments measuring IL-2 production. (d) In vitro T-cell suppression assays using S1P₁-deleted peripheral T_reg cells. Foxp3⁺ T_reg cells from the periphery of S1pr1^+/+ and S1pr1^fl/fl mice were transduced with Cre-expressing retrovirus (Cre-GFP), and sorted GFP⁺ T_reg cells were used in the T-cell suppression assays with different T_conv and T_reg ratios; freshly isolated T_reg cells were used as a comparison. The left panel is a representative of 3 independent experiments, and the right panel shows the percentage of suppression with the mean (+s.d.) calculated from 3 experiments. *, P < 0.01; **, P < 0.001 (Student’s t-test).