Figure 6. S1P₁ induces activation of Akt-mTOR to inhibit T_reg development and function.

(a) IL-2 activated signaling pathways in thymic T_reg precursors from WT and S1P₁-Tg mice. CD4⁺CD25⁺Foxp3⁻ cells were purified and stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 4 independent experiments. (b) Effects of drug treatments on IL-2 induced Foxp3 expression in T_reg precursors. CD4⁺CD25⁺Foxp3⁻ cells were treated with U0126, LY294002 and Rapamycin for 30 minutes, followed by IL-2 stimulation. Data are representative of 3 independent experiments. (c) IL-2 activated signaling pathways in peripheral T_reg cells from WT and S1P₁-Tg mice. T_reg cells were stimulated with medium alone or IL-2, and activation of Akt, STAT5, Erk and S6 ribosomal protein (S6) were examined by flow cytometry using phospho-specific antibodies. Data are representative of 5 independent experiments. (d) Suppressive activity of T_reg cells transduced with dn-Akt retrovirus. WT and S1P₁-Tg T_reg cells were transduced with control (MiT) and dn-Akt expressing (dn-Akt) retroviruses (non-transduced cells are shown on the right as a comparison), and transduced cells were sorted and used in the T-cell suppression assays with different T_conv and T_reg ratios. Data are representative of 3 independent experiments. *, P < 0.001 (Student’s t-test).