(A) Rapamycin (3 nM) or P4 (2 μg/ml or 6.4 μM) was examined for their effects on phosphorylation of ribosomal S6 protein (Ser235/236) in antigen-primed T cells. Treg-depleted naïve CD4+ T cells, isolated from lymph nodes and spleen, were activated for 2 hours with anti-CD3/28 beads and IL2 (200 U/ml) in the presence of rapamycin or P4. Staining with an isotype control antibody is shown in gray. Representative data obtained from at least 3 independent experiments are shown. (B) The S6 protein phosphorylation in PR (−/−) T cells is not suppressed well by P4. The data in the graph are shown as % of control groups. (C) Rheb over-expression decreased the function of P4 in induction of LAP-TGFβ1+ Tregs. Rheb was over-expressed in Treg-depleted naïve CD4+ T cells by a retroviral gene transfer method and cultured for 5 days. P4 was added to the culture as indicated, and T cell differentiation into LAP-TGFβ1+ Tregs was examined. Representative and combined data from 3 independent experiments are shown. Significant differences between indicated groups,* control and P4 groups,* or WT and PR(−/−) groups.**