Fig. 5.

Expression of the constitutively active form of FoxO3a blocks IL-2 ability to reverse T-cell anergy. Naïve CD4 T cells from wild-type and Sirt1^−/− mice were purified and cultivated with anti-CD3 plus anti-CD28 for 24 h followed by infection with retrovirus that carry (A and B) GFP only (GFP) or GFP with the constitutively active form FoxO3a (FoxO3a/Act), or (C and D) with control shRNA or with the shRNA against FoxO3a. Three days after infection, GFP⁺ cells were sorted out, rested, and then treated with or without ionomycin for 16 h to induce tolerance. Cells were restimulated with anti-CD3 and anti-CD28 in the absence or presence of IL-2. (A and C) Sirt1 (Top) and FoxO3a (Middle) expression levels were determined by Western blotting using β-Actin as a control (Bottom). (B and D) The proliferation was determined by 3H-thymidine (3H-TdR) incorporation. Error bars represent data from three independent experiments.