Figure 4.

Impaired activation of AKT in PKC-θ^−/− T cells is responsible for enhanced iTreg differentiation. (A) Impaired AKT activation in PKC-θ^−/−. Naïve WT and PKC-θ^−/− T cells were either untreated or stimulated with 1 µg/ml anti-CD3, 1 µg/ml anti-CD28 and 5 ng/ml TGF-β1. Phosphorylation of AKT (T308), expression of total AKT, and the β-actin control were detected by Western blot analysis. The ratio between phosphorylated-AKT and total AKT bands were averaged (mean ± SEM) from three experiments; *, p<0.01; error bars indicate ±SD. (B) Normal activation of ZAP70 in PKC-θ^−/− T cells. WT and PKC-θ^−/− T cells were either untreated or stimulated with similar conditions as in (A) and phosphorylation of ZAP70 (Tyr493), expression of total ZAP70, and the β-actin control were detected by Western blot analysis. (C) Forced expression of constitutively active AKT (AKT*) inhibits iTreg differentiation. WT and PKC-θ^−/− T cells were either untreated or infected with control virus (MIT) or virus expressing activated AKT (MIT-AKT*). T cells were then differentiated into iTreg cells with anti-CD3/28, TGF-β1 and IL-2. Thy1.1 was a marker for successful retroviral transduction, and thus expression of the protein of interest. Percentages of Foxp3 negative or positive cells are indicated. (D) LY294002 prevents AKT phosphorylation. WT and PKC-θ^−/− T cells were either untreated or stimulated with the conditions described in (A) in the presence or absence of 10 µM LY294002. Phosphorylation of AKT (T308), expression of total AKT and the β-actin control were detected by Western blot analysis. (E) LY294002 potentiates iTreg differentiation. Naïve WT and PKC-θ^−/− T cells were differentiated into iTreg cells with or without 10 µM LY294002. Tregs were detected by flow cytometric analysis of intracellular Foxp3 detected with a specific antibody. Results shown are representative of at least three independent experiments. (F) Summary of (E) averaged from three independent experiments.