AKT* impairs de novo induction of Foxp3 by TGF-β. Naive CD4⁺CD25⁻ T cells were stimulated with anti-CD3/CD28 beads in the presence of TGF-β and IL-2. 24 h after activation, cells were transduced with retrovirus encoding either empty vector control (CTRL-RV) or a constitutively active form of AKT (AKT*-RV; A). After a total of 4 d in culture, cells were analyzed for intracellular levels of Foxp3 by flow cytometry. The percentages of cells positive for Foxp3 are shown. (B) Summary of eight independent experiments quantifying Foxp3 expression by flow cytometry analysis on the indicated populations. Horizontal lines indicate the mean value for each sample. (C) Real-time PCR analysis for Foxp3 mRNA levels in the indicated populations. Foxp3 mRNA is normalized to HPRT mRNA. Data represent the mean ± SEM for three independent experiments. (D) The experiment was performed as in A, and cell numbers were determined at the end of culture. (E) Naive CD4⁺CD25⁻ T cells were stimulated in the presence of TGF-β and IL-2. 24 h after activation, cells were transduced with retrovirus expressing AKT* or a kinase dead (KD) AKT. Expression of Foxp3 was determined after a total of 4 d of culture. The percentages of cells positive for Foxp3 are shown. (F) Similar to the experiment in A, except that T cells were transduced with retrovirus expressing Bcl-2 or c-myc.

AKT* specifically targets a subset of the TGF-β signature. (A) Comparison of the expression values of TGF-β–activated cells transduced with CTRL or AKT* retrovirus (left) compared with a randomized dataset (right). (B) Fold change versus fold change (FcFc) plot comparing the TGF-β–induced signature in AKT*- versus CTRL-transduced cells. Gene expression values for cells activated in the absence of TGF-β and transduced with a CTRL retrovirus were used as the denominator for the Fc. Regions R1 and R3 highlight TGF-β–regulated genes that are refractory to AKT* expression. Regions R2 and R4 highlight TGF-β–regulated genes that are affected by AKT* expression.

Impact of AKT* on Foxp3-dependent and -independent genes. (A) Similar to the experiment in Fig. 3 B, except that genes up-regulated (red) or down-regulated (blue) by Foxp3 retroviral transduction are highlighted. Regions R1, R2, R3, and R4 are outlined. Naive CD4⁺CD25⁻ T cells were activated as before and transduced with the indicated retrovirus. (B) Naive CD4⁺ T cells were activated in the presence with CD3/CD28 beads in the presence of TGF/IL-2 and transduced with AKT*-Thy1.1 and Foxp3-GFP retrovirus. (C) Real-time PCR analysis for Foxp3, CTLA-4, and Gpr83 mRNA was performed. Data represent the mean ± SEM.

Molecular mechanisms of AKT-mediated suppression of Foxp3 induction. Naive CD4⁺CD25⁻ T cells were stimulated in the presence of TGF-β and IL-2. 24 h after activation, cells were transduced with retrovirus and cultured for an additional 72 h in the presence or absence of 100 nm rapamycin. Foxp3 expression was determined by flow cytometry. The percentages of cells positive for Foxp3 are shown. Data are representative of three or more experiments.

Differential effect of AKT on established levels of Foxp3. (A) Naive CD4⁺CD25⁻ T cells were activated as before and transduced with retrovirus at the indicated time points. Foxp3 expression was determined by flow cytometry analysis. The percentages of cells positive for Foxp3 are shown. (B) Naive CD4⁺CD25⁻ T cells or CD4⁺CD25⁺ T reg cells were stimulated in the presence of TGF-β. T reg cell stimulation was done in the presence of 2,000 U IL-2. Cells were transduced with retrovirus 72 h after activation and analyzed for Foxp3 by flow cytometry after an additional 72 h of culture. Data are representative of three or more experiments.

AKT* significantly impairs a subset of T reg signature. Volcano plot representation (fold change vs. p-value) of the effect of AKT* expression (ratio of TGF-AKT* to TGF-GFP) on a common T reg signature (red, up; blue, down). Numbers indicate the probes present in either of the regions.

AKT* impairs differentiation of CD4⁺Foxp3⁺ T cells in vivo. (A) Schematic of the intrathymic transfer experiments. CD4⁻CD8⁻ DN thymocytes from CD45.1 mice were transduced with retrovirus encoding AKT* or CTRL vector and injected into sublethally irradiated CD45.2 B6 mice. 2 wk after transfer, donor CD45.1⁺ cells were analyzed for their ability to give rise to CD4⁺Foxp3⁺ cells. (B) Flow cytometry profile of the thymocyte populations gated on donor CD45.1⁺ cells transduced either with CTRL or AKT* retrovirus. Intracellular staining for Foxp3 was performed on electronically gated CD45.1⁺CD4⁺Thy1.1^−/+ populations. The percentages of cells positive for Foxp3 are shown. (C) Summary of independent experiments (n = 6–8 mice) for the percentage of CD4⁺Foxp3⁺ cells gated on donor cells. Horizontal lines indicate the mean. (D) CD4/CD8 profile of infected (identified by a Thy1.1 retrovirally encoded marker) donor cells. The percentages of cells in the indicated regions are shown. Summary of the ratio of (E) the sum of SP to DP cells and (F) of the CD4SP to CD8SP infected cells from various mice. Horizontal lines indicate the mean.