FIGURE 4.

Akt-1 inhibits, but Akt-2 facilitates, the proliferation of tTregs during EAE induction. (A) The spleen cells and lymph node cells from the mice (n = 5) described in Figure 2 were surface-stained with anti-CD4 and anti-CD25, and intracellularly stained with anti-Foxp3 and anti-Helios. *p < 0.05, **p < 0.01; Student t test. (B) 5 × 10⁶ CD4⁺ T cells from WT, Akt1^–/–, and Akt2^–/– mice were transferred by i.v. injection into Tcrb^–/– mice (n = 5). After 30 days equilibration, the recipient mice were immunized with MOG_35–55 in CFA. 10 days after immunization, the mice were sacrificed. The draining lymph node cells were surface-stained with anti-CD4 and anti-CD25, and intracellularly stained with anti-Foxp3 and Helios. **p < 0.01; Student t test. (C) WT, Akt1^–/–, and Akt2^–/– mice (n = 5) were immunized with MOG_35–55 in CFA. At day 8, we collected the draining lymph node cells from each group, labeled with CFSE, and cultured them in the presence of 20 μg/ml MOG_35–55 for three days. The cells were then surface-stained with anti-CD4 and anti-CD25, and intracellularly stained with anti-Helios and anti-Foxp3. The expression of Helios within CD4⁺CD25⁺Foxp3⁺ T cells was determined. *p < 0.05, ***p < 0.001; Student t test. (D and E) WT, Akt1^–/–, and Akt2^–/– mice (n = 5) were immunized with MOG_35–55 in CFA. On day 5, the mice were injected with BrdU at 1 mg/mouse every 12 h for three consecutive days. The mice were sacrificed on day 8. The BrdU incorporation in CD4⁺CD25⁺Foxp3⁺Helios⁺ or CD4⁺CD25⁺Foxp3⁺neurophilin⁺ T cells in the draining lymph nodes (D) and CNS (E) of WT, Akt1^–/–, and Akt2^–/– mice was determined. **p < 0.01, ***p < 0.001; Student t test. The data shown are one representative of three independent experiments.