CD27 signaling maintains G₁ and inactivates Foxo1 via PI3K and Akt. (A) Purified splenic CD8 T cells were incubated with or without anti-CD27 for the indicated times. Cells were pretreated with wortmannin (100 nM) or LY294002 (10 μM) 1 hour before stimulation. (B) Akt phosphorylation in the high-density naive or intermediate-density memory splenic CD8 T cells before and after 48 hours of culture under the indicated conditions with or without wortmannin or LY294002. Cell lysates were prepared, and phospho-Ser⁴⁷³ Akt and total Akt were detected by immunoblotting. (C) Splenocytes were sorted for CD8⁺CD44^high and CD8⁺CD44^low, and lysates of equal numbers of cells were loaded on SDS-PAGE and immunoblotted for Foxo1 and β-actin. (D) Purified splenic CD8 T cells were stimulated with plate-bound anti-CD27 for the indicated times, after which nuclear and cytosolic fractions were prepared. Tubulin and lamin A were used as controls for the efficacy of the fractionation. (E) Purified splenic CD8 T cells were fractionated on Percoll, and the intermediate-density cells were analyzed at time 0 or after 48 hours of culture under the indicated conditions. Wortmannin or LY294002 was added during culture with either DCs or plate-bound anti-CD27. Cell cycle was analyzed as in Figure 1. Cell viability after 48 hours was 58% to 73%. (F) The intermediate-density CD8 T cells were lysed immediately or after 48 hours of culture under the indicated conditions. Equal protein concentrations were loaded on SDS-PAGE gels and immunoblotted for cyclin D3 or β-actin. (G) Cyclin D3 levels in intermediate-density CD8 T cells before and after incubation under the indicated conditions. Cell lysates were prepared, and cyclin D3 and β-actin levels were determined by immunoblotting.