FIG. 3.

PD-1's ITSM motif is required to mediate PD-1 suppression of the PI3K/Akt pathway. CD4 T cells were activated with CD3/CD28-coated beads and transduced with lentiviral vectors expressing mCD28-PD-1 wild type (WT), mCD28-hPD-1 Y223F, mCD28-PD-1 Y248F, and the double mutant mCD28-hPD-1 Y223F, Y248F. (A) Expression of each construct was examined by staining the cells with an Ab specific for mCD28 3 days after transduction. (B) Transduced cells were left unstimulated or were restimulated with the indicated aAPCs for 30 min. Cells were lysed, and proteins were resolved by SDS-PAGE. Akt activity was assessed by immunoblotting for serine-phosphorylated Akt (upper panel). The blot was then stripped and reprobed with anti-Akt antibody (lower panel) to demonstrate equal loading. Data are representative of three independent experiments.