Impaired activation of Pten-mutant NKT cells in vitro and in vivo. (A) Impaired proliferation. NK1.1⁺TCRβ⁺ cells were purified from the thymi of Pten^flox/flox or LckCrePten^flox/flox mice and cocultured with DCs loaded with vehicle (DC) or αGalCer (DC-αGC). After 48 hours, cells were pulsed with [³H]-thymidine for 12 hours and thymidine uptake was measured. (B-C) Impaired cytokine production. IL-4 and IFNγ in the supernatants of the cultures in panel A were measured by ELISA. (D-E) Impaired response of Vα14iNKT cells to αGalCer administration in vivo. αGalCer was intraperitoneally injected into Pten^flox/flox, Pten^+/−, or LckCrePten^flox/flox mice, and serum concentrations of IL-4 (D) and IFNγ (E) were evaluated at 2 hours and 12 hours after αGalCer injection, respectively. For all panels, data are expressed as the mean value ± SEM for 3 mice per group. **P < .01.