T-bet mediates IFNγ and granzyme B induction in Vα14i NKT cells. T-bet^–/– Vα14i NKT cells were infected as described in Figure 3 and sorted. The cells were placed on anti-CD3/anti-CD28–coated plates for 48 hours, washed, and expanded in IL-2 (40 U/mL) for a week. (A) Expression levels of T-bet, CCR5, Fas ligand, CxCR3, IFNγ, IL-4, CD122, perforin, and RANTES were analyzed by quantitative PCR. Results are shown as mean ± standard deviation. (B) The cells were then stimulated with PMA/ionomycin for a total of 4 hours with the last 3 hours of the stimulation in the presence of golgi block, and analyzed by intracellular staining. At the end of the activation period, the cells were washed in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA) and surface-stained with CD1d tetramers. The cells were then fixed and stained for intracellular IFNγ. (C) The cells were treated as in panel B, and were analyzed for granzyme B expression. (D) NK1.1 expression on T-bet^–/– Vα14i NKT cells infected with the control virus or the T-bet–encoding virus. Data are representative of 3 independent experiments. Percentages of positive cells are indicated.