Expression of DN T-bet in mature Vα14i NKT cells. C57BL/6 thymocytes were depleted of CD8⁺ cells using anti-CD8 magnetic beads. The cells were then placed in culture in complete media supplemented with 50 ng/mL IL-15. On day 2 and 3 the cells were spin-infected using a control retrovirus (empty GFP), a dominant-negative T-bet–encoding retrovirus (DN T-bet), or a control virus encoding an irrelevant dominant-negative T-box gene (Tbx13) and replaced in culture with IL-15 for 2 to 3 more days. At the end of the culture period, the cells were harvested and stained with the CD1d tetramer and anti-TcRβ mAb. CD1d tetramer⁺ TcRβ⁺ GFP⁺ cells were sorted (A), placed on anti-CD3/anti-CD28–coated plates for 48 hours, washed, and further expanded in complete media with IL-2 for a week. Percentages of tetramer-positive, TCRβ⁺ cells are indicated. (B) Total RNA was prepared and expression levels of CCR5, FasL (CD95L), IFNγ, granzyme B, and CxCR3 were measured by quantitative PCR as in Figure 4. Results are shown as mean ± standard deviation. (C-D) Alternatively, the cells were restimulated with PMA/ionomycin and analyzed as described in Figure 4. Data are representative of 2 independent experiments.