Expression of T-bet in immature Vα14i NKT cells. (A) T-bet^–/– thymocytes were depleted of CD8⁺ cells using anti-CD8 coated magnetic beads. The cells were placed in culture in complete media with 10 ng/mL IL-7. On days 2 and 3 the cells were spin-infected using a control retrovirus (empty GFP) or a T-bet–encoding retrovirus (Tbet-GFP) and replaced in culture with IL-7 for 2 to 3 more days. At the end of the culture, the cells were harvested and stained with the CD1d tetramer and anti-TcRβ mAb. CD1d tetramer⁺ TcRβ⁺ GFP⁺ cells were sorted and total RNA prepared. Percentages of tetramer-positive, TCRβ⁺ cells are indicated. (B) Expression levels of T-bet, CCR5, Fas ligand, CxCR3, IFNγ, and CD122 in T-bet^–/– Vα14i NKT cells infected with the control virus or the T-bet–encoding virus were analyzed by quantitative PCR using specific primers and probes. The amount of transcript in each sample was normalized to the amount of HPRT. Results are shown as mean ± standard deviation. Data are representative of 3 independent experiments. (C) C57BL/6 thymocytes were depleted of CD8⁺ and NK1.1⁺ cells using anti-CD8 and anti-NK1.1 magnetic beads. Depletion of NK1.1⁺ cells was 90% effective. The cells were placed in culture with rmIL-7 and infected and sorted as in panel A. Percentages of tetramer-positive, TCRβ⁺ cells are indicated. (D) Gene-expression level analysis was performed as in panel B. Results are shown as mean ± standard deviation.