Both NKT cells and NK cells contribute to the rapid production of IFN-γ after vaccination. WT mice were challenged with 1 × 10⁵ Eμ-myc 299 tumor cells and some treated on day 5 with irradiated, α-GalCer–loaded tumor cells. At 24 hours after vaccination, direct ex vivo IFN-γ production was assessed by intracellular staining of NKT cells, NK cells, and CD8 T cells isolated from peripheral lymph nodes (A) and spleens (B). Data are mean ± SEM (n = 5) of the percentage of IFN-γ–producing cells (left panels) and levels of IFN-γ (mean fluorescence intensity [MFI], right panels). ΔMFI = change in MFI after subtracting values from a naive, non–tumor-bearing WT mouse. (C) Representative flow cytometry plots of IFN-γ expression in NK cells (top panels) and NKT cells (bottom panels) from spleen. Data are representative of 2 independent experiments.