(A) Spleen cells were prepared from mice treated 4 weeks earlier with α-GalCer or the indicated bacteria, cultured in vitro (2 × 10⁵ per well) for 6 hours in plain medium (alone), 100 ng/ml α-GalCer, or a combination of 10 ng/ml PMA and 1 μM ionomycin (PMA + IONO), in the presence of Golgi­Plug to allow intracellular accumulation of cytokines. Cells were then harvested and surface stained with tetramer-PE and anti-B220–PerCP, followed by intracellular staining with anti–IFN-γ–FITC and anti–IL-4–allophycocyanin. Data are shown for B220^–tetramer⁺ cells. Numbers indicate the percentage of cells within each quadrant. Data shown are representative of 3 independent experiments with 2 mice in each group per experiment. (B) IL-2 overcomes the proliferative defect of hyporesponsive iNKT cells in vitro. Spleen cells from naive mice or from mice injected 1 month earlier with α-GalCer or heat-killed E. coli were labeled with CFSE. Cells (2 × 10⁵ per well) were then cultured with α-GalCer (100 ng/ml) for 24 hours in the presence or absence of IL-2 (10 ng/ml). Cells were then washed and cultured for an additional 96 hours without α-GalCer in the presence or absence IL-2. At the end of the culture period, cells were harvested, stained with tetramer-allophycocyanin and anti-B220–PerCP, and analyzed by flow cytometry. CFSE dilution was analyzed on B220^–tetramer⁺ cells. Representative data from 2 independent experiments are shown.