(A, C, E, and G) At the indicated times after injection with heat-killed E. coli (A), live L. monocytogenes (C), heat-killed S. aureus (E), or heat-killed S. typhimurium (G), mice were rechallenged in vivo with vehicle or α-GalCer (1 μg/mouse, i.p.). Mice were sacrificed 3 days later, and spleen cells were stained with anti–TCR-β–FITC, anti-B220–PerCP, and tetramer-allophycocyanin and analyzed by flow cytometry. Numbers indicate the percentage of TCR-β⁺tetramer cells among B220^– cells. Data represent results obtained in at least 2 separate experiments involving 5–7 mice per group. (B, D, F, and H) Total spleen iNKT cells calculated from the experiments shown in A, C, E, and G, respectively. *P < 0.05 compared with naive mice rechallenged with α-GalCer. (I) Hyporesponsive iNKT cells are defective in transactivating B cells, DCs, and NK cells in vivo. Mice were injected with the indicated bacteria and rechallenged with α-GalCer (1 μg/mouse, i.p) 3 weeks later. Mice were then sacrificed at the 24-hour time point, and spleen mononuclear cells were stained with different combinations of anti-CD86–PE, anti-B220–PerCP, anti-CD11c–allophycocyanin, anti-CD69–FITC, anti-NK1.1–allophycocyanin, and anti–TCR-β–PE. For IFN-γ staining on NK cells, mice were sacrificed 6 hours following α-GalCer rechallenge, and spleen mononuclear cells were cultured 2 hours in the presence of GolgiPlug. Cells were then stained with anti–IFN-γ–FITC, anti-NK1.1–allophycocyanin, and anti–TCR-β–PE. Data shown are representative of 6 mice per group from 2 separate experiments.