(A) Liver MNCs from B6 or FcγR^–/– mice were cultured with or without (–) aggregated IgG (10 μg/ml), and CD25 and CD69 expression was analyzed 24 hours after stimulation. Open histograms represent negative controls, in which cells were stained with anti-NK1.1 and -TCRβ mAbs and isotype-matched IgG for anti-CD25 or -CD69 mAb. Numbers indicate MFI ± SD, from 3 independent experiments. (B) DN32-D3 cells were stimulated with or without aggregated IgG for 2 hours, lysed, and immunoprecipitated with anti-Syk or -Lyn mAb followed by anti-phosphotyrosine (anti–p-Tyr). The same cell lysates were directly blotted using anti-Syk or -Lyn mAb. Sorted NKT and DN32-D3 cells were stimulated with and without aggregated IgG for 24 hours, and the total proteins were separated by SDS-PAGE and immunoblotted using anti–T-bet or –GATA-3 mAbs. (C) Sorted NKT cells from B6 or FcγR^–/– mice (1 × 10⁵ cells/well) were stimulated with aggregated IgG for 48 hours. The amounts of IL-4, IFN-γ, IL-10, and IL-13 in the culture supernatant were measured using ELISA. (D) Sorted NKT cells from B6 mice (1 × 10⁵ cells/well) were cultured with aggregated IgG or anti-CD3 mAb at the indicated concentrations, and the amounts of IL-4, IFN-γ, IL-10, and IL-13 measured using ELISA. (E) The same cells as in D were cultured with aggregated IgG (10 μg/ml), anti-CD3 mAb (100 ng/ml), or both for 48 hours. The amounts of IL-4 and IFN-γ were measured using ELISA. Results are representative of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.