Repeated alpha-galactosylceramide (α-GalCer) injection induces anergy in NKT cells to α-GalCer. (a) Serum IFN-γ levels 12 hours after α-GalCer injection and (b) IL-4 levels 3 hours after α-GalCer injection in (NZB/NZW) F1 (BWF1) and C57BL/6 J (B6) mice. The mice were injected with 2 μg/body of α-GalCer once a week for 4 weeks, and cytokine levels were evaluated after every injection (n = 4 in each group). *p < 0.05, **p < 0.01 compared with B6 mice. ††p < 0.01 compared with the levels after the first α-GalCer injection. †p < 0.05 compared with the levels after the second α-GalCer injection. Microphotographs of liver mononuclear cells (MNCs), stimulated by α-GalCer in the (c) vehicle and (d) α-GalCer groups (original magnification, 100×). In vitro production of (e) IFN-γ and (f) IL-4 from liver MNCs in BWF1 mice treated with α-GalCer or vehicle, and B6 mice treated with α-GalCer. Liver MNCs were obtained 8 weeks after the last α-GalCer or vehicle injection and were stimulated with α-GalCer for 48 hours (n = 4 in each group). **p < 0.01 compared with the vehicle group of BWF1 mice.