Next, we assessed the cytokine responses under α-GalCer stimulation. The repeated administration of α-GalCer reduced the elevation of serum IFN-γ and IL-4 levels following α-GalCer injection in mice, and the levels of both cytokines decreased to undetectable levels (Fig. 6a,b). These results were also confirmed in vitro. Thus, α-GalCer-stimulated liver MNCs strongly proliferated in the vehicle group (Fig. 6c), whereas the proliferation was poor in the α-GalCer group (Fig. 6d). In addition, the IFN-γ and IL-4 levels produced by liver MNCs following α-GalCer stimulation were significantly lower in the α-GalCer group, even at 8 weeks after the last α-GalCer injection (Fig. 6e,f). Furthermore, not only BWF1, but also C57BL/6 J (B6) mice undergoing repeated α-GalCer administration showed similar cytokine responses both in in vivo and in vitro (Fig. 6a,b,e and f). These results suggested that the repeated administration of α-GalCer induced a state of anergy to α-GalCer, which was not specific to BWF1 mice.Figure 6Repeated 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.