The role of mouse liver NK1.1 Ag+ T (NKT) cells in the antitumor effect of α-galactosylceramide (α-GalCer) has been unclear. We now show that, whereas α-GalCer increased the serum IFN-γ concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of α-GalCer. Injection of Abs to IFN-γ also inhibited the α-GalCer-induced increase in antitumor cytotoxicity of MNCs. α-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas α-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCs. The α-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-γ. Depletion of CD8+ T cells did not affect the α-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of α-GalCer on the survival of B6 mice. Thus, IFN-γ produced by α-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8+ T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate α-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.

We recently reported that NK cells and CD8+ T cells contribute to the antimetastatic effect in the liver induced by α-galactosylceramide (α-GalCer). In the present study, we further investigated how CD8+ T cells contribute to the antimetastatic effect induced by α-GalCer. The injection of anti-CD8 Ab into mice 3 days before α-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-γ production nor did it reduce the NK activity of liver mononuclear cells after α-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after α-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8+ T cells did. CD122+CD8+ T cells in the liver increased after α-GalCer injection, and antitumor cytotoxicity of CD8+ T cells in the liver gradually increased until day 6. These CD8+ T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122+CD8+ T cells. The critical, but bystander role of CD122+CD8+ T cells was further confirmed by adoptive transfer experiments into CD8+ T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/α-GalCer injection for the mice to generate CD8+ T cells that can reject s.c. rechallenged B16 cells. These findings suggest that α-GalCer activates bystander antitumor CD122+CD8+ T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8+ CTLs.