Alpha
-galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC-based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of ␣-Galactosylceramide-pulsed DCs (␣GCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of ␣GCDC administration in comparison with p53 peptide-pulsed DCs using a well-established murine CMS4 tumor model. Injection of ␣GCDC into CMS4 liver tumors resulted in complete tumor rejection and established long-term survival of the animals, while injection of p53 232-240 peptidepulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN-␥ in sera of ␣GCDC-treated mice were significantly higher than those of pepDCtreated mice. Hepatic NK cells were efficiently activated by ␣GCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody-mediated NK cell depletion study. Injection of ␣GCDC into liver tumor led to higher p53 232-240 peptide- specific CD8؉ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by ␣GCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. Conclusion: These results demonstrate that ␣GCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor. (HEPATOLOGY 2007