The ability of vaccination with plasmids coding for the extracellular and the transmembrane domain of the product of transforming rat Her-2/neu oncogene (r-p185) to protect against r-p185+ transplantable carcinoma (TUBO) cells and mammary carcinogenesis was evaluated. In normal BALB/c mice, DNA vaccination elicits anti-r-p185 Ab, but only a marginal CTL reactivity, and protects against a TUBO cell challenge. Massive reactive infiltration is associated with TUBO cell rejection. In BALB/c mice transgenic for the rat Her-2/neu gene (BALB-neuT), DNA vaccination elicits a lower anti-r-p185 Ab response, no CTL activity and only incompletely protects against TUBO cells, but markedly hampers the progression of carcinogenesis. At 33 wk of age, when control BALB-neuT mice display palpable tumors in all mammary glands, about 60% of immunized mice are tumor free, and tumor multiplicity is markedly reduced. Tumor-free mammary glands still display the atypical hyperplasia of the early stages of carcinogenesis, and a marked down-modulation of r-p185, along with a massive reactive infiltrate. However, BALB-neuT mice protected against mammary carcinogenesis fail to efficiently reject a TUBO cell challenge. This suggests that the mechanisms required for the rejection of transplantable tumors may not coincide with those that inhibit the slow progression of carcinogenesis.

Recent studies have revealed significant efficacy of the marine sponge glycolipid, ␣-galactosylceramide (␣-GalCer), in treatment of experimental metastatic cancers, infections, and autoimmune diseases. However, the capacity of ␣-GalCer to prevent tumor development had never, to our knowledge, been evaluated in mouse models of chemical-and oncogene-dependent carcinogenesis. In this study, we demonstrate that long-term administration of soluble ␣-GalCer, spanning the time of tumor initiation, inhibits primary tumor formation in three different models: methylcholanthrene-induced sarcomas, mammary carcinomas in Her-2͞neu transgenic mice, and spontaneous sarcomas in p53 ؊/؊ mice. Weekly treatment of mice with ␣-GalCer maintained lymphoid tissue natural killer cell and T cell activation and elevated serum IFN-␥ and IL-4 concentrations. Consistent with the antimetastatic activity of ␣-GalCer, prevention of methylcholanthrene-induced sarcoma was IFN-␥-and tumor necrosis factor-related apoptosisinducing ligand dependent, but not perforin-dependent. Taken together, our results demonstrate that NK1.1 ؉ ␣␤TCR ؉ cell-based immune therapy can inhibit primary tumorigenesis. K RN7000, a marine sponge glycolipid with a novel ␣-galactosylceramide (␣-GalCer) structure, was found to exhibit potent antitumor activity in a variety of experimental and spontaneous tumor metastasis models (1-7). The antitumor effects of ␣-GalCer were first shown to be attributable to natural killer (NK) cell activation and the antigen presentation function of dendritic cells (8). ␣-GalCer was then identified as a ligand presented by CD1d to the invariant V␣14 T cell antigen receptor of mouse NK1.1 ϩ ␣␤TCR ϩ (NKT) cells (6, 9) and the invariant V␣24J␣QV␤11 TCR of human NKT cells (10). NKT cells have a range of characteristics that distinguish them from conventional T and NK cells (reviewed in refs. 11 and 12). In particular, upon stimulation, NKT cells have a remarkable capacity to produce immunoregulatory cytokines, including IL-4, IL-10, 13), that can powerfully influence the nature of adaptive immune responses. NKT cells also express a wide variety of cell death-inducing effector molecules and have been demonstrated to kill tumor target cells in vitro. Several studies have demonstrated the importance of NKT cells in immunoregulation, tumor immunity, and the prevention of autoimmune diseases in mice (reviewed in refs. 11 and 14-16).The antitumor activities of ␣-GalCer resemble those of IL-12, and a series of in vitro and in vivo studies (17)(18)(19)(20) have suggested that the initial steps that follow NKT cell TCR and CD1d͞␣-GalCer interaction involve IL-12 secretion by the antigenpresenting cell (21). In addition to these immediate events between NKT cells and dendritic cells, there are a series of consequences downstream, some of which result in the antitumor activity of ␣-GalCer (22-24). ␣-GalCer administration leads to rapid Th1 and Th2 cytokine production by NKT cells, and the activation of NKT cell IFN-␥ production rapidly results in NK cell act...

An assessment was made of the effectiveness of DNA vaccination in prevention of the mammary adenocarcinomas of BALB/c female mice transgenic for the activated rat Her-2/neu oncogene. Atypical hyperplasia is evident in their mammary glands when they are 6 weeks old and in situ carcinoma by the 13th week. Palpable invasive carcinomas appear around the 17th week and are always evident in all 10 glands by the 33rd week. Intramuscular vaccinations with 100 g plasmid DNA encoding the extracellular domain of the Her-2/neu p185 (ECD) performed at the 6th, 12th, 18th and 24th week provided no significant protection, whereas those ECD plasmids in which the DNA coding for the

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