BACKGROUND: CD1d-restricted iNKT cells are protective against murine melanoma 
B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from 
the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d 
glycoproteins are related to the class I MHC molecules, and are involved in the 
presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT 
cells. In the present work we attempted to identify the endogenous lipid 
mediator expressed in melanoma cells inducing such immunesurveillance response 
and study the possibility of protecting animals challenged with tumor cells with 
lipid-primed DC.
RESULTS: Crude cytosolic and membrane fractions from in vivo growing melanoma 
contained iNKT-stimulating substances. Lipids were then extracted from these 
cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived 
dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to 
produce IL-2. The active fraction was analyzed by electrospray ionization-mass 
spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. 
When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally 
produced indicating iNKT cell activation. GM3 consistently inhibited this 
response. To assess the antitumor response-induced by iGb3, a cytotoxicity assay 
in vitro was used with [3H]-thymidine labeled B16F10-Nex2 cells. At 
target/effector (iGb3-activated iNKT) cell ratio of 100(-1)-100(-4) tumor cell 
lysis was shown. The antitumor activity in vivo was tested in mice challenged 
i.v. with B16F10-Nex2 cells and treated with iGb3- or 
alpha-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was 
observed with either treatment.
CONCLUSION: Our results show the expression of globo and 
isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 
and its precursor, iGb4, on tumor cells may prime an effective iNKT 
cell-dependent antitumor response, modulated negatively by GM3 which is also 
produced in these cells. iGb3-primed BMDC exerted a significant iNKT 
cell-mediated anti-tumor activity in mice challenged with melanoma cells.