Identification of iGb3 and iGb4 in melanoma B16F10-Nex2 cells and the iNKT cell-mediated antitumor effect of dendritic cells primed with iGb3

Background: CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57B1/6 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 [H-3]-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 antitumor activity in mice challenged with melanoma cells.