Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

Invariant natural killer T (iNKT) cells are unconventional T cells recognizing lipid antigens in a CD1d-restricted manner. Among these lipid antigens, alpha-galactosylceramide (alpha-GalCer), which was originally identified in marine sponges, is the most potent antigen. Although the presence of alpha-anomeric hexosylceramide and microbiota-derived branched alpha-GalCer is reported, antigenic alpha-GalCer has not been identified in mammals. Here, we developed a high-resolution separation and detection system, supercritical fluid chromatography tandem mass spectrometry (SFC/MS/MS), that can discriminate hexosylceramide diastereomers (alpha-GalCer, alpha-GlcCer, beta-GalCer, or beta-GlcCer). The B16 melanoma tumor cell line does not activate iNKT cells; however, ectopic expression of CD1d was sufficient to activate iNKT cells without adding antigens. B16 melanoma was unlikely to generate iNKT cell antigens; instead, antigen activity was detected in cell culture serum. Activity-based purification and SFC/MS/MS identified dihydrosphingosine-based saturated alpha-GalCer as an antigenic component in serum, bile, and lymphoid tissues. These results show the first evidence for the presence of potent antigenic alpha-GalCer in mammals.