Lysosomal processing of sulfatide analogues alters target NKT cell specificity and immune responses in cancer

In a structure-function study of sulfatides, that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogues with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by CD1d monomer on plastic stimulated type II, not type I, NKT-cell hybridomas as expected. Unexpectedly, when presented by bone-marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT-cell hybridomas, mimicking the corresponding βGalCer without sulfate. It induced IFNγ-dependent immunoprotection against CT26 colon-cancer lung metastases, skewed the cytokine profile, and activated cDC1s. This was abrogated by blocking lysosomal processing with bafilomycin A1, or sulfite-blocking or deletion of arylsulfatase A that cleaves off sulfate. Thus, C24:2 is unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery of antigen processing of glycosylceramides altering the specificity for the target cell that reverses its function from stimulating type II to stimulating type I NKT cells, introducing protective functional activity in cancer. It also uncovers a new role for antigen processing, not to allow MHC loading but to alter the cell responding.