Previous studies have shown that presentation of natural CD1d-bound ligands requires lysosomal trafficking of CD1d molecules and several lysosomal proteins, including proteases and lipid-transfer proteins, leading to the hypothesis that the endogenous ligands might be lysosomal glycosphingolipids. So, Zhou et al. analysed mice deficient in the B subunit of the lysosomal-glycosphingolipid-degrading enzyme B-hexosaminidase (HEXB) and found that the number of Vα14+ NKT cells was decreased by 95%. In addition, thymocytes that were isolated from HEXB-deficient mice could not stimulate interleukin-2 (IL-2) production by an autoreactive CD1d-restricted Vα14+ NKT-cell hybridoma, indicating that HEXB-deficient cells have a specific defect in generating lysosomal Vα14+ NKT-cell ligands.
HEXB-dependent enzymes remove the β-linked N-acetylgalactosamine (GalNAc) residues from several distinct types of glycosphingolipid, such as isogloboglycosphingolipids. However, Vα24+ NKT cells present in freshly isolated peripheral-blood mononuclear cells (PBMCs) clonally expanded only in the presence of one of these glycosphingolipid types, iGb3. CD1d presentation of iGb3 also stimulated interferon-γ and IL-4 production by Vα24+ NKT cells and IL-2 production by the Vα14+ NKT-cell hybridoma. Furthermore, although HEXB-deficient bone-marrow-derived dendritic cells (BMDCs) presented iGb3 to the Vα14+ NKT-cell hybridoma as efficiently as wild-type BMDCs, they could not present the iGb3 precursor iGb4, indicating that iGb4 processing by HEXB-dependent enzymes is required for iGb3 recognition by the Vα14+ NKT cells.
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