Our original characterization of endogenous ligands of invariant NKT cells suggested strongly the existence of two separate classes of ligands, one belonging to ɑ galactosylceramides, the second one to ɑ glucosylceramides. To confirm the existence of ɑ glucosylceramides (ɑGlucer), rabbit polyclonal antibodies were produced and purified on ɑGlucer columns after exhaustion for ɑGalcer binding. These antibodies detected the presence of ɑGlucer in cells and in the thymic cortex of wild type mice in a pattern distinct from ɑGalcer staining. The differential reactivity of NKT cells towards ɑGlucer and ɑGalcer was further explored by producing ɑGlucer- and ɑGalcer-CD1d tetramers. Whilst the overlap of staining was important, as expected, small distinct populations reactive with only one ligand could be detected in mice. Members of the panel of NKT cell hybridomas that we tested exhibited the same exquisite specificity for staining and activation. The systematic study of biosynthesis and degradation pathways of ɑGlucer are currently under way in cell lines using the CRISPR/Cas9 gene knockout approach. Early results have identified acid glucosidase (GAA) as essential for the degradation of ɑGlucer. The conventional NKT compartment of GAA deficient animals was shown to be profoundly affected both quantitatively and qualitatively, confirming the in vitro results.

The study of ɑGlucer should allow the development of new reagents and a better understanding of the thymic selection of NKT cells.