Mechanisms of Endogenous Alpha-Glycosylceramide Generation for Development and Activation of NKT Cells

Abstract

Invariant natural killer T (iNKT) cells recognize lipid antigens loaded on CD1d molecules and modulate downstream immune responses by rapidly releasing multiple cytokines. Their most potent antigen is the lipid α-galactosylceramide (α-GalCer) from a marine sponge, which has been thought not to exist in mammals. Despite its importance in iNKT cell development and activation, the nature of the endogenous iNKT cell antigen remained unclear. The recent development of antibodies specific for α-glycosylceramides demonstrated that α-linked glycolipids are produced in mammals. Here, we discuss the mechanisms of endogenous α-glycosylceramide generation and how they influence iNKT cell biology. Based on its structural similarity to fatty acid binding protein and its ability to distinguish galactose anomers, we hypothesized that galactose mutarotase (Galm) is involved in endogenous α-GalCer synthesis. Galm is an enzyme known for its role in the Leloir pathway of galactose metabolism. We demonstrated that in the absence of Galm, both α-GalCer synthesis and iNKT cell autoreactivity are abrogated. Furthermore, we have generated mice deficient in Galm and confirmed that α-GalCer is lacking in these mice. Galm-deficient mice develop normally and have normal frequencies of iNKT cells. However, iNKT cells in these mice exhibit increased activation when challenged, suggesting a role of endogenous α-GalCer in regulation of iNKT cell activation. In addition, we demonstrated that α-glucosylceramide (α-GluCer) is another endogenous α-anomeric antigen, which is dependent on UDP-glucose ceramide glucosyltransferase (Ugcg). Importantly, mice deficient in both Galm and Ugcg have reduced frequencies of iNKT cells, indicating that α-GalCer and α-GluCer cooperate in iNKT cell development. However, these glycolipids may be serving non-redundant functions in iNKT cell selection based on their distinct localization patterns in the thymus. Together, these studies suggest a distinct role for the newly discovered endogenous lipid antigens in iNKT cell development and activation. Moreover, we identified the key enzymes that generate these ligands and are vital for iNKT cell biology, which can be targeted in order to regulate iNKT cells in autoimmunity and other disease settings.