Structural determination of lipid antigens captured at the CD1d-T-cell receptor interface

Glycolipid antigens recognized by alpha beta T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with alpha(-)linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual alpha-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as alpha-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished alpha- from beta-anomeric monohexosylceramides. These data provide direct biochemical evidence for alpha-linked lipid antigens from a common dietary source.