Invariant natural killer T cells (iNKT cells) respond to CD1d-presented glycolipids from Borrelia burgdorferi , the causative agent of Lyme disease. Although mouse and human iNKT cells respond to different antigens based on subtle differences in their fatty acids, the mechanism by which fatty acid structure determines antigenic potency is not well understood. Here we show that the mouse and human CD1d present glycolipids having different fatty acids, based in part upon a difference at a single amino acid position that is involved in positioning the sugar epitope. CD1d also can bind nonantigenic lipids, however, but unexpectedly, mouse CD1d orients the two aliphatic chains of a nonantigenic lipid rotated 180°, causing a dramatic repositioning of the exposed sugar. Therefore, our data reveal the biochemical basis for the high degree of antigenic specificity of iNKT cells for certain fatty acids, and they suggest how microbes could alter fatty acid biosynthesis as an immune evasion mechanism.

Glycolipids presented by the major histocompatibility complex class I (MHC I) homolog CD1d are recognized by natural killer T (NKT) cells characterized by either a semi-invariant (type I or iNKT) or a relatively variable (type II) T cell receptor (TCR) repertoire. Here we describe the first structure of a type II NKT TCR complexed with CD1d-lysosulfatide (LSF). Both TCR α and β chains contacted the CD1d molecule with a diagonal footprint, typical of MHC-TCR interactions, while the antigen was recognized exclusively with a single TCR chain, similar to the iNKT TCR. Type II NKT cells, therefore, recognize CD1d-sulfatide complexes with a distinct recognition mechanism characterized by features of both iNKT cells as well as conventional peptide-reactive T cells.

Background: Antibodies that recognize glycolipids presented by the antigen-presenting molecule CD1d are useful tools in studying natural killer T cell biology. Results: The CD1d-␣-galactosylceramide specific antibody L363 has TCR-like binding properties. Conclusion: Glycolipid-reactive antibodies are more antigen-specific than the TCR of iNKT cells. Significance: This is the first crystal structure of a glycolipid-reactive TCR-like antibody.

The CD1 family of Ag-presenting molecules is able to display lipids to T cells by binding them within a hydrophobic groove connected to the protein surface. In particular, the CD1b isotype is capable of binding ligands with greatly varying alkyl chain lengths through a complex network of interconnected hydrophobic pockets. Interestingly, mycobacterial lipids such as glucose monomycolate exclusively bind to CD1b. We determined the crystal structure of one of the three expressed bovine CD1b proteins, CD1b3, in complex with endogenous ligands, identified by mass spectrometry as a mixture of phosphatidylcholine and phosphatidylethanolamine, and analyzed the ability of the protein to bind glycolipids in vitro. The structure reveals a complex binding groove architecture, similar to the human ortholog but with consequential differences. Intriguingly, in bovine CD1b3 only the A’, C’ and F’ pockets are present, whereas the T’ pocket previously described in human CD1b is closed. This different pocket conformation could affect the ability of boCD1b3 to recognize lipids with long acyl chains such as glucose monomycolate. However, even in the absence of a T’ tunnel, bovine CD1b3 is able to bind mycolates from Rhodococcus ruber in vitro.

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