The CD1 family of glycosylated cell-surface receptors constantly surveys the lipid content of APCs by intracellular trafficking through the various endosomal compartments. The number and different classes of lipids that are now known to act as ligands at the cell surface of APCs for specific T cells have increased significantly in the last few years and include phosphoglycerolipids, sphingolipids, diacylated glycerolipids, lipopeptides, mycolates, phosphomycoketides, and small hydrophobic compounds (for review see references 13–15). Some of these CD1 ligands promiscuously bind to all CD1 isotypes (e.g., sulfatide), whereas others are isoform-specific. For example, mycolates are only bound to CD1b, whereas phosphomycoketides bind to CD1c. These observations were at first surprising. Sulfatides and lipopeptides, which have quite different chemical composition, can both bind to CD1a but do so in a way that does not alter the structure of the CD1a-binding groove. Both ligands maneuver into the binding groove and attain an optimal fit without inducing any changes in the CD1 conformation (7
). In contrast, structures of mCD1d in complex with either PC, sulfatide, or the short-chain α-GalCer PBS-25 show interesting structural changes at the T cell–recognition surface of CD1. Compared with the first crystal structure of mouse CD1d, to which an endogenous PC ligand was bound (5
), binding of the short-chain α-GalCer variant results in an alteration at the CD1 surface, which leads to the formation of a roof above the F′ portal (11
). This induced fit, however, has not been observed in the structure of mCD1d–sulfatide. Although both sulfatide and α-GalCer are glycosphingolipids, they differ in their chemical structure, not only in the linkage to their galactosyl headgroups, but also in their ceramide lipid backbone. α-GalCer is based on phytoceramide, which has 3′ and 4′ OH groups, whereas the ceramide of sulfatide has a 4′ OH group and a 4′–5′ unsaturation. This additional 4′ OH group of α-GalCer forms a second hydrogen bond with Asp80, and the α-anomeric galactose forms tight hydrogen bonds with Asp153 of the α2-helix. We propose that this tighter hydrogen bond interaction of α-GalCer with both α-helices results in the structural changes observed in the mouse CD1d–α-GalCer complex. PC is a glycerolipid and its lipid backbone diacylglycerol, as well as the phosphorylcholine headgroup, interestingly form fewer hydrogen bonds with CD1 and, therefore, do not appear to induce any structural changes, although no truly “empty” structure is known. However, the crystal structure of human CD1d with bound full-length α-GalCer shows that under certain experimental conditions, such as refolding without ligands, the protein can undergo structural changes that partially close the binding groove. Two CD1d molecules were observed in the crystal structure: one was loaded with α-GalCer, whereas the other was empty. However, whether this capacity of human CD1d to close the binding groove in absence of any detectable ligand is biologically significant remains elusive. Under physiological conditions, such as during folding in the ER, lipids are always present to stabilize the hydrophobic CD1d binding groove, thereby rendering any requirement for gross structural changes unnecessary.
All of the four lipid ligands that have been crystallized in complex with either human (12
) or mouse (6
) CD1d so far show a common mode of lipid binding. The C18
sphingosine chain is always inserted in the F′ pocket, and fatty acid (C8
) is always inserted in the A′ pocket. This orientation is similar for PC, where the shorter fatty acid chain (C12
) is inserted in the A′ pocket and the longer fatty acid (C24
) in the F′ pocket. Nevertheless, a sphingolipid with a C18
fatty acid, as found in sulfatides from natural sources, could insert its two alkyl chains in either pocket if the length of the alkyl chain is the only factor responsible for ligand binding. But the crystal structure of the CD1d–α-GalCer (PBS-25) complex revealed that the short fatty acid (C8
) is surprisingly inserted only in the A′ pocket. The orientation still needs to be determined for the recently identified family of α-anomeric microbial ligands, the glycuronosyl ceramides (C14
; references 24
). Thus, we propose that the N-amide linkage between the fatty acid and the sphingosine together with the hydroxyl groups of the sphingosine chain ( C, highlighted in yellow), which forms a precise hydrogen-bonding network with CD1, constrains the ceramide backbone that orients the respective tails into the A′ and F′ pockets.
It is not known whether the immunodominance of cis-tetracosenoyl sulfatide is due to a more efficient binding to CD1d or to the presence of specific TCR repertoires that are stimulated preferentially by individual sulfatide species. In this regard, it is interesting to note that the T cell hybridoma specific for lyso-sulfatide does not recognize any of the other sulfatides examined so far (unpublished data). Whether NKT cells specific for each individual sulfatide express a unique TCR or whether overlapping TCR repertoires exist is currently not clear and should be determined by generating T cell hybridomas reactive to each of the individual sulfatides. Of interest, however, is that although the hydrogen bonding network formed between the lipid backbone and CD1d is responsible for the orientation of the different lipid ligands in the binding groove, slight differences in the positioning of sulfatide and α-GalCer can be observed when both structures are superimposed (not depicted). The same is true when the binding of α-GalCer to CD1d is compared in both mouse and human crystal structures. The exchange of one residue at the CD1d surface (Trp153 in human and Gly155 in mouse) results in a different positioning of the galactose, whereas the lipid backbone is bound in the same orientation (26
). Therefore, it is possible that the biological differences between the different sulfatide species shown in A are not only the result of differential loading characteristics of the ligands, but rather the consequence of subtle structural differences upon ligand binding due to either differences in fatty acid chain length and/or saturation (C16
), or, in case of lyso-sulfatide, the complete lack of a fatty acid chain. However, it cannot be completely ruled out that the stronger NKT cell activation by cis-tetracosenoyl sulfatide is a result of the increased solubility of the mono-unsaturated and more polar nervonyl (C24:1
) fatty acid versus the fully saturated tetracosanoyl (C24:0
) fatty acid, which could lead to an increased efficiency of loading onto CD1.
Recognition of sulfatide by CD1d-restricted NKT cells has important implications in autoimmune diseases of the CNS, as sulfatides are one of the major glycolipid components of the myelin membranes that are targeted in such diseases as multiple sclerosis (MS). MS is a demyelinating disease mediated by a T cell–guided immune response that is either initiated from antigen-presenting events in the CNS or induced after the peripheral activation by a systemic molecular mimicry response (27
). Indeed, in MS patients, increased serum levels of glycolipids (29
) and antibodies directed against them have been reported (31
). T cells specific for glycolipids have been isolated from MS patients. Their frequency in five active MS patients was three times higher compared with five normal individuals (35
). Recently, it has been demonstrated that sulfatide binds promiscuously to all of the CD1a, CD1b, CD1c, and CD1d isoforms (36
). Because CD1 molecules are up-regulated on macrophages in areas of demyelination in chronic-active MS lesions but not in silent lesions in the brain (37
), it is possible that self-glycolipids from myelin could be presented during local inflammation to T cells. Microglia, as well as infiltrating macrophages, could either engulf myelin components or internalize them by Fc receptors or by complement receptor–mediated phagocytosis after binding to myelin-specific antibodies. Thus, activated APCs in the CNS could not only present peptides (MHC) to T cells, but also glycolipids (CD1). Thus, myelin glycolipid–reactive T cells could potentially influence the inflammation and demyelination in the CNS. It is clear from our data (19 and unpublished data) that the peripheral activation of sulfatide-reactive T cells after adjuvant-free administration of sulfatide ameliorates experimental autoimmune disease of the CNS. Because CD1 molecules, unlike the classical MHC molecules, are nonpolymorphic, insight into the molecular recognition of sulfatide by the CD1d molecules and their interaction with a unique CD1d-restricted NKT cell population will be extremely valuable in the potential development of non-HLA–dependent therapeutic approaches for autoimmune demyelinating diseases in humans.