Natural killer T (NKT) cells express a semi-invariant V?14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to ?- and ?-anomeric glycosphingolipids, as well as microbial ?-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen ?GalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 ? resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the ?GalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented ?GalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3? residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.
Invariant NKT (iNKT) cells expressing a semi-invariant V?14 TCR recognize self and foreign lipid Ags when presented by the nonclassical MHCI homolog CD1d. Whereas the majority of known iNKT cell Ags are characterized by the presence of a single ?-linked sugar, mammalian self Ags are ?-linked glycosphingolipids, posing the interesting question of how the semi-invariant TCR can bind to such structurally distinct ligands. In this study, we show that the mouse iNKT TCR recognizes the complex ?-linked Ag isoglobotrihexosylceramide (iGb3; Gal?1-3-Gal?1-4-Glc?1-1Cer) by forcing the proximal ?-linked sugar of the trisaccharide head group to adopt the typical binding orientation of ?-linked glycolipids. The squashed iGb3 orientation is stabilized by several interactions between the trisaccharide and CD1d residues. Finally, the formation of novel contacts between the proximal and second sugar of iGb3 and CDR2? residues of the TCR suggests an expanded recognition logic that can possibly distinguish foreign Ags from self Ags.
Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.
Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic ?-galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1-biasing glycolipid, ?-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.
Invariant natural killer T cells (iNKT cells) rapidly produce effector cytokines. In this study, we report the first crystal structures of the iNKT cell T cell receptor (TCR) bound to two natural, microbial glycolipids presented by CD1d. Binding of the TCR induced CDR3-?-dependent structural changes in the F roof of CD1d; these changes resemble those occurring in the absence of TCR engagement when the highly potent synthetic antigen ?-galactosylceramide (?-GalCer) binds CD1d. Furthermore, in the Borrelia burgdorferi ?-galactosyl diacylglycerol-CD1d complex, TCR binding caused a marked repositioning of the galactose sugar into an orientation that closely resembles ?-GalCer. The TCR-dependent reorientation of the sugar, together with the induced CD1d fit, may explain the weaker potency of the microbial antigens compared with ?-GalCer. We propose that the TCR of iNKT cells binds with a conserved footprint onto CD1d, regardless of the bound glycolipid antigen, and that for microbial antigens this unique binding mode requires TCR-initiated conformational changes.
Values and life goals are associated with ones general well-being and quality of life. However, there is insufficient documentation about issues that are linked with the notions of spiritual values and life goals among college students in Asia. This study addressed this lack of research by focusing on the spiritual values and life goals among students in Singapore.
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