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iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo . Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, β-ManCer, a glycolipid with a β-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized β-ManCer-CD1d complexes and could inhibit β-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with β-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another β-linked glycosylceramide, β-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the β-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain β-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution., (Copyright © 2019 Clark, Yau, Bloom, Wang, Venzon, Suzuki, Pasquet, Compton, Cardell, Porcelli, Painter, Zajonc, Berzofsky and Terabe.)

CD1d-restricted T (natural killer T; NKT) cells are important for controlling herpesvirus infections. Interestingly, herpes simplex virus (HSV) can down-regulate CD1d-mediated activation of NKT cells. We have previously shown that the Thr322 residue in the cytoplasmic tail of human CD1d is important for its intracellular trafficking and functional expression. We proposed that the phosphorylation of T322 is a signal for CD1d lysosomal targeting and subsequent degradation. In the current study, we generated dual mutants by substituting the T322 and S323 residues of wild-type (WT) CD1d with Ala (non-phosphorylatable) or Asp (mimicking phosphorylation) and ectopically expressed them in human embryonic kidney 293 cells. We found that the surface expression levels of the CD1d mutants was in this order: T322AS323A > WT > T322A > S323A > S323D > T322D > T322DS323D. Our results therefore suggest that mimicking the phosphorylation of both T322 and S323 has a cumulative negative effect on the functional expression of CD1d. As previously reported, we also found that upon an HSV infection, antigen presentation by WT CD1d is reduced and the CD1d molecule is degraded. Interestingly, the T322A/S323A double mutation inhibited CD1d degradation and rescued CD1d-mediated antigen presentation following an HSV-1 infection. This suggests that the T322/S323 dyad may be phosphorylated, which then targets CD1d for lysosomal degradation post-infection as a means of immune evasion, explaining (at least in part) the reduced antigen presentation observed. Hence, our findings strongly suggest that T322 and S323 form a dual residue motif that can regulate the functional expression of CD1d during a viral infection., (© 2013 John Wiley & Sons Ltd.)

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

Invariant NKT (iNKT) cells expressing a semi-invariant Vα14 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 α-linked sugar, mammalian self Ags are β-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 β-linked Ag isoglobotrihexosylceramide (iGb3; Galα1-3-Galβ1-4-Glcβ1-1Cer) by forcing the proximal β-linked sugar of the trisaccharide head group to adopt the typical binding orientation of α-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 CDR2α residues of the TCR suggests an expanded recognition logic that can possibly distinguish foreign Ags from self Ags.

NKT cells recognize lipid Ags presented by CD1d molecules and play an important role in the regulation of innate and adaptive immune responses. In this study, we report the identification of a membrane-associated protein, Ig-like transcript 4 (ILT4), as a novel human CD1d receptor that inhibits CD1d-mediated immune responses. We found that native CD1d tetramer generated by mammalian cells was able to specifically bind human monocytes in the peripheral blood, and this binding was at least partly mediated by monocyte-expressed ILT4. The interaction between ILT4 and CD1d involves the two N-terminal domains of ILT4 and the Ag-binding groove of CD1d (alpha1/alpha2 domain). This interaction has been identified on the cell surface as well as in the endosomal and lysosomal compartments. Functional analysis showed that ILT4 could block the loading of lipid Ags such as alpha-GalCer, and consequently inhibited NKT recognition. The interaction between ILT4 and CD1d may provide new insights into the regulation of NKT-mediated immunity.