Abstract
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.
Highlights
Type I natural killer (NK)T cells, or invariant NKT cells, express a semi-invariant TCRα Vα14-Jα18 rearrangement paired with a limited Vβ repertoire
Given the consistent capability of monoclonal antibodies specific for CD1d-α-GalCer complex (L363 and L317) to inhibit βManCer-stimulation of invariant NKT cells (iNKT) cells in a variety of biological assays, the direct measurement of binding affinity of anti-CD1dα-GalCer to CD1d-β-ManCer complex by plasmon resonance, and flow cytometry, we concluded that these monoclonal antibodies, which were developed to be reactive to the CD1d-α-GalCer complex, recognize and functionally bind to the CD1d-β-ManCer complex
Anti-CD1d-α-GalCer would be unable to capture CD1d-presented β-ManCer. This finding is intriguing considering the nature of the β-linked mannose sugar moiety of β-ManCer relative to the α-linked galactose head group of α-GalCer
Summary
Type I natural killer (NK)T cells, or invariant NKT cells (iNKT), express a semi-invariant TCRα Vα14-Jα18 rearrangement paired with a limited Vβ repertoire. Though α-GalCer is the most well-characterized iNKT cell ligand, the iNKT cell TCR binds a diverse assortment of structurally distinct antigens [11] and recognizes several self-glycosphingolipid antigens and β-linked mammalian lipid molecules, such as isoglobotrihexosylceramide (iGb3) and βgalactosylceramide (β-GalCer, Figure 1) [17,18,19]. The capacity of anti-CD1d-α-GalCer mAbs to quench iNKT cell reactivity to β-ManCer is nearly equivalent to their inhibition of α-GalCer-induced iNKT cell activation This finding cannot be explained by αanomer contamination. This further suggests that the iNKT cell TCR does not need to “force” the β-linked mannose sugar moiety into a favorable conformation to enable binding, helping to explain why β-ManCer exhibits properties unlike other β-linked ligands
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