Quantitative Microarray Analysis of Intact Glycolipid−CD1d Interaction and Correlation with Cell-Based Cytokine Production

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Abstract
The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates TH1 and TH2 chemokine release. In this study, a variety of glycolipid ligands were attached to a microarray surface and their binding with dimeric CD1d was investigated. An α-galactosyl ceramide (α-GalCer) bearing a carbamate group at the 6′-OH position was tethered to the surface, and the dissociation constant on surface with CD1d was determined to reflect the multivalent interaction. Competition assays were then used to determine the dissociation constants (Ki) of new and intact glycolipids in solution. The 4-fluorophenyloctanoyl-modified α-GalCer (18) was found to bind most strongly with CD1d (Ki 0.21 μM), 2 orders of magnitude stronger than α-GalCer and more than three times more selective than α-GalCer for IFN-γ release from NKT cells. Various α-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-γ production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the TH1 pathway.

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