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

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 R-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.

Design of a potent CD1d-binding NKT cell ligand as a vaccine adjuvant

“…Biological studies on α-GalCer analogues have shown that the modification of the lipid chain determines Th1/Th2 balance (32,33). Therefore, attempts have been made to synthesize α-GalCer analogues with modifications in the lipid portion aiming to selectively induce Th1 cytokine secretions.…”

Avidity of CD1d-ligand-receptor ternary complex contributes to T-helper 1 (Th1) polarization and anticancer efficacy

“…Thus, it was not surprising that the phase I clinical trial of α-GalCer showed meager antitumor responses, perhaps as a consequence of counteraction of Th1 cytokines by the Th2 cytokines (11)(12)(13). It has been reported that α-GalCer analogs with higher binding affinity for CD1d induced more Th1-polarized immune response (14) and analogs with the phenyl ring on the acyl chain displayed better anticancer activity than α-GalCer (15). To account for the Th1 biased immune modulatory activities of phenyl analogs, we explored the structure-activity relationships between iNKT TCR and CD1d-glycolipid complex to examine the following two possibilities: The first possibility was that each glycolipid might activate a particular repertoire of iNKT cells bearing specific beta chain.…”

Structure-Activity Relationship Studies of Novel Glycosphingolipids That Stimulate Natural Killer T-Cells

“…[56][57][58] They expected that their analogs would possess additional -stacking and other specific interactions with aromatic amino acid residues Tyr73 or Trp40 at the bottom of the hydrophobic pocket of human CD1d, leading to increased complex stability. 56) In fact, their aromatic acyl analogs, C8Ph (7) and relatives of it, exhibited a stronger Th1-biased cytokine response in humans in vitro.…”

“…56) In fact, their aromatic acyl analogs, C8Ph (7) and relatives of it, exhibited a stronger Th1-biased cytokine response in humans in vitro. [56][57][58] They performed quantitative microarray analysis to investigate the correlation of the binding affinity of the aromatic analogs to CD1d with the Th1/Th2 cytokine balances produced. 58) Among their aromatic acyl analogs, C8PhF (8) showed highest affinity with human CD1d and the largest Th1/Th2 ratio.…”

“…[56][57][58] They performed quantitative microarray analysis to investigate the correlation of the binding affinity of the aromatic analogs to CD1d with the Th1/Th2 cytokine balances produced. 58) Among their aromatic acyl analogs, C8PhF (8) showed highest affinity with human CD1d and the largest Th1/Th2 ratio. A docking model study of these aromatic analogs of CD1d confirms these results of affinity analyses.…”