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Published on: October 29, 2018
Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine
Pi-Hui Liang1, Masakazu Imamura, Xiangming Li
1The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang Dist., Taipei, Taiwan 11529.
Insights
This study shows that modified alpha-galactosyl ceramide (alpha-GalCer) ligands bind strongly to CD1d, enhancing T(H)1 immune responses. Tighter binding correlates with increased IFN-gamma, suggesting a pathway bias.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- CD1d protein presents glycolipids to T cells, influencing immune responses.
- Understanding CD1d-glycolipid interactions is crucial for immune modulation.
Purpose of the Study:
- To investigate the binding affinity of various glycolipid ligands to CD1d.
- To determine how binding strength affects NKT cell cytokine release.
Main Methods:
- Glycolipid ligands were immobilized on a microarray surface for CD1d binding assays.
- Surface plasmon resonance and competition assays were used to quantify binding constants (Kd and Ki).
- Interferon-gamma (IFN-γ) and Interleukin-4 (IL-4) release from NKT cells were measured.
Main Results:
- A modified alpha-galactosyl ceramide (alpha-GalCer) derivative exhibited significantly higher binding affinity to CD1d compared to native alpha-GalCer.
- The binding affinity of glycolipids to CD1d strongly correlated with IFN-γ production by NKT cells.
- A poor correlation was observed between binding affinity and IL-4 secretion, indicating a T(H)1 bias.
Conclusions:
- Enhanced binding affinity of glycolipids to CD1d can preferentially activate T(H)1 immune responses.
- Modified alpha-GalCer analogues represent potent tools for modulating NKT cell activity.
- These findings have implications for the development of targeted immunotherapies.
Abstract:
The protein CD1d binds self and foreign glycolipids for presentation to CD1-restricted T cells by means of TCR recognition and activates T(H)1 and T(H)2 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 alpha-galactosyl ceramide (alpha-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 alpha-GalCer (18) was found to bind most strongly with CD1d (Ki 0.21 microM), 2 orders of magnitude stronger than alpha-GalCer and more than three times more selective than alpha-GalCer for IFN-gamma release from NKT cells. Various alpha-GalCer analogues were analyzed, and the results showed that the binding affinity of glycolipids to CD1d correlates well with IFN-gamma production but poorly with IL-4 secretion by NKT cells, suggesting that tighter binding ligands could bias cytokine release through the T(H)1 pathway.
