NKT TCR recognition of CD1d-α-C-galactosylceramide

Abstract

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vβ8.2 and Vβ7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vβ8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.

FIGURE 1. Structures of the glycolipid Ags used

α-GalCer, α-C-GalCer, α-1C-GalCer, α-C-alkyne-GalCer, α-C-O-GalCer, α-N-Cyc-Cer.

FIGURE 2. In vitro stimulation of spleen NKT cells with α-C-GalCer and associated analogues

Spleen cells were cultured with 100, 10, and 1 ng/ml of the glycolipids as indicated. A. After 8 hours, cell cultures were harvested and surface labelled with anti-TCR-β and α-GalCer-loaded CD1d tetramer then fixed and permeabilised for ICS for IFN-γ production. B. The mean ± SEM from three independent experiments testing IFN-γ production by ICS (as per A) are shown. C. Cytokine production at 72 hours from spleen cells cultured with the various analogues was determined by cytometric bead array. The mean ± SEM from three independent experiments are shown. The exception is IL-10, which was only measured in 2 experiments. D. Representative data showing the proliferation of NKT cells in response to the different ligands as determined by CFSE labelled spleen cells, gating on α-GalCer-CD1d tetramer + cells after 72 hr. E. The CFSE proliferation results for NKT cells (as depicted in C) from four independent experiments (mean ± SEM) are shown. F. Cytokines detected in the serum of mice determined by cytometric bead array following the i.p. administration of 1 μg of glycolipid. The combined results from three independent experiments (10 mice per group) are shown. G. The ratio of serum IFN-γ (24 hr) to serum IL-4 (2 hr). For B, C and E, the data readings for each individual experiment were the mean of duplicate cultures. Error bars represent SEM.

FIGURE 3. Binding analysis of Vβ8.2 and Vβ7 to CD1d-Ag

Equilibrium binding response curve for Vβ8.2 TCR to CD1d-α-GalCer (A), CD1d-α-C-GalCer (C), CD1d-α-C-alkyne-GalCer (E) and CD1d-α-N-Cyc-Cer (G) and Vβ7 TCR to CD1d-α-GalCer (B), CD1d-α-C-GalCer (D), CD1d-α-C-alkyne-GalCer (F) and CD1d-α-N-Cyc-Cer (H). The equilibrium dissociation constant (K_Deq) by fitting to one-site binding model. All results are shown as one experiment performed in triplicate (representative of 2–3 experiments).

FIGURE 4. Structure of Vα14-Vβ8.2 NKT TCRs in complex with CD1d-α-C-GalCer

(A)Vα14-Vβ8.2 NKT TCR in complex with CD1d-α-C-GalCer. α-C-GalCer, blue; CD1d heterodimer, grey; Vα14, cyan; Vβ8.2, green. CDR1α, purple; CDR3α, yellow; CDR1β, teal; CDR2β, ruby; CDR3β, not modelled. B, Vα14-Vβ8.2 NKT TCR in complex with CD1d-α-GalCer. α-GalCer, magenta; CDR3β, orange; CD1d, Vα14, Vβ8.2, CDR1α, CDR3α, CDR1β, CDR2β colour coding as in A. C, Footprint of Vα14-Vβ8.2 on the surface of CD1d-α-C-GalCer. α-C-GalCer is shown in spheres. CD1d, α-C-GalCer and CDR loops colour coding as in A. D, footprint of Vα14-Vβ8.2 on the surface of CD1d-α-GalCer. CD1d, α-GalCer and CDR loops colour coding as in A and B.

FIGURE 5. Vα14-Vβ8.2 NKT TCR mediated interactions with mouse CD1d-glycolipid interface

A, Vα14-Vβ8.2 NKT TCR CDR3α and mCD1d mediated contacts with α-C-GalCer. α-C-GalCer, blue; CDR3α, yellow; CD1d, grey. B, Vα14-Vβ8.2 NKT TCR CDR3α and CD1d mediated contacts with α-GalCer. α-GalCer, magenta; CDR3α, and mCD1d colour coding as in A. H-bonds are shown in black dashed lines. C, superposition of mouse Vα14-Vβ8.2 NKT TCR-CD1d-α-C-GalCer and Vα14-Vβ8.2 NKT TCR-CD1d-α-GalCer complexes. Vα14Vβ8.2 NKT TCR-CD1d-αC-GalCer is shown in cyan and Vα14-Vβ8.2 NKT TCR-CD1d-α-GalCer is shown in pink. The replacement of the glycosidic -O- linkage in α-GalCer to a hydrophobic -CH₂- linkage in α-C-GalCer does not result in a major head group movement. D, Vα14-Vβ8.2 NKT TCR CDR1α and CDR3α mediated contacts with α-C-GalCer. α-C-GalCer, blue; CDR1α, purple; CDR3α, yellow; mCD1d, grey. H-bonds are shown in black dashed lines.