Recognition of β-linked self glycolipids mediated by natural killer T cell antigen receptors

Abstract

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens.

Figure 1

Hierarchical recognition of multiple CD1d-antigen complexes by NKT cell TCRs. Flow cytometry of the TCRαβ⁻ 5KC mouse hybridoma transduced with the V_α14J_α18 invariant chain and various engineered TCRβ chains and stained with five different CD1d tetramers: α-GalCer, β-GalCer, β-LacCer, iGb3 or Gb3. Above, CDR2β and CDR3β sequences of the TCRβ chains; ‘Self’ (top row), CD1d tetramers made of CD1d monomers affinity-purified from the supernatants of human 293 cells transduced with a lentivirus encoding mouse CD1d with no external antigen added before the formation of tetramers; right, structures of antigens used in CD1d tetramers. β-GalCer was purified from bovine galactocerebrosides; β-LacCer was purified from porcine red blood cells; both contain heterogeneous fatty acid length and saturation (R). Numbers in top right quadrants indicate percent CD1d tetramer–positive cells. Data are representative of two independent experiments.

Figure 2

Overview of the structures of NKT cell TCR–CD1d–β-linked antigens. (a) Auto-V_α24 NKT cell TCR–CD1d–β-GalCer. C_α, α-chain constant region; C_β, β-chain constant region; β₂m, β₂-microglobulin; hCD1d, human CD1d. (b) Auto-V_α14 NKT cell TCR–CD1d–β-GalCer. mCD1d, mouse CD1d. (c) Mouse NKT cell TCR–CD1d–α-GalCer12. (d) Auto-V_α14 NKT cell TCR–CD1d–iGb3. (e) Auto-V_α14 NKT cell TCR–CD1d–Gb3. (f) Auto-V_α14 NKT cell TCR–CD1d–β-LacCer. Below (a–f), associated footprints on the CD1d-antigen complexes: pink, CDR1α loops; purple, CDR3α loops; red, CDR2β loops; orange, CDR3β loops; black, framework (FW) contributions. (g,h) CDR3β loops in the binding of auto-V_α14 and auto-V_α24 NKT cell TCRs to mouse (g) or human (h) CD1d: gold, human NKT cell TCRα chain; green, human NKT cell TCRβ chain; blue, mouse NKT cell TCRα chain; brown, mouse NKT cell TCRβ chain; yellow, β-linked ligands and α-GalCer.

Figure 3

Recognition of β-GalCer by human and mouse NKT cell TCRs. (a) Auto-V_α14 NKT cell TCR–CD1d–β-GalCer. (b) Auto-V_α24 NKT cell TCR–CD1d–β-GalCer. (c) V_α14-V_β8.2 NKT cell TCR–CD1d–α-GalCer. (d) V_α24-V_β11 NKT cell TCR–CD1d–α-GalCer. (e) Conformational adjustments of the β-GalCer head group (relative to sulfatide; Protein Data Bank accession code 2AKR) after ligation of the NKT cell TCR.

Figure 4

Recognition of bulky β-linked ligands by NKT cell TCRs. (a) Auto-V_α14 NKT cell TCR–CD1d–iGb3. (b) Auto-V_α14 NKT cell TCR–CD1d–Gb3. (c) Auto-V_α14 NKT cell TCR–CD1d–β-LacCer. (d) Conformational adjustments of the iGb3 head group after ligation of the NKT cell TCR (gray, not ligand bound; yellow, ligand bound). The position of the third sugar head group of iGb3 in the binary complex (green) was not resolved in the crystal structure of the CD1d-iGb3 complex.