Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells

Abstract

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist alpha-galactosyl ceramide (alpha-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, alpha-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to alpha-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-gamma secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with alpha-GalCer. Because alpha-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.

Fig. 1.

NKT cells are reactive to CD1d-bound α-GalCer. This stimulation results in the production of large amounts of cytokines such as IL-4 and IFN-γ.

Fig. 2.

Structures of glycolipids and analogs.

Fig. 3.

Synthesis of sphingosine. Reagents and conditions are as follows. a, (i)C₂H₃MgBr, THF, Anti:Syn 3.5:1 61%; (ii) Grubbs catalyst second generation, CH₂Cl₂, pentadecene, 71%. b, (i) BzCl, pyridine, 90%; (ii) amberlyst 15 H⁺ form, MeOH, 70%.

Fig. 4.

Synthesis of glycolipids. Reagents and conditions were as follows. a, 22, TMSOTf, 67%. b, (i) TFA, DCM; (ii) HBTU, myristic acid or (S)-2-acetoxymyristic acid, n-morpholine, ≈92%, two steps. c, H₂, 20% Pd(OH)₂, (ii) LiOH, H₂O:THF:MeOH, 38%, two steps. d, 26, TMSOTf, 60%. e, (i) TFA, DCM, (ii) HBTU, myristic acid or (S)-2-acetoxymyristic acid, n-morpholine, ≈92%, two steps. f, (i) NaOMe, MeOH, (ii)Pd/C, H₂, EtOH, 90%, two steps. g, 30, TMSOTf, 62%. h, (i) TFA, DCM, (ii) HBTU, nervonic acid, n-morpholine, 60%, two steps. i, (i) NaOMe, MeOH, quant; (ii) Bu₂SnO, MeOH; (iii) Me₃N·SO₃, THF, 95%. j and k, (i) LDA, TMSOOTMS, (ii) H⁺, MeOH, 30%, two steps; then LiOH, H₂O:MeOH, 81%; l, novozyme 435, CH₂=CHOAc, 54% based on S isomer.

Fig. 5.

IL-2 secretion by murine 1.2 hybridomas when stimulated by the indicated glycolipids loaded on CD1d-coated plates. (A) IL-2 secretion profile of 3″-O-sulfo-α-GalCer compared with α-GalCer and analogs. (B) Dose-dependent secretion of IL-2 by Sphingomonas GSLs and analogs. CD1d molecules (10 μg/ml in PBS) were coated in a 96-well plate by incubation for 1 h at 37°C. IL-2 release was measured after 16 h of culture in a sandwich ELISA.

Fig. 6.

Human Vα24i NKT cells release IL-4 and IFN-γ in response to the indicated glycolipids. Flow cytometric analyses also show that Vα24i NKT cells bind to the various glycolipids in response to the indicated glycolipids. (A) Human Vα 24i NKT cells respond to synthetic Sphingomonas and sulfatide glycolipids. IFN-γ and IL-4 release after 16 h of culture by 1 × 10⁵ Vα24i human NKT cell line in response to culture with 4 × 10⁵ autologous immature CD14⁺ dendritic cells pulsed with the indicated glycolipid antigens at 10 μg/ml. The negative control wells contained similar numbers of NKT cells and dendritic cells, cultured without added glycolipid. Data are mean ± SD of duplicate wells. (B) Human Vα 24i NKT cells bind to glycolipids in the context of CD1d. Flow cytometric analysis of a human Vα24i human NKT cell line with human CD1d dimers that were unloaded or loaded with 10 M indicated glycolipid antigen. The cells were also stained with anti-human CD3-PerCP.

Fig. 7.

Computer modeling of GSL-1 docked to the crystal structure of mCD1d. The two acyl tails fit nicely into the hydrophobic pockets of the protein, allowing for the sugar head group to be presented for NKT cell recognition.