Implications for invariant natural killer T cell ligands due to the restricted presence of isoglobotrihexosylceramide in mammals

Abstract

Development of invariant natural killer T (iNKT) cells requires the presentation of lipid ligand(s) by CD1d molecules in the thymus. The glycosphingolipid (GSL) isoglobotrihexosylceramide (iGb3) has been proposed as the natural iNKT cell-selecting ligand in the thymus and to be involved in peripheral activation of iNKT cells by dendritic cells (DCs). However, there is no direct biochemical evidence for the presence of iGb3 in mouse or human thymus or DCs. Using a highly sensitive HPLC assay, the only tissue where iGb3 could be detected in mouse was the dorsal root ganglion (DRG). iGb3 was not detected in other mouse or any human tissues analyzed, including thymus and DCs. Even in mutant mice that store isoglobo-series GSLs in the DRG, we were still unable to detect these GSLs in the thymus. iGb3 is therefore unlikely to be a physiologically relevant iNKT cell-selecting ligand in mouse and humans. A detailed study is now warranted to better understand the nature of iNKT cell-selecting ligand(s) in vivo.

Fig. 1.

GSL biosynthetic and catabolic pathways and Galα1–3 biosynthetic enzymatic pathways. (a) Synthetic pathway of GSLs from ceramide with the full structures of the globo- and isoglobo-series GSLs. (b) Catabolic pathways of the globo- and isoglobo-series GSLs with the catabolic enzymes and the relevant lysosomal storage disorders highlighted. (c) The three different enzymes responsible for the addition of galactose in an α1–3 linkage are shown. R, glycoprotein or GSL.

Fig. 2.

NP-HPLC profile of mouse, rat, and commercial GSL-derived oligosaccharides. The iGb3 and iGb4 elution region of the chromatogram, for GSL-derived oligosaccharides from mouse DRG and rat stomach neutral GSLs, is shown. Dotted lines indicate the elution times of iGb3 and iGb4. α-Gal A^−/− mice were aged 40 wk, and hexb^−/− and controls were aged 11 wk.

Fig. 3.

NP-HPLC profile of mouse, human, and rat thymus neutral GSL-derived oligosaccharides. Mouse and human profiles are representative of three independent experiments, and the rat profile is representative of four independent experiments. Dotted lines indicate the elution times of iGb3 and iGb4 standards. α-Gal A^−/− mice were aged 38 wk, and hexb^−/− and control mice were 9 wk. (Insets) Profiles scaled to that of the rat thymus where iGb3 is detectable.

Fig. 4.

NP-HPLC profile of Gb3 and iGb3 standard oligosaccharide mixtures. (a) The profile shows the detection of 10 fmol of iGb3 standard oligosaccharide. (b) Gb3 was coinjected with different quantities of iGb3 (5% and 1%), as shown. The final image is the overlay of Gb3 alone (solid line) and Gb3 with 1% iGb3 (dashed line). This demonstrates that the presence of iGb3 at 1% the level of Gb3 is readily detected. The profiles are the overlay of three independent injections of standards.

Fig. 5.

NP-HPLC profile of mouse and human DC neutral GSL-derived oligosaccharides. Mouse profiles are representative of three independent experiments. Dotted lines indicate the elution times of iGb3 and iGb4 standards. α-Gal A^−/− and control mice were aged 16 wk, and hexb^−/− mice were aged 7 wk.

Fig. 6.

Q-PCR analysis of GSL synthetic and degradative enzyme expression in different organs. Δ-Ct values (inversely proportional to transcript levels) were calculated as described in Materials and Methods. The average value with the standard error of the mean is displayed from three experiments.

Fig. 7.

Stimulation of a human iNKT cell line with C1R-CD1d cells pulsed with different concentrations of iGb3 or αGalCer. IFN-γ release from iNKT cells in picograms per milliliter is plotted against the αGalCer (nanograms per milliliter in vehicle) or iGb3 (micrograms per milliliter in methanol) concentrations pulsed onto C1R-CD1d cells. No toxicity was observed at any concentration of either compound. NKT denotes iNKT cells alone with no C1R-CD1d cells, and 0 is the vehicle (no added αGalCer/iGb3) control showing background recognition of endogenous ligands. The experiment was performed twice in duplicate, and the average with the standard error of the mean is displayed.