Figure 3.

iNKT cell reactivity to a β-GlcCer panel with differing N-acyl chains. (a) A β-GlcCer panel in co-culture with RAW or CD1d-transfected RAW cells and the iNKT cell hybridoma DN32. IL-2 production was determined by ELISA to a 5-fold dose titration of the indicated lipid with a top concentration of 10 μg/ml, or 10 ng/ml for α-GalCer. (b) IFN-γ production by a primary murine iNKT cell line in co-culture with wild-type CD11c⁺ BMDC, comparing β-GlcCer C_24:1, reported iNKT cell lipid self-antigens, and a microbial GSL antigen. IL-2 and IFN-γ ELISA data are presented as mean and range in duplicate wells, and are representative of three separate experiments. (c) IFN-γ and (d) IL-4 cytokine capture assay in liver mononuclear cells following intravenous injection of the indicated lipid. Mice were injected intravenously with 25 μg of the indicated lipid, or 1 μg for α-GalCer. The TCRβ⁺PBS-57 (α-GalCer analog)-loaded tetramer⁺ gate is shown except for the last plot in each panel, where the total TCRβ⁺ gate is shown for a β-GlcCer C_24:1-injected CD1d-deficient mouse. The percentage of iNKT cells producing IFN-γ or IL-4 is indicated for each plot. Results are representative of at three independent experiments. The structures of the synthetic lipids used here are depicted in Supplementary Fig. 4, and as all contained a d_18:1 shingenine base, they are abbreviated in this and other figures with only the N-acyl chain composition listed. For example, “β-GlcCer 24:1” represents β-D-glucopyranosylceramide d_18:1-C_24:1.