FIGURE 6.

Biological assays with GSLs. A, cell-based assay in which an A20 B cell lymphoma cell line transfected with CD1d was pulsed with 100 ng of the indicated GSL and co-cultured with a Vα14Vβ8.2 NKT cell hybridoma (1.2). TCR engagement was measured after 24 h through IL-2 production in serum measured with a sandwich ELISA. Data represent samples in triplicate and are representative of three independent experiments. B, human type I NKT cells were activated by human PBMCs pulsed with the indicated glycolipid concentrations for 24 h. Cytokine levels for human GM-CSF were measured in the supernatant using a sandwich ELISA. Representative data from one of two experiments performed in triplicate wells using multiple human cell lines are shown. C, C57Bl/6 mice were injected with 1 μg of the indicated GSL, and sera were analyzed at 24 h. Serum samples were measured for IFN-γ cytokine levels by ELISA. Data are representative of two independent experiments from three mice per group. Error bars represent ± S.E. D. Cell-free antigen presentation assay. 96-Well plates were coated with recombinant CD1d and incubated overnight with the indicated concentration of glycolipids. After the initial incubation, Vα14Vβ8.2 NKT cell hybridoma 1.2 was added overnight. Serum IL-2 was measured using a sandwich ELISA. Representative data from 1 of 2 different hybridoma lines are shown.