Figure 5. Synergistic effect of concurrent stimulation by α-GSL-loaded CD1c and CD1d molecules. (A) Stimulation by the combination of both CD1c and CD1d. Microtiter plate wells were coated with 0.5 μg/well CD1c-Fc fusion protein, or 0.05 μg/well CD1d-Fc fusion protein, or a combination of 0.05 μg/well CD1d-Fc and 0.5 μg/ well CD1c-Fc fusion protein, incubated with α-GSL, and used to stimulate iNKT cell clones. GM-CSF production was quantitated by ELISA. Similar results were obtained in 13 independent experiments. (B) Compiled results from all 13 experiments showing the amount of GM-CSF produced in response to the combination of CD1c-Fc and CD1d-Fc divided by the sum of the responses to each CD1-Fc molecule alone. The p value comparing the results to a hypothetical median of 1 was calculated using a two-tailed Wilcoxon signed-rank test. (C) Comparison of the effect of concurrent stimulation by α-GSL-loaded CD1c and CD1d on iNKT cell production of four different cytokines. The plot shows the amount of each cytokine produced in a given experiment in response to the combination of CD1c-Fc and CD1d-Fc divided by the sum of the responses to each CD1-Fc molecule alone. Results are from independent analyses of two different iNKT cell clones.