NKG2 and CD28 regulate activation of naive or primed iNKT cells by α-GalCer. (A) Mice were primed with intraperitoneal injection of α-GalCer (□) or OCH (○). Splenic MNCs were prepared on the indicated days after priming and stimulated with α-GalCer or OCH in vitro for 24 hours. Data are represented as the mean ± SD of triplicate wells. Similar results were obtained from 3 independent experiments. (B) Mice were primed with intraperitoneal injection of α-GalCer, OCH, or vehicle on day –2.5. Then, splenic MNCs were prepared on day 0 and stimulated with α-GalCer in vitro for 24 or 48 hours in the presence or absence of the indicated mAbs. Data are represented as the mean ± SD of triplicate wells. Similar results were obtained from 3 independent experiments. (C) Liver iNKT cells and splenic DCs were isolated from naive mice or primed with α-GalCer or OCH 2.5 days before and then cocultured with α-GalCer for 48 hours in the presence or absence of anti-NKG2 mAb. Data are represented as the mean ± SD of triplicate wells. Similar results were obtained from 2 independent experiments.