Efferocytosing macrophages increased CD1d expression and activate iNKT cells. (A) Day 0, day 3, and day 4 peritoneal macrophages (F4/80+) were assessed for surface expression of CD1d, and mean fluorescence intensity was compared. Data represent at least 3 independent experiments. (B) Activation of N38-2C12 or N37-1A12 NKT cells by day 0 or day 4 peritoneal macrophages was determined by measuring IL-2 production by the NKT cells 24 hours after co-culture. Data represent at least 3 independent experiments. (C-D) Day 4 WT and Il4^−/− peritoneal macrophages were assessed for surface expression of CD1d (C) and their ability to activate N38-2C12 NKT cells to produce IL-2 (D). Data represent 2 independent experiments. (E) Day 0 and day 4 WT and Il4ra^−/− macrophages were co-cultured with N38-2C12 NKT cells and IL-2 from culture media were measured as in (B). Data represent 2 independent experiments. (F) Day 3 peritoneal exudate macrophages were co-cultured with apoptotic mouse neutrophils for 24 hours, and surface expression of CD1d on these macrophages was compared with that on macrophages without AC. (G) Macrophages from (F) were used to co-culture with N38-NKT cells and IL-2 in supernatants was measured by ELISA. Data represent 3 independent experiments.