Figure 3. Ganglioside treatment alters CD1d-mediated NKT cell activation.

(A) GD3 (B) GT1b or (C) GD2 at the indicated concentrations for 4 h, washed extensively and co-cultured with NKT cell hybridomas, DN32.D3, N37-1A12, and N38-3C3 overnight. (D) LCD1dwt cells were treated with the indicated gangliosides (5μg/ml) for 4 h, washed, and co-cultured with NKT cell hybridomas. Pretreatment with the indicated purified gangliosides altered CD1d-mediated NKT cell activation (E) Treatment of α-GalCer loaded CD1d-Ig aAPC with GD3 inhibits antigen presentation. α-GalCer - loaded aAPC were incubated for 4 h with either medium, GD3 (5μg/ml), or ascites fluid. The aAPC were washed extensively and then cocultured with the Vα14⁺ NKT cell hybridoma, N38-3C3. (F) Addition of anti-GD3 mAb to tumor associated ascites restores NKT cell recognition of CD1d molecules. LCD1dwt cells were treated with ascites (OC-91) or the ascites pretreated with an antibody specific for GD3 (clone R24) for 4 h, then washed extensively and cocultured with a panel of NKT cell hybridomas. IL-2 was measured, as an indication of NKT cell activation, by standard cytokine ELISA. Data are shown as mean ±S.E.M. of one experiment set up in triplicate. ** P≤0.01, *** P≤0.001. The experiments were performed three times.