CD34⁺CD1d⁺ cells present antigen to NKT cells. (A) Fresh NKT cells from cord-blood samples (i) were purified by flow sorting (ii) after staining with anti–TCR Vα24 and -Vβ11 mAbs. (B) MACS-selected CB CD34⁺ cells (i) were further flow-sorted into total CD34⁺ (ii) and CD1d^–CD34⁺ (iii) fractions and cocultured with highly purified fresh NKT cells. (C) Analysis of surface activation markers HLA-DR and CD69 after 24 hours revealed that, in the presence of αGalCer, NKT cells cocultured with total CD34⁺ cells were highly activated compared with NKT cells cocultured with CD1d^–CD34⁺ cells. In the presence of vehicle, NKT cells cultured either with total CD34⁺ or CD1d^–CD34⁺ cells showed little evidence of activation. Plots are gated in NKT cells identified by staining with the NKT-cell–specific mAb Vα24Jα18 after dead-cell exclusion with propidium iodide. Data are representative of 2 independent experiments. (D) Activation of NKT cells cocultured with CD34⁺ or CD34⁺ CD1d^– cells with or without αGalCer as determined by intracellular staining for IFNγ. As with surface activation markers, NKT-cell production of IFNγ was highest when they were cocultured with total CD34⁺ cells and in the presence of αGalCer. Data are representative of 2 independent experiments.