CD117 and CD94 define 2 separate populations of differentiating NK cells. (A) CD117 defines 2 populations of CD56⁺ cells. FACS staining with CD56-APC versus CD117-PE shows 2 populations: CD56⁺CD117^high and CD56⁺CD117^low/–. (B) Expression of inhibitory receptors on CD56⁺CD117^high and CD56⁺CD117^low/– cell populations. Cells were stained with a monoclonal antibodies (mAbs) directed against CD56, CD117 in combination with CD161, KIR (CD158a and CD158b), NKG2A, and CD94 at day 28 of culture. Shown are histograms generated by gating on either the CD56⁺CD117^high or CD56⁺CD117^low/– populations. Results are representative of more than 20 donors. (C) Expression of activating receptors on CD56⁺CD117^high and CD56⁺CD117^low/– cells. FACS staining for NKp44, NKp46, NKp30, NKG2D, NKG2C, CD16, and CD8 after gating on either CD56⁺CD117^high or CD56⁺CD117^low/– cells. Results were representative of more than 20 donors. (D) CD117 and CD94 define 2 discrete populations of differentiating NK cells. FACS staining for CD117 and CD94 after gating on the CD56⁺ fraction. Results were representative of more than 20 experiments. (E) Adaptor protein expression in CD94^–CD117^high and CD94⁺CD117^low/– populations. Cells were FACS sorted into CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– fractions at days 21 to 28 of culture. RT-PCR was performed using primers specific for DAP12, DAP10, CD3ζ, FcϵRIγ, and actin. Results were representative of 3 or more experiments.