Functional differences between CD94^–CD117^high and CD94⁺CD117^low/– NK cells. (A) CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– populations were FACS sorted at days 21 to 28 of culture, rested for 24 hours, and then used in a ⁵¹Cr release assay with K562 cells as targets. Error bars depict standard deviation. Results are representative of 3 or more experiments. (B) Expression of cytotoxic molecules by RT-PCR in developing NK cells. mRNA was isolated from the FACS-sorted cell populations and was used for RT-PCR with primers specific for perforin, granzyme B, Fas ligand, TRAIL, and actin. Results were representative of 3 or more experiments. (C) Differential expression of perforin, granzyme B, and FasL in CD56⁺CD94^–CD117^high and CD56⁺CD94⁺CD117^low/– cell populations. At days 21 to 28, cultures were analyzed for the expression of perforin and granzyme B using intercellular staining. FasL was assayed using surface staining. High levels of perforin, granzyme B, and FasL could be detected in the CD56⁺CD94^–CD117^low/– population, whereas these proteins were either absent or present at minimal amounts in the CD56⁺CD94^–CD117^high population. Results were representative of 3 or more experiments. (D) Interferon-γ secretion. HPC-derived NK cells were challenged with IL-12 (10 μg/mL) and IL-18 (100 μg/mL) for 18 hours, and cultures were assayed for intracellular interferon-γ. In the gated CD56⁺ population, intracellular interferon-γ was detected only in CD94⁺ cells representing the CD56⁺CD94⁺CD117^low/– population. Results were representative of 3 or more experiments.