UCB primitive progenitors differentiate into CD94⁺/KIR⁺ NK cells when cultured in direct contact with AFT024.

UCB CD34⁺/Lin⁻/CD38⁻ cells (300 per well) were cultured in direct contact with AFT024 (▪), separated from the feeder by a Transwell membrane (▨), or in cytokines alone in the absence of AFT024 (░). All cultures were supplemented with weekly FL in combination with KL alone, or with the addition of a lymphocyte-stimulating cytokine (IL-7, IL-15, or IL-2). IL-3 was only added once at culture initiation. After 30 to 34 days, cell progeny were harvested and evaluated for the absolute number of CD56⁺, CD56⁺/CD94⁺, and CD56⁺/KIR⁺ NK cells. (A) The absolute number of NK cells was greatest in the presence of IL-15 or IL-2 when progenitors were cultured in direct contact with AFT024. Under this condition, NK cells accounted for more overall cell growth after the addition of IL-15 (49% ± 5.6%) or IL-2 (56% ± 7.3%) compared with the addition of IL-7 (1.6% ± 0.6%) or in the absence of a lymphocyte-stimulating cytokine (0.6% ± 0.2%). (B) The absolute number of NK cells expressing CD94 (clone HP-3B1) accounted for 38% ± 9.1% of total NK cells for IL-15 and 49% ± 8.5% for IL-2 cultures in direct contact with AFT024. (C) The absolute number of NK cells expressing at least one of the known KIRs (using a PE-cocktail of 3 monoclonal antibodies, DX9, GL183, and EB6) accounted for 2.8% ± 0.8% of total NK cells for IL-15 and 4.9% ± 0.8% for IL-2 cultures in direct contact with AFT024. There were no (FL, KL, IL-3 ± IL-7) or few (+IL-15 or +IL-2) KIR-expressing NK cells in cultures containing cytokines alone in the absence of AFT024.P values are listed for significant differences between progenitors separated from stroma or with cytokines alone compared with NK cell differentiation in contact with AFT024. Each bar represents the mean ± SEM from 6 to 9 UCB progenitor populations sorted for each condition. The insert to the right of the graph shows IL-7 data with a smaller scale to show differences.