BMP signaling influences human NK differentiation. (A) The levels of BMP4 were determined by ELISA in the supernatants obtained culturing human thymic CD34⁺CD1a⁻BMPRIA⁻ precursors with SCF and IL-15 for different days. Data represent the mean ± SD from 3 to 4 independent experiments. Histograms show intracellular staining for BMP4 on immature CD161⁺CD56⁻ and mature CD161⁺CD56⁺ NK cells generated after 7 days of culture. (B) The scatter plots show the effects on cell recovery when the BMP inhibitor dorsomorphin was added to the cultures. The number of cells recovered in each experiment was divided by the mean number of cells recovered from the control cultures, to give the relative cell number from 6 individual experiments (**P ≤ .01 by t test). (C) Dot plots show CD161 versus CD56 expression from control and dorsomorphin-treated cultures after 9 days. As shown, the addition of BMP4 neutralized the inhibitory effect of dorsomorphin on human NK cell differentiation. (D) Determination of the proliferation rate of differentiating NK cells after 5 days of culture. Cells were pulsed for 12 hours with BrdU. A specific kit was used to measure BrdU incorporation into newly synthesized DNA. Results are the mean ± SD of 4 independent experiments, each with 2 cultures per point (**P ≤ .01 by t test). (E) Annexin V staining was measured by flow cytometry in NK cells harvested from control and dorsomorphin-treated cultures on day 5. Data are representative results from 3 independent experiments. (F) Equal numbers of NK cells generated in dorsomorphin-treated cultures (black circles) and untreated cultures (gray squares) were stimulated with IL-15 + IL-12 for 12 hours and assayed in a 4-hour standard cytotoxicity assay against K562 target cells at different effector-to-target ratios (E:T) in duplicate wells. The mean percentage of specific lysis ± SD is represented (n = 6 different individuals) for each E:T ratio (*P ≤ .05, **P ≤ .01 by t test). (G) NK cells (3 × 10⁵ cells) recovered from control (gray bars) and dorsomorphin treated-cultures (black bars) were stimulated for 12 hours. Supernantants were collected and analyzed for the presence of IFN-γ, IL-10, and TNFα (*P ≤ .05, **P ≤ .01, and ***P ≤ .001 by t test).