Transient Notch stimulation allows generation of cyCD3⁺CD56⁺ NK cells. (A) Notch receptor triggering of CB progenitor cells by DL1 for 4 days is sufficient to generate cyCD3⁺ NK cells. Comparative frequencies of and numbers of cyCD3⁺CD56⁺ NK cells derived from CD34⁺ progenitor cells that did or did not receive a Notch signal in preculture. CD34⁺ CB progenitor cells received a Notch signal in vitro by preculture on OP9-DL1 cells during 4 days with Flt-3L, SCF, and IL-7. Both CD34⁺ CB cells without preculture (D0) and CD34⁺CB cells precultured on OP9-DL1 for 4 days (D4) were used to generate NK cells by coculture for 22 or 18 days, respectively, on OP9-DL1 stromal cells with Flt-3L SCF, IL7, and IL-15 in the absence or presence of DAPT. Left bar diagram shows the frequency of cyCD3⁺ NK cells generated from CB cells without preculture (D0) or precultured (D4) and further cultured on OP9-DL1 in the absence (■) or presence (□) of 5 μM DAPT. The right bar diagram shows the influence on the absolute number of NK cells obtained in the absence (■) or presence (□) of DAPT as indicated. Each time, the left bar indicates the absolute number of all CD56⁺ NK cells, whereas the right bar indicates the absolute number of cyCD3⁺CD56⁺ NK cells. Results represent means (+SD) of 2 independent experiments. (B) NK cells derived from thymic precursors express cyCD3, even without further Notch signaling. CD34⁺ progenitors from the thymus were cultured on OP9-DL1, OP9-DL1 in the presence of 5 μM DAPT, and OP9-control. The left bar diagram represents the frequencies of cyCD3⁺CD56⁺ NK cells. The right bar diagram represents the absolute number of NK cells in different stromal coculture conditions as described on the x-axis. Each time, the left bar indicates the number of all CD56⁺ NK cells, whereas the right bar indicates the absolute number of cyCD3⁺CD56⁺ NK cells. Dot plots in lower panel represent the flow cytometric analysis of cyCD3 (y-axis) versus CD56 (x-axis) of cells generated from thymic progenitor cells under different stromal coculture conditions as indicated. Numbers in the dot plots show the percentage of cells in the respective top right and bottom right quadrants. R% indicates the frequency of CD56⁺CyCD3⁺ NK cells within the CD56⁺ cell population. Note the high number of cyCD3⁺CD56⁺ NK cells even when the Notch pathway is not triggered by DL (OP9-control) or inhibited by the γ-secretase inhibitor DAPT. The bars represent means (+ SD) of 3 independent experiments, of which 1 is shown as a representative dot plot.