Notch signaling favors the differentiation of NK cells with cyCD3 expression. Numbers in the dot plots show the frequency of cells in the respective top right and bottom right quadrants. R% indicates the frequency of cyCD3⁺ cells within the CD56⁺ cell population. (A) The generation of cyCD3⁺ NK cells in FTOC can be inhibited by DAPT. Dot-plot analysis of cyCD3 (y-axis) versus CD56 expression (x-axis) in human CD56⁺ cells generated in FTOC starting from human CD34⁺Lin⁻ CB cells after 14 days of culture with different concentrations of DAPT. Note the dose-dependent decrease in both the frequency and the absolute number of cyCD3⁺CD56⁺ cells when the Notch pathway is inhibited by the γ-secretase inhibitor. The left bar diagram shows the frequencies of cyCD3⁺CD56⁺ NK cells; the far right bar diagram shows the absolute number of CD56⁺ NK cells (□) or cyCD3⁺CD56⁺ NK cells (■) obtained in FTOC with different concentrations of DAPT as indicated on the x-axis. Bars represent means and SD of 3 independent experiments. (B) Notch signaling by DL1 favors the generation of cyCD3⁺ NK cells on stromal cell coculture. Dot-plot analysis of cyCD3 (y-axis) versus CD56 expression (x-axis) in human CD56⁺ cells generated on stromal cell line OP9-control or OP9-DL1 as indicated starting from human CD34 CB cells after 14 days of culture. Note the high frequency and absolute number of cyCD3⁺CD56⁺ cells when the Notch pathway is triggered by DL1 (OP9-DL1), and the low frequency when the DL1 signal is absent (OP9-control) or inhibited by the γ-secretase inhibitor DAPT. Left bar diagram shows the frequencies of cyCD3⁺CD56⁺ NK cells; the far right bar diagram shows the absolute number of CD56⁺ NK cells (□) or cyCD3⁺CD56⁺ NK cells (■) in different culture conditions as described on the x-axis. Bars represent means and SD of 3 independent experiments. (C) ICN1-transduced CB cells generate cyCD3⁺ NK cells on coculture with the MS-5 stromal cell line. Dot-plot analysis of cyCD3 (y-axis) versus CD56 expression (x-axis) in human CD56⁺ cells generated on MS-5 stromal cells starting from human CD34⁺ CB cells either transduced with LZRS-IRES-EGFP (LIE) or ICN1 after 14 days of coculture on the MS-5 stromal cell line with the mix of Ftl-3L, SCF, IL-7, and IL-15. Note the high frequency and total numbers of cyCD3⁺CD56⁺ NK cells when the Notch pathway is triggered by ICN1. Left bar diagram shows the frequencies of cyCD3⁺CD56⁺ NK cells; far right bar diagram shows the absolute number of CD56⁺ NK cells (□) or cyCD3⁺CD56⁺ NK cells (■) for the cells generated from either ICN1- or LIE-transduced cells as described on the x-axis. Bars represent means and SD of 2 independent experiments. (D) DNMAML1 inhibits the generation of cyCD3⁺ NK cells starting from CB on OP9-DL1 cells. Dot-plot analysis of cyCD3 (y-axis) versus CD56 expression (x-axis) in human CD56⁺ cells generated on OP9-DL1 stromal cells starting from human CD34⁺ CB cells either transduced with control EGFP murine stem cell virus vector (MSCV) or DNMAML1 after 18 to 20 days of coculture with Flt-3L, SCF, IL-7, and IL-15. Note the low frequency and numbers of cyCD3⁺CD56⁺ NK cells when the Notch-CSL complex is inhibited by DNML1. Left bar diagram shows the frequencies of cyCD3⁺CD56⁺ NK cells, and the 2 far right bar diagrams show the absolute number of CD56⁺ NK cells (□) or cyCD3⁺CD56⁺ NK cells (■) generated from control (MSCV) or DNMAML1-transduced CB cells as described on the x-axis. Due to the large interindividual variation of the 2 independent experiments, the results are shown separately.