Fig 1: Differentiation of ILC1-like NKP lead to formation of complex NK cell repertoires via NOTCH signaling.CB-derived ILC1-like cells and CD56bright NK cells were flow cytometrically sorted and subsequently cultured for 8 days on OP9, OP9-DL1, or without feeder cells (a, b) or cultured on OP9-DL1 for 8 and 14 days (c–e). (a) Frequency of CD94+NKG2A+ (n = 3–13) and (b) KIR2DL2/L3/S2+ NK cells (n = 3–9). (c) Representative dot plots for NKG2A and KIR (comprising antibodies against KIR2DL2/L3/S2, KIR2DL1/S1/S3/S5, and KIR3DL1) of ILC1-like-derived NK cells (left hand side) and CD56bright-derived NK cells (right hand side). (d) Frequency of total KIR+ (left hand side) and KIR+NKG2A- (right hand side) NK cells derived from ILC1-like cells and CD56bright NK cells, respectively (n = 6) on day 8 (top) and day 14 (bottom). (e) Dissection of NK cell repertoire diversity of NK cells derived from ILC1-like cells and CD56bright NK cells, respectively by combinatorial analysis of the major inhibitory receptors KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A at day 8 and 14 (n = 5–6, one donor missing KIR3DL1). DN refers to NKG2A-KIR- NK cells. Color coding: ILC1-like-derived NK cells (day 8: light green and yellow, day 14: dark green with yellow) and CD56bright-derived NK cells (day 8, light red, day 14 dark red). Height of the bars represent mean ± SEM. Levels of significance were calculated with a One-Way ANOVA with a multiple comparison post-test (Kruskal-Wallis test) (a, b) and an unpaired t test (Mann Whitney U) comparing both populations at the same time point. Data represent at least three independent experiments with each dot representing an individual donor (see Figure 6—figure supplement 1 for individual KIR/NKG2A expression), * p-value<0.05, ** p-value<0.01, *** p-value<0.001.