Figure 5. Eomes^hiNK^neg cells generate functional NK cells in vitro.

a, Representative flow cytometry plots showing ILCP- and Eomes^hiNK^neg cell-derived NK1.1^–ICOS⁺ ILC2/ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1s and NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK cells on day 7 of co-culture with OP9 cells with IL-2, IL-7 and SCF. b, Pie chart showing the average frequency of NK1.1^–ICOS⁺Rorc-Thy1.1^– ILC2s, NK1.1^–Rorc-Thy1.1⁺ ILC3s, NK1.1⁺ICOS^–TRAIL⁺KLRG1^– ILC1s, NK1.1⁺ICOS^–TRAIL^–KLRG1⁺ NK cells and NK1.1⁺ICOS^–TRAIL^–KLRG1^– undifferentiated cells in ILCP or Eomes^hiNK^neg cell-derived progenies at day 7 as in a. Data are representative of three independent experiments. c, Bar graph showing the expression of TRAIL and KLRG1 on NK1.1⁺ICOS^– cells derived from single ILCP (n=37) or Eomes^hiNK^neg cells (n=19) at day 7 as in a. d, Intracellular staining of Tbet and perforin in ILCP- or Eomes^hiNK^neg cell-derived NK1.1⁺ICOS^–Nkp46⁺ cells at day 7 of culture as in a. e, Percentage of efficiency of killing of YAC1 tumor cells cultured at 1:1 ratio with NK1.1⁺ICOS^– cells derived from Eomes-GFP⁻NK1.1⁺IL-7Rα⁺ ILC1s (n=3), α4β7⁺CD244⁺IL-7Rα⁺CD90⁺PD1⁺ ILCPs (n=3), Eomes^hiNK^neg cells (n=3) or Eomes-GFP⁺NK1.1⁺ NK cells (n=3) sorted from the BM of Eomes^GFP mice, co-cultured with OP9 cells with IL-2, IL-7, SCF for 6 days and stimulated with IL-15 overnight. Statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test. Data represent mean ± s.e.m. ***P<0.001, ****P < 0.0001