Emergence and generation of tNK cells in the mouse embryo. (A) Representative FACS profiles of Lin⁻CD3⁻CD25⁻CD122⁺NK1.1⁺Ly49D⁻IL-7Rα⁺ tNK cells in the fetal thymus. (B) Total number of fetal thymic cells and (C) total number of tNK cells. Data represent mean ± SD values from 5 to 13 thymi analyzed in 14.5- to 18.5-day-old fetuses from 2 to 3 litters. Text above the plots and arrows indicate the gating. Numbers in FACS profiles show mean frequencies of populations within the indicated gates of total fetal thymocytes. Profile from the adult thymus is included as a control; 0 indicates the values below the detection level. Five Lin⁻CD122⁺NK1.1⁻DX5⁻ NKPs were sorted from the liver from 18.5-day-old fetuses and cultured on OP9-DL1 stroma for 21 days with KL, IL-7, FL, and IL15. Double-negative fetal thymocytes (CD4/CD8-depleted) were used as a positive control. Generated clones were evaluated by FACS for the presence of tNK cells (TCRβ⁻CD25⁻Ly49D⁻NK1.1⁺CD122⁺IL-7Rα⁺) and BM-dependent cNK (TCRβ⁻NK1.1⁺CD122⁺IL-7Rα⁻) cells. (D) Representative FACS profiles of cells generated from fetal liver NKPs and CD4⁻CD8⁻ thymocytes cultured on OP9-DL1 stroma. The specific gates are indicated in the text above the plot and by arrows. (E) Proportion of total positive wells and wells containing thymic NK (tNK) and conventional NK (cNK) cells generated from E18.5 fetal liver NKPs. Data from one experiment, 20 wells were analyzed. *P < .05, **P < .01, and ***P < .0001.