An NKp46⁺ cell–specific mouse model to study the oncogenic role of STAT5B^N642H in NK cells. (A) Schematic overview of the generation of N642H^NK/NK, STAT5B^NK/NK, and GFP^NK/NK mice. (B) pYSTAT5, STAT5A/B, and V5 immunoblot analysis of IL-2–cultured NK cells from GFP^NK/NK, STAT5B^NK/NK, and N642H^NK/NK mice. IL-2–cultured NK cells were either directly lysed (+IL-2), lysed after being starved off IL-2 for 3 hours (starv.), or lysed after IL-2 starvation and restimulation with IL-2 and IL-15 (restim.). β-actin served as a loading control. Absolute numbers of NK cells (CD3^–NK1.1⁺NKp46⁺) in blood (C) and spleen (D) of 8- to 12-week-old GFP^NK/NK, STAT5B^NK/NK, N642H^NK/NK, and Cre neg control mice (n ≥ 5 per genotype; mean ± standard deviation [SD]). (E) Absolute numbers of NK cells (lineage [Lin] negative [CD3^–CD19^–Gr1^–Ter119^–] CD122⁺ cells) in BM of 8- to 12-week-old GFP^NK/NK, STAT5B^NK/NK, N642H^NK/NK, and Cre neg mice (n ≥ 6 per genotype; mean ± SD). (F) Representative gating of NK-cell developmental stages among Lin^–CD122⁺ cells within the BM, including NK1.1^–NKp46^– NK-cell precursors (NKPs), NK1.1⁺NKp46^– immature (iNKs), and NK1.1⁺NKp46⁺ mature NK cells (mNKs) (left). Percentages of NKPs, iNKs, and mNKs among Lin^–CD122⁺ BM cells (n ≥ 6 per genotype; mean ± SD) (right). (G) Schematic overview on splenic NK-cell maturation stages based on CD27 and CD11b expression (left). Percentages of CD27⁺CD11b^–, CD27⁺CD11b⁺ and CD27^–CD11b⁺ NK cells in the spleens of GFP^NK/NK, STAT5B^NK/NK, N642H^NK/NK, and Cre neg mice (n ≥ 6 per genotype; mean ± SD) (right). (H) Apoptosis staining of splenic NK cells from GFP^NK/NK, STAT5B^NK/NK, N642H^NK/NK, and Cre neg mice (n ≥ 4 per genotype; mean ± SD). Levels of significance were calculated using the 1-way analysis of variance (C-H). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.