Lack of STAT3 does not influence NK cell development, maturation, and proliferation. (A) Bar graphs depict absolute numbers of splenic NK cells (CD3⁻NK1.1⁺NKp46⁺) of Stat3^Δ/ΔNcr1-iCreTg mice and Stat3^fl/fl controls as determined by flow cytometry. Five independently conducted experiments with n ≥ 14 per genotype are pooled. Data represent mean ± standard error of the mean (SEM). (B) Flow cytometric gating scheme for identification of NKPs, iNKs, and mature NKs (mNKs) in the bone marrow. (C) For the analysis of NK cell development, BM cells of Stat3^Δ/ΔNcr1-iCreTg mice and Stat3^fl/fl controls were counted and analyzed via flow cytometry by gating on Lin⁻ (CD3, CD19, Ter119, Ly6C/G) CD122⁺ cells. NKPs are defined as Lin⁻CD122⁺NK1.1⁻DX5⁻, iNKs as Lin⁻CD122⁺NK1.1⁺DX5⁻, and mNKs as Lin⁻CD122⁺NK1.1⁺DX5⁺. Bar graphs depict mean ± SEM (n ≥ 6 per genotype). (D) Stat3 deletion efficiency of MACS-purified NK cells was analyzed ex vivo by qPCR. Data represent mean ± SEM (3 replicates per group); gene expression is calculated relative to the housekeeping gene (HKG) Gapdh and normalized to Stat3^fl/fl. (E-F) For detection of NK cell maturation stages, splenocytes of Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were analyzed for CD27 and CD11b expression after previous gating on CD3⁻NK.1.1⁺ cells. Statistical analyses are summarized (F) (n ≥ 8 per genotype). (G) Splenocytes of Stat3^fl/fl and Stat3^Δ/ΔNcr1-iCreTg mice were stained for individual NK cell receptors after gating on NK cells (CD3⁻NK1.1⁺). One representative histogram is shown (4 independent experiments, with n ≥ 10 in total per genotype). There are significantly more Stat3^Δ/ΔNcr1-iCreTg NK cells expressing DNAM-1: 57 ± 0.8 (Stat3^fl/fl) vs 66 ± 1.5 (Stat3^Δ/ΔNcr1-iCreTg) %DNAM-1⁺ NK cells. Statistics are included in supplemental Figure 1D.