Figure 2: IL-12 induces eNK cells in lungs of naive Rag2^−/−Il2rg^−/− mice.

(a) Representative plots and absolute numbers of CD45⁺CD3⁻CD122⁺CD49b⁺ cells in lungs from IL-12 or Fc-treated (Ctrl) WT or Rag2^−/−Il2rg^−/− mice. The expression of NK1.1 on CD45⁺CD3⁻CD122⁺CD49b⁺ cells in lungs from IL-12 or Ctrl-treated WT or Rag2^−/−Il2rg^−/− mice is shown. Data represent five experiments (n≥4 mice per group per experiment). (b) Cytospin ( × 100) of sorted CD45⁺CD3⁻CD122⁺CD49b⁺ cells from lungs of IL-12-treated WT or Rag2^−/−Il2rg^−/− mice stained with May–Grünwald–Giemsa solution. Two independent experiments were performed. Scale bar, 20 μm. (c) Expression levels of Eomes, NKG2D, NKp46 and DNAM-1 on the NK1.1⁺ subsets of CD45⁺CD3⁻CD122⁺CD49b⁺ cells isolated from lungs of Ctrl (NK) and IL-12-treated WT mice (NK1.1⁺ NK+IL-12), the NK1.1^low subset of CD45⁺CD3⁻CD122⁺CD49b⁺ cells isolated from lungs of IL-12-treated WT mice (NK1.1^low NK+IL-12) and eNK cells from Rag2^−/−Il2rg^−/− mice. Three independent experiments were performed with at least three mice per group. (d) Ki-67 and Bcl-2 expression levels were quantified in lung NK, NK1.1⁺ NK+IL-12, NK1.1^low NK+IL-12 and eNK cells. Data shown represent three independent experiments (n=3–5 mice per group per experiment). (e) Leukocytes isolated from lungs of WT and Rag2^−/−Il2rg^−/− mice either treated with IL-12 or Ctrl were cultured for 4 h with PMA/ionomycin and CD45⁺CD3⁻Gr-1⁻ cells were analysed for their IFN-γ production. NK1.1 and NKp46 expression was examined on IFN-γ expressing cells. (f) NK cells and eNK cells were isolated from lungs of WT mice and Rag2^−/−Il2rg^−/− mice, respectively. Their cytotoxicity was tested using YAC-1 cells at an effector:target ratio of 6:1. Data are representative of two independent experiments with three mice per group each. Data are expressed as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001 as determined by unpaired Student’s t-test with Welch’s correction or one-way analysis of variance with Bonferroni post-test.