Protection of mice mediated by memory NK cells is IFNγ independent.

(A-B) Mouse infections are carried out as in the scheme in Fig 3A. Organs were collected at 24h and 40h post-infection for flow cytometry analysis and ELISA assays. (A) Flow cytometry analysis of IFNγ expression in CD45.1⁺ endogenous NK cells and CD45.2⁺ transferred NK cells (hatched) into both mice having received D21PBS NK cells (black) and mice having received D21SPN NK cells (red). Violin plot where each dot represents an individual mouse, lines are the median. Data are pooled from two repeats with n ≥ 2 mice/group. (B) IFNγ ELISA assays of lung supernatants from infected mice having received either D21PBS NKs (black) or D21SPN NKs (red) at 24h and 40h post-infection. Bars are the mean of at least four experiments with n ≥ 4 mice/group, each dot represents an individual mouse and error bars are the standard error of the mean (SEM). (C) Mouse infections are carried out as in the scheme in Fig 3A, with the exception that WT mice were used as donor mice and Ifngr KO mice as recipient mice. Bacterial counts at 40h post-infection in the lungs and spleen of Ifngr KO mice having received D21PBS WT NK cells (black symbols) or D21SPN WT NK cells (red symbols). Box plots where each dot represents an individual mouse, lines are the mean, error bars show min to max and dotted lines represent limit of detection. Data are pooled from two repeats with n ≥ 4 mice/group. (D) WT and Ifngr KO mice were intranasally infected with a lethal dose of S. pneumoniae (1x10⁷ CFU). Lungs were collected at 40h post-infection for CFU counts and flow cytometry analysis. Percentages of neutrophils (Ly6G⁺ CD11b⁺), NK cells (NK1.1⁺ CD3^-) and CD69⁺ NK cells. Box plots where each dot represents an individual mouse, lines are the mean, error bars show min to max and dotted lines represent limit of detection. Data are pooled from two repeats with n ≥ 3 mice/group ns, not significant. * p < 0.05, ** p < 0.01. 2way ANOVA (A,B) and Mann-Whitney (C,D) tests for statistical significance.