Fig 2: Natural killer (NK) cells require hypoxia-inducible factor-1a (HIF1a) for an optimal response to virus infection.(A) 1aKO or FL control mice were infected with 50K plaque forming unit (pfu) then monitored for weight loss over 7 days. Each data represents four mice from two independent experiments. (B) Splenic viral load in 1aKO or FL control mice were infected with 50K pfu then spleens harvested day 5 post-infection (pi). Data are pooled from four biologically independent experiments with a total of 5–7 mice in the indicated groups. (C) Number of bulk NK cells was quantified at days 1.5 and 3 pi from 1aKO or FL control mice. Data are from 2–3 independent experiments with 4–6 mice per group. (D) 1aKO or FL control mice were infected with 50K pfu and Ly49H+ NK cell expansion was determined at days 1.5, 3, 7, 14, and 24 pi. Data are from three independent experiments with 4–9 mice per group. (E) Quantification of 1aKO or Flox Ly49H- NK cells at days 1.5, 3, 7, 14, and 24 post murine cytomegalovirus (MCMV) infection. Data are from 3–4 independent experiments with 3–4 mice per group. Data depict mean ± SEM, with each data set containing data indicated number of mice per group from independent experiments. Unpaired t-test was performed on (A–D). Statistical significance indicated by n.s., no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 3: Cell division in hypoxia-inducible factor-1a (HIF1a) KO natural killer (NK) cells is normal but numbers are reduced.(A, B) 1aKO or FL control mice infected with murine cytomegalovirus (MCMV) received a dose of 50K plaque forming unit (pfu) and analyzed as indicated. (A) Ki67 expression with representative histograms (left) and frequency quantification (right) in 1aKO or FL control bulk NK cells at day 1.5 and day 3 post-infection (pi). Data are from three independent experiments with 5–7 mice per group. (B) Expression of Ki67 in Ly49H negative and positive NK cells shown in histogram (left) and frequencies quantified (right). Data are from two independent experiments with three mice per group. (C, D) Rag-?Rc mice analyzed for CFSE + splenic NK cells from 1aKO or FL control mice 4 days post transfer, showing representative histogram (C, left) and quantification of total proliferation (C, right), and frequencies (D). Data are from two independent experiments with four mice per group. (E) Rag-?Rc mice analyzed on day 14 since post splenocyte transfer for numbers of NK cells in the spleen from 1aKO or FL control mice. Data are from two independent experiments with three mice per group. (F, G) In vitro proliferation assays were done using human recombinant IL-2 at different concentrations and time points indicated below. (F) Representative histograms (left), quantification of total proliferated cells (middle), and number of CFSE peaks (right) of CFSE labeled NK cells from 1aKO or FL control mice stimulated with different concentrations of IL-2 for 6 days. (G) Numbers of NK cells were counted from (F) and graphed in cells per 1 ml. Data are from two independent experiments with four mice per group. Data depict mean ± SEM, with each data set containing data indicated number of mice per group from independent experiments. Unpaired t-test was performed on (A–G). Statistical significance indicated by n.s., no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig 4: Phenotype of NK cells from WT and PD-1-/- mice(A) Frequency of NK cells in the spleens of WT and PD-1-/- mice (*p<0.01 Mann-Whitney test, n = 18–20 mice/group data represent mean ± SD).(B) Expression of CD11b and CD27 on NK cells from WT (open boxplots) and PD-1-/- (shaded boxplots) mice (*p<0.01 Mann-Whitney test, n = 18–20 mice).(C) Expression of KLRG1 on NK cells from WT (shaded plot) and PD-1-/- (open plot ) mice (*p<0.01 Mann-Whitney test, n = 18–20 mice/group data represent mean ± SD).(D) Expression of CD62L on NK cells from WT and PD-1-/- mice.(E) Expression of DNAM-1 on NK cells from WT (shaded plot) and PD-1-/- (open plot) mice, bar graphs represent percent expressing cells and the mean fluorescent intensity of expression (*p<0.01 Mann-Whitney test, n = 18–20 mice/group data represent mean ± SD).(F) Expression of inhibitory Ly49 molecules and NKG2A on NK cells from WT (open bars) and PD-1-/- (shaded bars) mice (*p<0.01 Mann Whitney n = 18–20 mice/group data represent mean ± SD).(G) Expression of activating Ly49 molecules on NK cells from WT and PD-1-/- mice. (*p<0.01 Mann-Whitney test, n = 18–20 mice/group data represent mean ± SD).(H) Expression of Ly49D and Ly49H populations on NK cells from WT and PD-1-/- mice (*p<0.01 Mann-Whitney test, n = 18–20 mice/group data represent mean ± SD).