Fig 2: Iron uptake by NK cells upon FV infection.C57BL/6 mice were infected i.v. with FV for 7 days or used as uninfected, naive control. Transferrin uptake assay and controls (no transferrin, competition with holo-transferrin, uptake at 4 °C) are shown as representative histograms for splenic NK cells (a). Experiments were repeated independently twice with similar results. Transferrin uptake of NK cells (b) and CD71+ NK cells (c) from spleen and bone marrow (BM) are displayed as bar graphs ± SEM. Statistically significant differences were analysed by two-tailed Mann–Whitney test (b) and two-tailed unpaired t-test (c). Minimum of six mice per group from two independent experiments was used for the analysis. An experimental overview of the induction of low serum iron concentrations using mini-hepcidin (mHep) is shown in box d. Serum iron was analysed in e with a minimum of five mice from two independent experiments. Data are presented as mean values ± SEM. A representative histogram of CD71 expression on NK cells of FV-infected vehicle-treated and FV-infected mHep-treated mice is shown in f. Experiments were repeated independently twice with similar results. Activation of NK cells was analysed by the early activation marker CD69 (g) and the percentage of IFN?+ NK cells (h) after mHep treatment are displayed as bar graphs ± SEM. At least six mice from two independent experiments were used. IFN? concentration in mouse serum (i) as well as IL-12, IL-15 and IL-18 concentrations in spleens (j) were analysed using a custom-made Legendplex kit. Cytokines were measured in a minimum of seven mice from two independent experiments (i) or four mice from one experiment (j) were used for the analyses. In i serum from seven mice from two independent experiments were used. At least four mice were used to measure cytokines in the spleen from one experiment (j). Data are presented as mean values ± SEM (i, j). mRNA levels of cytokines were measured in splenocytes by quantitative real-time PCR and are displayed as spider plot (k). Statistically significant differences were analysed by an ordinary one-way ANOVA within groups of bone marrow or spleen. Significances are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file. ns not significant.

Fig 3: P. chabaudi chabaudi AS has no erythrocyte preference (A–C) and P. vinckei vinckei S67 prefers normocytes (D–F) in vivo. Flow cytometry data of peripheral blood taken from infected mice before the peak of infection (two days shown). Reticulocytes (blue circles) and normocytes (red circles) are defined as CD71+ CD98+ cells and CD71- CD98- cells, respectively. Infected cells are Hoechst-positive. (A, D) Data shown are infected reticulocytes and infected normocytes, as a percentage of total erythrocytes. (B, E) Data normalized to the relative frequencies of reticulocytes and normocytes, by expressing infected reticulocytes and normocytes as a percentage of total reticulocytes and total normocytes, respectively. (C, F) The in vivo tropism index is calculated as the ratio of percentage of infected reticulocytes (out of total reticulocytes) to percentage of infected normocytes (out of total normocytes). The mean tropism indices (n = 5 mice) are shown. Dotted lines at tropism index = 1 represent no erythrocyte preference. All error bars are standard deviations. ns, not significant; *p < 0.05; **p < 0.01.

Fig 4: Increased NK cell blastogenesis and augmented metabolic activity after acute FV infection.Splenic and bone marrow (BM) NK cells from naive mice or mice FV-infected for 7 days were analysed for cell size by analysing the FSC on NK cells. NK cells were gated on lymphocytes, single cells, viable, CD3-, NK1.1+ CD49b+ cells. A representative histogram of the FSC on NK cells from both organs are shown in a and displayed as a bar graph ± SEM in b. Statistically significant differences were analysed between naive and FV groups by a two-tailed unpaired t-test within the bone marrow or spleens. Experiments were repeated independently twice with similar results. The correlation between activated NK cells and NK cell size (FSC-A) was analysed and displayed in c. NK cells from naive mice are displayed in open circles whereas NK cells from FV-infected mice are displayed in grey circles. At least six mice per group from two independent experiments were used. FSChigh and FSClow NK cells were analysed for the expression of CD98 and CD71 (d). NK1.1 expression was analysed on FSChigh and FSClow NK cell population (d, right-hand side). Experiments were repeated independently twice with similar results. Differences between FSChigh and FSClow NK cells regarding CD71 and CD98 are shown in a bar graph in e. At least 14 mice from four independent experiments were used for the analysis. Statistically significant differences between FSChigh and FSClow NK cells were analysed with a two-tailed Mann–Whitney test within the bone marrow or spleens. Data are presented as mean values ± SEM. Representative histogram of cMyc of NK cells in the spleen and bone marrow are shown in f. Experiments were repeated independently twice with similar results. The MFI of cMyc on NK cells (g) is displayed as bar graphs ± SEM and were analysed by a two-tailed unpaired t-test. At least six animals from two independent experiments were used for the analysis. cMyc+ and cMyc- NK cells were further analysed for the cell size by measuring the FSC (h) and for proliferation by detecting KI-67 (i). At least six mice per group from two independent experiments were used. Data are presented as mean values ± SEM. Statistically significant differences in bone marrow or spleen were analysed with a two-sided unpaired t-test and displayed as *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig 5: Metabolism of NK cells after acute MCMV infection.C57BL/6 mice were infected with MCMV (Smith strain) or used uninfected and spleens were harvested at 1.5 days. NK cells (CD3−NKp46+) were analysed for activation (CD69) and cell size (FSC-A) (a). Ex vivo restimulated NK cells were analysed for IFNγ (b). Cytotoxicity was detected by granzyme B expression (c). A representative histogram and bar graphs of CD71 and CD98 are shown in d and e. A minimum of four (e) or seven (a–d) mice per group were used and analysed by a two-sided unpaired t-test and displayed ± SEM. **p < 0.01, ***p < 0.001. f Volcano plots of nutrient transporters and glycolysis-related genes displaying differences in relative gene expression after MCMV infection. The plot represents the −log10 of the p-value against log2 fold change. The horizontal red line represents a p-value <0.05. Source data are provided as a Source Data file.