Fig 2: Linezolid-associated bone marrow suppression was not observed in macaques. Bone marrow was acquired from the sternum of all macaques at necropsy after 4 weeks LZD or LZD+IL-1Rn. A portion was used for flow cytometric analysis of erythroid progenitor populations and the rest for histopathology analysis. (A) A schematic of erythropoiesis indicating the stage of differentiation and corresponding expression levels of phenotyping markers (CD235a and CD71) and progenitor ratios (1:2:4:8). Included is the gating strategy to identify progenitor populations and an example of MitoTracker staining. (B) The ProE:EryC ratio (left) of each animal, including untreated TB only macaques (black) from other ongoing unpublished studies. A single uninfected animal (open circle) is shown as reference, the dotted line indicates the homeostatic ratio of 1:8. MitoTracker MFI (right) is shown for each macaque per treatment group. Kruskal-Wallis test was performed to compare the three groups (excluding uninfected macaque) and p-values are shown in the graphs. (C) Representative H&E images from sternal bone marrow acquired at necropsy, 20x (left) and 40x (right) are shown.

Fig 3: P. berghei sexual development occurs preferentially in reticulocytes.(A) UMAP of P. berghei colored by AP2-G expression strength. (B) ap-2g expression level in early stages, separated by host cell. (C) Proportion of ap-2g–positive cells in early stages, separated by host cell (box plot color). Individual dots are colored by organ (n = 6). (D) Gametocyte conversion rate after 28 hours of ex vivo culture of normocyte- or reticulocyte-enriched iRBC isolated from the spleen or blood. Samples from one organ were matched across host cell (n = 8, two-way ANOVA and Tukey’s post test). (E) Correlation between proportion of CD71+ cells 1 hpi and subsequent gametocyte rate after 28 hours of ex vivo culture. Same samples as in (D). (F) Gametocyte rate after ex vivo culture in nutrient-rich or nutrient-poor medium (n = 6, one-way ANOVA and Dunnett’s post test). (G) Gametocyte rate after ex vivo culture in minimal medium supplemented with varying concentrations of FCS (n = 4, one-way ANOVA and Dunnett’s post test). (C, D, F, and G) Center line, median; box limits, upper and lower quartiles; whiskers, minimum to maximum range; all points shown. *P < 0.05; **P < 0.01.

Fig 4: Cellular Iron Metabolism Is Remodeled upon T Cell Activation, and Iron Deficiency Disrupts T Cell Physiology(A) Left to right: Histograms of CD71 (Tfrc) expression on 4-OHT treated CD8 T cells from IRP1/2 floxed mice lacking (blue) or expressing Cre (red) at 24h (top) and 72h (bottom) after in vitro activation; relative CD71 MFI for CD8 T cells treated with 4-OHT measured by flow cytometry (72h after activation); relative CD71 MFI of 4-OHT treated compared to EtOH treated CD8 T cells (to control for effects of 4-OHT, 72h after activation). Mean+/- SD, Student's two tailed T-test, unpaired.(B) Percentage divided CD8 T cells of each genotype/EtOH or 4-OHT combination cultured with a titration of FeSO4, 72 h after activation. Percentage of divided determined as percentage of cells that had diluted out their CellTrace Violet signal once or more. Mean ± range. Two-way ANOVA, corrected for multiple comparisons, p values for difference between EtOH (control), and 4-OHT at each iron concentration.(C) Left to right: ATP content was measured using CellTiter Glo luminescent assay for equal numbers of WT CD8 T cells per condition and normalized to the control condition of each biological replicate. Total, glycolytic, and mitochondrial ATP production rates measured by Seahorse. Each data point is a biological replicate from a single mouse, representing the average of 2–5 technical replicate wells. Mitochondrial content (MitoTracker Green [MTG]) and mitochondrial inner membrane potential (MitoTracker Deep Red [MTDR]) were measured by flow cytometric analysis of 72-h activated divided cells and the ratio calculated. Mean ± range. One-way ANOVA, effect of iron, paired within each biological replicate.

Fig 5: Minihepcidin-Induced Serum Iron Deficiency Inhibits Immune Responses(A) Experimental design for investigation of the effect of combined minihepcidin injection and low iron diet on the primary immune response to immunization. Serum iron levels and liver hepcidin mRNA were measured 24 h after the last minihepcidin injection. Means ± SDs. Student’s 2-tailed t test, unpaired.(B) Left: representative flow cytometry plots showing frequency of OVA (SIINFEKL) tetramer-positive CD8 effector T cells (Tetramer+ CD44+ CD8 T cells) as a percentage of CD8s T cells. Right: number of splenic OVA-specific CD8 effector cells 7 days after immunization with AdHu5-OVA. Means ± SDs. Student’s 2-tailed t test, unpaired.(C) Number of splenic OVA-specific CD8 effector cells 7 days after OVA protein in adjuvant immunization. Means ± SDs. Student’s 2-tailed t test, unpaired.(D) Number of T follicular helper cells (PD-1+ CXCR5+ CD44+ CD4 T cells), germinal center (GC) B cells (IgD- CD95+ B220+ B cells), plasma cells (CD138+ IgD- cells), and anti-OVA IgG titer (a.u., arbitrary unit) 7 days after immunization with OVA in adjuvant. Means ± SDs. Student’s 2-tailed t test, unpaired.(E) Experimental design to determine whether parenteral iron supplementation can reverse the effect of minihepcidin injection; FAC = ferric ammonium citrate. Left: the frequency of antigen-specific splenic OT-I CD8 T cells; center: normalized relative mean fluorescence intensity (MFI) of surface CD71 expression by OT-I CD8 T cells 5 days after OVA and adjuvant immunization were detected with flow cytometry; right: liver hepcidin mRNA. Means ± SDs. One-way ANOVA with correction for multiple comparisons.