Fig 2: 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.

Fig 3: The effects of Rab11A silencing on cell surface levels of c-fms and RANK receptors in RAW-D cells stimulated with RANKL (300 ng/mL). (A) The endogenous levels of c-fms, RANK, and NFATc1 in Ctrl or Rab11A si-treated RAW-D cells stimulated with RANKL for 0 (left panels) or 3 days (right panels). (B) The endogenous levels of c-fms, RANK and NFATc1 in Ctrl or Rab11A si-treated BMMs stimulated with RANKL for 0 (left panels) or 3 days (right panels). (C,D) Quantitative analyses of Western blot for c-fms, RANK, NFATc1, in RAW-D cells (C) or BMMs (D). GAPDH was used as an internal control. * p < 0.05. (E) Upper: The biotinylated fractions were subjected to immunoblotting with anti-mouse c-fms and anti-mouse RANK antibodies, and whole cell lysates (WCLs) were subjected to immunoblotting with anti-GAPDH antibody as a loading control in RAW-D cells following RANKL stimulation for 3 days. Lower: Quantitative analyses of Western blot for c-fms, RANK. * p < 0.05. (F) Flow cytometric analyses of TfR using Ctrl or Rab11A siRNA-transfected RAW-D cells, followed by RANKL stimulation for 2 days. Data are representative of three independent experiments.

Fig 4: CD169 Reduces FVC Spread to Erythroblasts in the Red PulpFluorescence-activated cell sorting (FACS) plots showing the gating strategy and graphs depicting the numbers of FVC-infected cells, erythroblasts, B cells, and ratios of infected erythroblasts and B cells in splenocytes of B6 and CD169-/- mice (n = 4) 5 days after s.c. (A–E) or r.o. (F–J) administration (2,500 SFFU). Erythroblasts (CD71+ Ter119+ CD19-), B cells (CD19+), and FVC-infected cells (Glycogag+) were identified using the indicated fluorophore conjugates. p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line.

Fig 5: 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.