Fig 2: Iron Uptake via the Transferrin Receptor Is Cell-Intrinsically Essential for Immune Responses(A) Experimental design for establishing mixed bone marrow chimeras to investigate cell-intrinsic effect of TfrcY20H/Y20H allele on immune response in vivo. Data from TfrcY20H/Y20H ; WT chimeras are displayed in red, whereas data from WT;WT chimeras are in blue.(B) Ratio of the frequencies of CD45.2:CD45.1 cells within peripheral blood CD8 T cells, CD4 T cells, and B cells at 65 days after establishment of chimeras, determined by flow cytometry. Means ± SDs. Student’s 2-tailed t test, unpaired.(C) Comparison of the ratio of the frequencies of CD45.2:CD45.1 cells within naive and effector lymphocyte populations within each chimeric mouse, 72 days after establishment of chimeras and 1 week after MVA-OVA immunization. The data are displayed for naive (CD44-) and effector tetramer+ splenic CD8 T cells, dLN naive (CD44-) CD4 T cells and T follicular helper cells (PD-1+ CXCR5+ CD44+ CD4 T cells), and dLN naive follicular (IgD+, CD95-) and GC B cells (IgD- CD95+ GL7+ B cells). Student’s 2-tailed t test, paired.

Fig 3: SFPQ promotes the malignant phenotype of lung NSC cancer MSCs via regulating CD44v6 expression. (A) Control and lung NSC cancer MSCs isolated from 4 normal lung cell lines (Con210, Con205, Con215, Con249) and 4 lung NSC cancer cell lines (Can661, Can522, Can838 and A549) were used to evaluate the CD44v6 levels, Which were quantified with RT-PCR (left, control vs IPF: CD44 p<0.01; CD44v6 p<0.05) and western blot analysis (middle). Densitometry analysis of WB was shown in the right graph. (B) localizations of SFPQ and CD44v6 were analyzed by the confocal microscopy with anti-SFPQ (Abcam, USA) and anti-CD44v6 (Abcam, USA) in lung NSC cancer MSCs. CD44v6 is located in both cytoplasm and nucleus. Scale Bar=20µm. (C–E) Lung NSC cancer MSCs isolated from A549 and Can 661 cell lines were transduced with scramble or SFPQ shRNA or CD44v6 shRNA Lenti virus for 48 hours, and the cells were used for following analysis: (C) Colony Assay (left), Cell proliferation assay (middle) and Cell invasion assay (right). Colony number, cell proliferation rate and invaded cells in lung NSC cancer MSCs transduced with SFPQ shRNA or CD44v6 shRNA were reduced when compared with the control group. (D) SFPQ, CD44, and CD44v6 levels were analyzed by RT-PCR (left) and western blot analysis (middle) on the same cell groups. Densitometry values are shown in the right hand graph. (E) Expression levels of Sox2, Ki67 and MMP2 were analyzed with RT-PCR (left) and western blot analysis (middle) on the same cell groups. Densitometry analysis was shown in the graph on the right. (F) mRNA level of PARP1 was analyzed with RT-PCR (left), and western blot analysis (middle) of PARP1 protein and phosphorylated ?H2AX were conducted on the same cell groups. Densitometry analysis was shown in the right graph. All data are shown as mean ± S.E. (n=3 independent experiments).

Fig 4: Serum Iron Deficiency Suppresses Adaptive Immune Responses to Influenza Infection, Worsening Lung Inflammation and Preventing Recovery of Weight(A) Experimental scheme for investigation effect of minihepcidin injection on the outcome of infection with 0.08 hemagglutination units of influenza virus A/X-31 (H3N2).(B) Representative plots of the frequency of influenza nucleoprotein (NP)-specific CD8 effector cells as a percentage of CD8 T cells in the spleens of mice 8 days post-infection. Numbers of NP-specific CD8 effector cells in the spleen and lung at 8 days post-infection. Number of splenic granzyme B+ CD8 T cells. Means ± SDs. Student’s 2-tailed t test, unpaired.(C) Mediastinal lymph node T follicular helper cell number (PD-1+ CXCR5+ CD44+ CD4 T cells) and frequency of CD44+ antigen-experienced CD4 T cells as a percentage of CD4 were identified by flow cytometry. Representative plots of the frequency of mediastinal lymph node GC B cells as a percentage of total B cells and the number of mediastinal lymph node GC B cells at day 8 post-infection (IgD- CD95+ GL7+ B cells). Means ± SDs. Student’s 2-tailed t test, unpaired.(D) Half-maximal effective concentration (EC50) of neutralizing antibody from serum on day 10 post-infection. LOD, limit of detection. Means ± SDs. Student’s 2-tailed t test, unpaired.(E) Influenza NP RNA in homogenates of a whole lung lobe taken at day 10. Means ± SDs. Student’s 2-tailed t test, unpaired.(F) Leder staining of representative lung sections from mice on day 10 post-influenza infection from each group; 200×, scale bar 20 µm. Polymorphonuclear cells stained with pink cytoplasm indicated by arrows.(G) Lung inflammation and neutrophil infiltration scoring. Means ± SDs. Kruskal-Wallis test with correction for multiple comparisons.

Fig 5: Ablation of Hhex in GC B cells impairs memory B cell differentiation.(a) Experimental schematic. Hhex+/+ (WT), Hhexflox/+ (Het), and Hhexflox/flox (KO) S1pr2-ERT2cre TdTomato mice were infected with LCMV, treated with tamoxifen beginning at day 4, and then analyzed at day 30 p.i. (b) Representative FACS plots of the percentage of S1pr2-Tomato+ cells among splenic B220+IgDloGL7-CD38+ MBCs in the groups indicated in a. Data are representative of 4 independent experiments with at least 4 mice per group. (c, d) Number (c) or percentage (d) of GC B cells (B220+IgDloGL7+CD95+S1pr2-Tomato+) and MBCs (B220+IgDloGL7-CD38+S1pr2-Tomato+) in Hhex WT, Het, and KO mice at day 30 post LCMV infection. Data are representative of 4 independent experiments with at least 4 mice per group. Statistical analyses were performed using the ordinary one-way ANOVA with two-sided Dunnett multiple comparison testing (****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (e) Percentage of B cells that are GC B cells (left) and MBCs (right) in Hhex Het and KO mice at days 15 and 45 post LCMV infection. Data are representative of 2 independent experiments with 4–6 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test (****, p < 0.0001). Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (f) Percentage of MBCs that were CD62L- (left) and CD44+CD62L+ (right) in Hhex Het and KO mice at day 30 post LCMV infection. Data are pooled from 2 independent experiments with at least 2 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (g) Percentage of live splenic cells that are CD138+S1pr2-Tomato+ in Hhex WT, Het, and KO mice at day 30 post LCMV infection. Data are representative of 2 independent experiments with at least 4 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (h) Quantification of anti-LCMV IgG in the serum of Hhex WT, Het, and KO mice at day 30 post LCMV infection. Data are representative of 2 independent experiments with at least 4 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (i) Percentage of GC B cells that were BrdU+ (left) and Casp3+ (right) in Hhex Het and KO mice at day 30 post LCMV infection. Data are pooled from 2 independent experiments with 4 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (j) Representative FACS plot (left) and percentage (right) of CD138+ cells that are CD45.2+S1pr2-Tomato+ at day 5 post challenge with LCMV. Equivalent numbers of splenic CD45.2+ T and B cells from Hhex Het and KO LCMV immune mice at day 30 p.i. were transferred to naïve CD45.1+ recipients one day prior to challenge. Data are representative of 2 independent experiments with 3 mice per group. Statistical analyses were performed using the unpaired two-tailed Student’s t-test. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. (k) Percentage of CD138+ cells (left) and GC B cells (right) that were CD45.2+S1pr2-Tomato+ at day 5 post challenge with LCMV when normalized to the percentage of MBCs present in the transferred cells. Data are pooled from 2 independent experiments with 3 mice per group. Scatter plots indicate mean (middle line) with error bars indicating standard error mean. See also Figure S6.