Fig 2: PA T encounter with DCs results in limited activation involving upregulation ofIl2andBcl2. (A) PA T cells were co-cultured with DC1940 for 24 h, then CD4+ populations were gated into CD69+ and CD69- cells by FACS. The expression of CD103, CXCR5, CD62L, CCR9, CCR7, CD127 and CD83 between CD69+ and CD69- cells were compared. Three replicates in each group (n = 3), and results are representative of three independent experiments (N = 3). (B) CD69+ and CD69- cells cultured in complete RPMI for 24 h without additional treatment, then the cytokines released to the supernatant were measured by ELISA. Pooled data from three independent experiments are shown. (C) Gene expression of Il7r, Il2 and Bcl2 was determined by QPCR in PA T cells or in CD69 positive or negative populations purified by FACS sorting. Three replicates in each group (n = 3), and results are representative of three independent experiments (N = 3). GAPDH was used for normalization

Fig 3: 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 4: 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.