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: 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 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.