Fig 2: H1Y98F-VLP elicits robust CD4+ T cell responses with enhanced recruitment of antigen-specific CD4+ T cells to the bone marrow.Splenocytes and BM immune cells were stimulated for 18 h with 2.5 µg/ml H1WT-VLP. Flow cytometry was used to quantify H1-specific CD4+ T cells in a splenocytes isolated at 28 days post-vaccination (3 µg) and in b splenocytes and c BM immune cells at 28 days post-boost (0.5 µg/dose). Background values obtained from non-stimulated samples were subtracted from values obtained following stimulation with H1WT-VLP. a–c The left panel shows the mean frequency (±SEM) of CD4+ T cells expressing CD44 and at least one of IL-2, TNFα or IFNγ. The right panel shows the individual cytokine signatures of responding CD4+ T cells obtained by Boolean analysis. Color-matched pie charts depict relative distributions of cytokine-producing CD4+ T cell populations and IFNγ+ populations are highlighted by a green arc. Statistical significance was determined by Kruskal–Wallis test with Dunn’s multiple comparisons (total response) or two-way ANOVA with Tukey’s multiple comparisons (cytokine signatures) (*p < 0.033, **p < 0.01, ***p < 0.001). Spearman’s rank correlation technique was applied to evaluate the relationship between (d) the frequency of H1-specific CD4+ T cells in the spleen and BM, e the frequency of IL-2+TNFα+IFNγ- CD4+ T cells in the spleen and IgG avidity index (4 M urea) and f the frequency of IL-2+TNFα+IFNγ− CD4+ T cells in the BM and HI titer (*p < 0.033, **p < 0.01).

Fig 3: Virus-Specific T Cells Accumulate in WATVirus-specific T cells in adipose tissue and spleen were quantified at multiple times after acute infection by tetramer staining and ICCS.(A) Representative dot plots identify DbGP33+CD8+ T cells (left) and IAbGP67+CD4+ T cells (right) before infection and at days 8 and 70 post infection. The numbers indicate the frequency of the tetramer+ cells among all live cells in the spleen or among leukocytes isolated from the fat pads.(B) The number (mean ± SEM) of tetramer-positive T cells per spleen or fat pads at the indicated times post-infection. Data are drawn from two independent experiments with three to five mice per group.(C) Representative dot plots show GP33-specific CD8+ T cells or GP61-specific CD4+ T cells expression of IFNg after peptide stimulation. The analyses are from the indicated days after infection, with numbers indicating the percentage of IFNg+ cells among all cells.(D) The number (mean ± SEM) of IFNg+ epitopespecific T cells per tissue in spleen and fat pads as measured using ICCS assay at the indicated times post-infection. Data represent two experiments with five to nine mice per group.(E) T cell co-expression of IFNg with TNF or IFNg with IL-2 in the spleen or fat pads at day 8. CD8+ T cells were stimulated with GP33–41 peptide;CD4+ T cells were stimulated with GP61–80 peptide.See also Figure S1.

Fig 4: Aiolos deficiency results in acquisition of cytotoxic features by CD4 T cells in vivo.Naïve WT or Aiolos-deficient (Ikzf3-/-) mice were infected intranasally with 30 PFU influenza (A/PR8/34; “PR8”). After 8 days, draining lymph nodes (DLN) were harvested and viable CD4+ effector populations were analyzed via flow cytometry. a–c Analysis of of T-bet and Eomes expression by influenza nucleoprotein (NP)-specific CD4+. Data are compiled from 7 independent experiments (n = 22 ± s.e.m; **P < 0.01; two-sided, unpaired Student’s t test). d Single-cell suspensions from the DLN were incubated in culture medium in the presence of protein transport inhibitors for 3 h. Perforin production by Eomes+ populations was analyzed via flow cytometry. Data are compiled from four independent experiments (n = 11 ± s.e.m; *P < 0.05, **P < 0.01; two-sided, unpaired Student’s t test). e CD4+ NP-specific cells were enumerated. Data are compiled from 7 independent experiments (n = 22 ± s.e.m; *P < 0.05; two-sided, unpaired Student’s t test). f CD25 (IL-2Ra) surface expression was evaluated on viable antigen-specific CD4+CD44+CD62L-Foxp3- (effector) T cells from the lung-draining lymph nodes (DLN) via flow cytometry. Percentage of CD25+ cells are shown. Data are compiled from three independent experiments (n = 9 ± s.e.m; **P < 0.01, ****P < 0.0001; two-sided, unpaired Student’s t test). g Production of IL-2 by CD4+ T cell populations was analyzed in WT vs. Aiolos-deficient cells from the DLN by flow cytometry. Cells were cultured in the presence of PMA and Ionomycin and protein transport inhibitors for 3 h prior to analysis. Data are compiled from two independent experiments (n = 7 ± s.e.m; two-sided, unpaired Student’s t test). Source data are provided as a Source Data file.

Fig 5: Minihepcidin-Mediated Serum Iron Deficiency Inhibits the CD8 T Cell Response to a Diverse Range of Immunizations and Impairs the Quality of CD4 T Cell Responses.(A) Experimental scheme to examine effect of minihepcidin injection on immunization.(B) Antigen-specific T cell populations quantified by flow cytometry 7 days after immunization. Left to right: number of splenic endogenous OVA-specific CD8 T cells after AdHu5-OVA immunization, frequency of endogenous OVA-specific CD8 T cells as a percentage of total CD8s in peripheral blood after AdHu5-OVA immunization, number of splenic OT-I OVA-specific CD8 T cells after MVA-OVA immunization, number of splenic OT-I OVA-specific CD8 T cells after OVA and adjuvant immunization, and frequency of endogenous vaccinia (B8R epitope)-specific CD8 T cells as a percentage of total CD8s, resolved by IFN? production after ex vivo peptide restimulation, induced by MVA-OVA immunization. Means ± SDs. t test. Student’s 2-tailed t test, unpaired.(C) Left to right: relative MFI of IFN? and TNF-a for OT-I effector cells producing the respective cytokine, MFI normalized to average of vehicle group; percentage of OT-I effector cells that secrete IL-2. Cytokine-producing cells resolved by intracellular cytokine staining after ex vivo restimulation of splenocytes from mice with SIINFEKL peptide 7 days after MVA-OVA immunization. Means ± SDs. t test. Student’s 2-tailed t test, unpaired.(D) Frequency of endogenous vaccinia-specific IFN?, TNF-a, or IL-2 producing CD40L+ CD4 Th1 effector T cells as a percentage of total CD4s, resolved by intracellular cytokine staining after ex vivo restimulation of splenocytes with MVA-OVA-pulsed dendritic cells. Means ± SDs. t test. Student’s 2-tailed t test, unpaired.(E) Number of splenic OT-II T follicular helper cells induced by OVA and adjuvant immunization. Frequency of splenic TNF-a+ and IL-2+ OT-II effector cells induced by OVA and adjuvant immunization as a percentage of total OT-II CD4 T cells after ex vivo restimulation with peptide. Frequency of splenic GC B cells as a percentage of B cells after OVA and adjuvant immunization. All 7 days post-immunization. Means ± SDs. t test. Student’s 2-tailed t test, unpaired.