Fig 2: Homogeneous SCCs Elicit a Strong Immune Response(A) Flow cytometry analysis of the Granzyme B and CD107a population in total TCRß+ TILs on day 19. n = 4–5; data are mean ± SEM. **p < 0.01 for Granzyme B+ CD107a+ TILs, two-way ANOVA followed by Bonferroni’s post hoc test.(B) Flow cytometry analysis of interferon-? (IFN-?) in total TILs on day 19. n = 4–5, *p < 0.05, Kruskal-Wallis test followed by Dunn’s multiple comparisons test.(C) CYT score derived from RNA-seq data of sorted CD8+ TILs from UVB-irradiated B2905 and SCC 2 tumors on day 15. *p < 0.05, Mann-Whitney U test.(D) Pearson correlation between CYT score and weights of tumors in Figure 3C.(E) Quantitation of total CD8+ TILs in the indicated tumors. Four sections from each tumor and three tumors derived from each cell line were examined. A significant difference was observed between parental cells and SSC 2 but not between parental cells and UVB. Data are mean ± SEM. *p < 0.05, one-way ANOVA followed by Tukey’s post hoc test.(F) Relative quantitation of the average percentage of CD8+ TILs in the tumor core versus the margin of the tumors described in (E). Data are mean ± SD.(G) Representative immunohistochemical stain for CD8 in slides taken from tumors derived by parental, UVB and SCC 2 on day 10 after cell inoculation. The scale bars represent 100 µM.(H) Immunofluorescence stains of CD3 and Foxp3 in tumors derived from B2905 parental, UVB, and SCC 2, 16, and 11 on days 10-11 after cell inoculation. 3–4 sections from each tumor and two tumors derived from each cell line were examined. The scale bars represent 200 µM.(I) Relative quantitation of the percentage of Foxp3+ of CD3+ TILs described in (H). Data are mean ± SEM. *p < 0.05, one-way ANOVA followed by Tukey’s post hoc test.See also Figure S5.

Fig 3: Etifoxine decreased leukocyte infiltration and brain inflammation following MPTP treatment. C57BL/6 mice were administered etifoxine or vehicle immediately after saline or MPTP administration. After day 7, suspensions were prepared from substantia nigra and striatal tissues. (A) Flow cytometry plots showing the gating strategy for infiltrating leukocyte subsets, including T cells (CD45highCD3+), CD4+ T cells (CD45highCD3+CD4+), CD8+ T cells (CD45highCD3+CD8+), B cells (CD45highCD3–CD19+), NK cells (CD45highCD3–NK1.1+), macrophages (CD45highCD11b+LY6C+), and neutrophils (CD45highCD11b+Ly6G+). (B) Quantification of infiltrating lymphocytes, macrophages, and neutrophils in the groups of mice receiving the indicated treatments. T cells: F(3,20) = 36.01, P < 0.001; CD4+ T cells: F(3,20) = 12.48, P = 0.01; CD8+ T cells: F(3,20) = 66.51, P < 0.001; B cells: F(3,20) = 4.483, P = 0.51; NK cells: F(3,20) = 24.73, P = 0.09; macrophages: F(3,20) = 32.11, P = 0.009; neutrophils: F(3,20) = 25.53, P = 0.002, *P < 0.05, **P < 0.01. (C) Bar graphs showing the mRNA expression of IL-2, IL-4, IL-6, IL-8, IL-1ß, TNF-a, MIP-1a, iNOS, and IFN-? in substantia nigra and striatal tissues from groups of mice receiving the indicated treatment. IL-2: F(3,20) = 17.61, P = 0.001; IL-4: F(3,20) = 2.701, P = 0.086; IL-6: F(3, 20) = 96.75, P < 0.001; IL-8: F(3,20) = 4.170, P = 0.94; IL-1ß: F(3,20) = 43.86, P < 0.001; TNF-a: F(3, 20) = 20.50, P = 0.003; MIP-1a: F(3,20) = 17.81, P < 0.001; iNOS: F(3,20) = 12.82, P < 0.001; IFN-?: F(3,20) = 111.7, P < 0.001. All data are presented as means ± SEM. (n = 6 mice per group) *P < 0.05, **P < 0.01.

Fig 4: CD8 T Cell Recall Responses Are Disrupted by Serum Iron Deficiency during the Primary Immune Response(A) Experimental design to investigate the effect of serum iron deficiency during the primary immune response on the magnitude of the secondary recall CD8 T cell response.(B) Number of endogenous OVA-specific secondary CD8 effector cells in the spleen detected by OVA tetramer binding and percentage of splenic OVA-specific CD8 effector cells detected by IFN?+ production after ex vivo stimulation with SIINFEKL. Means ± SDs. Student’s 2-tailed t test, unpaired.

Fig 5: 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.