Fig 1: Iron promotes c-Maf expression in Treg cells by modulating HIF-2α expression.a, b WT mice were administered iron dextran (37.5 mg/e.a) every five days for 3 weeks. The percentages of c-Maf+ Treg cells in the spleen were determined by flow cytometry. a Representative flow cytometric plots; (b). Summary of the percentages of c-Maf+ cells in splenic Treg cells from three independent experiments. c, d CD4 T cells were activated under iTreg differentiation conditions (anti-CD3+anti-CD28 + TGFβ1 + IL-2+anti-IFNγ+anti-IL4) with the indicated concentrations of ferrous sulfate or DFO. The expression of c-Maf and Foxp3 was determined by intracellular staining four days after T-cell activation. c Representative flow cytometry analysis; (d). Summary of the percentages of c-Maf+ Tregs (n = 7 biologically independent experiments for mock, and n = 5 for FeSO4-treated mice). (e, f). As in (c), the transcriptomes of ferrous sulfate-treated iTreg cells were compared to those of iTreg cells without treatment by RNA-seq. e M-A plots showing the differentially expressed genes; Differentially expressed genes were determined by DESeq2 and are shown in red or blue (|LFC | ≥ 0.5, p < 0.05 in Wald test). f GO analysis of the differentially expressed genes using Metascape. p-values are calculated based on the cumulative hypergeometric distribution, and q-values are calculated using the Benjamini-Hochberg procedure. g The expression of HIF-1α and HIF-2α in YFP+Foxp3+CD4+ cells of Tfr1fl/fl Foxp3-CreYFP/+ mice (Tfrc cKO) was determined by flow cytometry and compared with that in their counterparts (WT) of Tfrc+/+Foxp3-CreYFP/+ mice. The data are representative of three independent experiments. h As in (c), one day after T-cell activation, iTregs were cultured with 50 μM FeSO4 for 24 h, and the expression of HIF-2α and HIF-1α in Foxp3+ cells was determined by intracellular staining. The data are representative of three independent experiments. i, j As in (a), iTreg cells were treated with 50 μM ferrous sulfate with or without 50 μM PT2385 (an HIF-2α inhibitor). The expression of c-Maf was determined by intracellular staining. i Representative results. j Summary of c-Maf+% cells in iTreg cells based on four independent experiments. b, d, j Data shown are mean ± SD, *p < 0.05, **p < 0.01,***p < 0.001 by two-tailed Student’s t test.
Fig 2: IF remodels the anti-tumor T cell immune response to EOC mouse tumors(A–R) (A) A representative t-SNE visualization of markers after gating on single, live, CD45+ CD3+, CD4+, CD8+, IFNγ, IL4, Granzyme B (Grz B) and perforin expression.(B) Histograms represent average counts of the main T cell subsets in RD and IF groups.(C) Heatmap represents marker expression of the main T cell subsets in individual samples. Bar graph represents the percentage of T cell subsets (D) CD4+, (E) CD4+IFNγ+, (F) CD + IL4+, (G) ratio of CD4+ IFNγ+ to CD4+IL4+, (H) CD8+, (I) CD8+IFNγ+, (J) CD8+GrzB+ and (K) CD8+perforin+. Immune profiling was performed in 5 individual mice per group. Bar graph represents the percentage of T cell subsets (L) CD4+, (M) CD4+IFNγ+, (N) CD + IL4+, (O) CD8+, (P) CD8+IFNγ+, (Q) CD8+GrzB+ and (R) CD8+perforin+ in the subcutaneous tumors. Immune profiling was performed in tumors from 4 individual mice per group.(S and U) Extracellular acidification rate (ECAR) in splenic CD4+ and CD8+ cells (n = 3).(T and V) Oxygen consumption rate (OCR) in splenic CD4+ and CD8+ cells (n = 3). The experiment was repeated twice in two different sets of mouse experiment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, IF compared to RD group by Student’s t test.
Fig 3: BHB inhibits tumor growth and promotes anti-tumor CD8+ T cell responses in EOC mouse models(A) Kaplan–Meier graph indicating overall survival (n = 12), p = 0.0085 by Gehan-Breslow-Wilcox test.(B) Average weekly body weight progression.(C) Average abdominal circumference.(D) Average ascites volume.(E) A representative t-SNE visualization of markers after gating on single, live, CD45+ CD3+, CD4+, CD8+, IFNγ+, IL4+ and Granzyme B + (GrzB) cells.(F) Histogram represents counts of the T cell.(G–O) Heatmap represents marker expression of the T cell subsets. Bar graph represents the percentage of T cell subsets (H) CD4+, (I) CD4+IFNγ+, (J) CD + IL4+, (K) ratio of CD4+ IFNγ+ to CD4+IL4+, (L) CD8+, (M) CD8+IFNγ+, (N) CD8+Grz B+ and (O) ratio of CD8+ to CD4+ cells. Immune profiling was performed in 4 individual mice per group.(P and R) Oxygen consumption rate (OCR) in (P) CD4+ and (R) CD8+ cells (n = 3).(Q and S) Extracellular acidification rate (ECAR) in (Q) CD4+ cells and (S) CD8+ cells (n = 3). The bar graph represents basal and stressed OCR and ECAR. The experiment was repeated twice in two different sets of mouse experiment (n = 3).(T–V) percentage of live and dead ID8p53−/- and CD8+ cells.(W) IFNγ levels (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, BHB compared to C, assessed by Student’s t-tests.
Fig 4: IF improves EOC survival and anti-tumor immune response better than BHB(A) Kaplan–Meier graph depicting overall survival (n = 12/group), p < 0.0001 IF compared to RD, p < 0.0001 BHB compared to RD and p = 0.0016 BHB compared to IF by Gehan-Breslow-Wilcoxon test.(B) Average weekly body weight progression.(C) Average abdominal circumference.(D–M) (D) Average ascites volume. Growth factors and cytokines were measured in ascites collected at week 5 by ELISA (n = 3/group), (E) Insulin, IGF-1, leptin, and adiponectin, (F) IFNγ, (G) TNFα, (H) IL-6, (I) IL-4, (J) IL-1β, (K) MCP-1 and (L) GMCSF. (M) A t-SNE visualization of markers after gating on single, live, CD45+ CD3+, CD4+, CD8+, IFNγ+, IL-4+ and Granzyme B+ (GrzB) cells.(N) Histogram represents average counts of the T cell subsets measured.(O–U) Heatmap represents marker expression of the measured T cell subsets in individual samples.(P–Y) Percentages of T cell subsets (P) CD4+, (Q) CD4+IFNγ+, (R) CD4+IL4+, (S) CD8+, (T) CD8+IFNγ+, and (U) CD8+Gnz B+. Immune profiling was performed in 4 individual mice per group. BHB levels measured in (V) plasma, (W) ascites, (X) CD8+ and (Y) CD4+ cells.(Z) mRNA expression of GPR109a measured by RT-PCR on CD8+ and CD4+ cells isolated from mice on RD, IF or treated with BHB. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = non-significant by one-way ANOVA, followed by Sidak multiple comparison test.
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