Fig 2: Reduction of Usp18 delays cancer progression.a Representative pictures of embryos (E14.5) of Usp18+/+, Usp18+/-, and Usp18-/- (Top) and Usp18f/f, Usp18+/f Vav-iCre, and Usp18f/f Vav-iCre (Bottom) mice. b Western blots showing phosphorylated and total STAT1 protein and ß-actin in Usp18 WT and Heterozygous bone marrows. c Ratio of live cells from Usp18+/? BMDM or Usp18+/? AE9a cells treated with different doses of IFNß for 72 h in one representative set of three independent repeats. p-value was determined by one-way ANOVA test. (Usp18+/? BMDM n = 3, Usp18+/? AE9a n = 3). d Percentage of Annexin V+ GFP+Lin-c-Kit+ splenocytes from Usp18+/f and Usp18+/? AE9a recipient mice as analyzed by FACS. p-value was determined by two-tailed Student t-test. (Usp18+/f n = 6 mice, Usp18+/? n = 6 mice). e Absolute numbers of CD8+, activated T cells (CD8+/CD69+, CD8+/CD25+, CD8+/CD71+), Tregs (CD4+CD25+Foxp3+) and B cells (CD19+) in viable host splenocytes per 10,000 GFP+ cells from Usp18+/f and Usp18+/? AE9a recipient mice. Information on other immune cell populations is also shown in source data. P-value was determined by two-tailed Student t-test. (Usp18+/f n = 7 mice, Usp18+/? n = 5 mice). f Kaplan–Meier survival curve for recipients of Usp18+/f or +/? UBCER-Cre AE9a-GFP AMLs. p-value was determined by log-rank test. (Usp18+/f n = 6 mice, Usp18+/? n = 6 mice). g Percentage of GFP+ cells in the PB of mice from (f). p-value was determined by one-way ANOVA test. h Timeline of treatments combining chemotherapy with heterozygous deletion of Usp18 to treat AML. i Kaplan–Meier survival curve for mice treated as in (h). (Oil (+/f) n = 6 mice, Tam (+/?) n = 4 mice). p-value was determined by log-rank test. j Percentage of GFP+ cells in the PB of mice from (i), analyzed weekly by FACS. All data represent mean ± s.d, except where indicated. n.s. = not statistically significant. Source data are provided as a Source Data file.

Fig 3: Cellular Iron Metabolism Is Remodeled upon T Cell Activation, and Iron Deficiency Disrupts T Cell Physiology(A) Left to right: Histograms of CD71 (Tfrc) expression on 4-OHT treated CD8 T cells from IRP1/2 floxed mice lacking (blue) or expressing Cre (red) at 24h (top) and 72h (bottom) after in vitro activation; relative CD71 MFI for CD8 T cells treated with 4-OHT measured by flow cytometry (72h after activation); relative CD71 MFI of 4-OHT treated compared to EtOH treated CD8 T cells (to control for effects of 4-OHT, 72h after activation). Mean+/- SD, Student's two tailed T-test, unpaired.(B) Percentage divided CD8 T cells of each genotype/EtOH or 4-OHT combination cultured with a titration of FeSO4, 72 h after activation. Percentage of divided determined as percentage of cells that had diluted out their CellTrace Violet signal once or more. Mean ± range. Two-way ANOVA, corrected for multiple comparisons, p values for difference between EtOH (control), and 4-OHT at each iron concentration.(C) Left to right: ATP content was measured using CellTiter Glo luminescent assay for equal numbers of WT CD8 T cells per condition and normalized to the control condition of each biological replicate. Total, glycolytic, and mitochondrial ATP production rates measured by Seahorse. Each data point is a biological replicate from a single mouse, representing the average of 2–5 technical replicate wells. Mitochondrial content (MitoTracker Green [MTG]) and mitochondrial inner membrane potential (MitoTracker Deep Red [MTDR]) were measured by flow cytometric analysis of 72-h activated divided cells and the ratio calculated. Mean ± range. One-way ANOVA, effect of iron, paired within each biological replicate.

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.