Fig 2: Engrafted murine cells in chimeric animals include hematopoietic progenitors and myeloid lineage cells. Mouse bone marrow cells were blue-labeled and transplanted into zebrafish blastulae. At 2 dpf, 100 chimeric animals were selected, and a whole-embryo cell suspension was prepared and analyzed by flow cytometry. Representative contour plots from color-based analysis show (A) 77% of viable/blue violet+/CD45+ events as Gr1+, (B) 33% as c-kit+, (C) 5% of events as CD11b/F4-80+, (D) 2% of events as Ter-119+ and (E) less than 1% of events as CD3+/CD19+. (F) Table summarizing mouse cell phenotypes in chimeric animals at 2 dpf. Results are representative from 2 independent experiments consisting of 3 biological replicates.

Fig 3: Pharmacological inhibition of IL-1ß decreased myelofibrosis in MPN mice.a Experimental setup of the drug treatment. b Grade of reticulin fibrosis was determined before therapy in groups of n = 6 mice killed at 12-, 16-, and 20-weeks after transplantation. c Time course of body weights (n = 12 mice per treatment group). Two-way ANOVA followed by uncorrected Fisher’s LSD test was performed. d Blood counts and mutant cell (% GFP) chimerism in the peripheral blood of vehicle (n = 12); ruxolitinib (n = 11); anti-IL-1ß (n = 12); combo (n = 12) treated mice in erythroid (Ter119), megakaryocytic (CD61), granulocytic (Gr1), and monocytic (CD11b) lineages. Two-way ANOVA followed by uncorrected Fisher’s LSD test was performed. Two-way ANOVA followed by Dunnett’s multiple comparisons test was performed for GFP chimerism. e Spleen weights of vehicle (n = 12); ruxolitinib (n = 11); anti-IL-1ß (n = 12); combo (n = 12) treated mice after 8 weeks of drug treatment. Two-tailed unpaired t-test was performed. Grey area represents normal range. f Representative images of reticulin fibrosis and H&E staining is shown and histological grade of reticulin fibrosis in the BM is illustrated in the bar graph. Similar results were obtained with other mice in each condition. Stacking bar graph showing the percentage of mice with osteosclerosis in the BM of vehicle (n = 12); Ruxolitinib (n = 11); anti-IL-1ß (n = 12); and combo (n = 12). Two-tailed unpaired t test was performed for comparisons of fibrosis grades between different groups. p value is computed using Fisher’s exact test for presence or absence of osteosclerosis in bone marrow. g Heatmap plot showing the inflammatory cytokine levels in the BM lavage and plasma of mice after 8 weeks of drug treatment. Vehicle (n = 12); Ruxolitinib (n = 11); anti-IL-1ß (n = 12); combo (n = 12). The color bars indicate treatment groups. Heatmap shows Z scores. Two-tailed unpaired t-tests without correction for multiple comparisons was performed. Green-colored asterisk is used for comparison of vehicle vs. anti-IL-1ß; salmon for vehicle vs. ruxolitinib; plum for vehicle vs. combo. All data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also Supplementary Figs. 9–11. Source data and exact p values are provided as a Source Data file.

Fig 4: Adtrp deficiency leads to increased expression of matrix metallopeptidase-9 (Mmp9) mRNA, protein, and activity levels in zebrafish embryos and newborn mice. A, Whole-mount in situ hybridization for mmp9 (blue) in 72 hours postfertilization (hpf) control and adtrp zebrafish morphants. Blue arrow: specific signal accumulation. Black arrow: caudal vascular plexus (CVP). n, biological replicates. Bars, 100 µm. B, Immunofluorescence staining with anti-Mmp-9 (Cy3, red) IgG on cross-sectioned CVP (green) of 72-hpf Tg(fli1:EGFP)y1 (EGFP, fluorescent vascular reporter) control and adtrp morphants±human ADTRP mRNA. Bars, 100 µm. C, Total lysates of 72-hpf zebrafish embryos incubated ±100 µmol/L of pan-MMP inhibitor GM6001 for 24 hours and analyzed by 10% gelatin gel electrophoresis. Left: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitometry and statistics (mean±SEM by t test). *P<0.05. Each marker represents a group of at least 10 embryos. D, In situ zymography with MMP fluorescence substrate QXL™ 570-KPLA-Nva-Dap(5-TAMRA)-AR-NH2 (red, dequenched fluorescence) on cross-sectioned CVP (green) of 48-hpf Tg(fli1:EGFP)y1 embryos injected with Control or adtrp morpholinos (MOs). Arrows: MMP activity. E, Fluorescence confocal microscopy of 72-hpf Tg(fli1:EGFP)y1 embryos injected with control or adtrp MOs (left panels; EGFP, fluorescent vascular reporter)±GM6001 as for C. Bars, 100 µm. Quantification of vascular defects (right panels): scatter plots and statistics (1-way ANOVA with Bonferroni's multicomparison test) of the percentage of defective intersegmental vessels (ISVs) and percentage area of CVP dilation normalized to tail area. Data are mean±SEM of biological replicates (n). ****P<0.0001 between all groups. F, Double immunofluorescence and whole-mount confocal microscopy with anti-MMP-9 (Cy3, red) and either anti-CD31 or anti-GR-1 (FITC, green) in cephalic skin; or with anti-MMP-9 (Cy3, red) and anti-mast cell (MC) Tryptase (FITC, green) in lung of wild-type (WT) and Adtrp -/- P0 pups. MMP-9 associates with both CD31 and GR-1–positive cells in the skin (lower panels, white arrow and inset), and with MC (lung panels, yellow arrows). Bars, 100 µm. G, Zymography (as in C) of conditioned media of primary fibroblasts cultured from P0 WT and Adtrp -/- cephalic skin explants. Upper panel: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitometry and statistics using t test. Data are mean±SEM of biological replicates. ****P<0.0001. Adtrp indicates androgen-dependent tissue factor pathway inhibitor regulating protein; EGFP, enhanced green fluorescent protein; IgG indicates immunoglobulin G.

Fig 5: Stimulation of Tnfrsf4-signaling reduces the capacity of Tregs to protect LSCs from CD8+ CTL–mediated killing.(A) Representative FACS plots for the expression of Tnfrsf4 on CD4+Foxp3-GFP+ T cells (Tregs), CD4+Foxp3– T cells, CD8+ CTLs, L-Gr-1+ cells, and LSCs in the BM of Foxp3DTR CML mice. One representative out of 9 plots is depicted. Staining, black; isotype control, gray. (B) Frequency of CD4+Foxp3-GFP+ T cells, CD4+Foxp3– T cells, CD8+ CTLs, L-Gr-1+ cells, and LSCs in the BM of CML mice expressing Tnfrsf4 (n = 9 mice/cell subset). (C) LSCs from the BM of Foxp3DTR CML mice were cultured with BM CD8+ T cells and/or BM Tregs of the same mice pretreated for 2 hours with a Tnfrsf4 antibody (clone OX-86, 30 µg/mL) or respective control antibody at a ratio of 1:1:1 in triplicate followed by plating in methylcellulose. Colonies were enumerated 7 days later; 1-way ANOVA followed by Tukey’s post hoc test. (D and E) BL/6 CML mice were randomized to control IgG or anti-Tnfrsf4 antibody treatment (OX-86, 200 µg/mouse, i.p, for 6 times every second day, starting at day 12), and leukemia development and survival was monitored. (D) Number of L-Gr-1+ cells in the blood of IgG-treated and Tnfrsf4 antibody–treated BL/6 CML mice; 2-way ANOVA followed by Sidak’s multiple comparison test (n = 5 mice/group). (E) Kaplan-Meier survival curves of control IgG– and TNFRSF4 Ab–treated CML mice (IgG, n = 5; Tnfrsf4, n = 5); log-rank test. (F–J) CD8/Treg ratio in the BM, Treg numbers in the BM, spleen weight, numbers of L-Gr-1+ cells, and LSCs in the BM of control IgG– and TNFRSF4 Ab–treated CML mice (IgG, n = 5; Tnfrsf4, n = 4) 18 days after CML transplantation. t test. Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. One representative out of 2 independent experiments is shown.