JMML cells produce excessive IL-1β that plays a key role in driving HSC attrition. (A) Purified WT HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻Flk2⁻) were cocultured with BM cells isolated from Ptpn11^E76K/+LysM-Cre⁺ and Ptpn11^+/+LysM-Cre⁺ mice in 2-chamber transwell systems in StemSpan medium supplemented with thrombopoietin (TPO; 50 ng/mL), Flt3 ligand (50 ng/mL), and stem cell factor (SCF; 50 ng/mL) for 8 days. Percentages of Mac-1⁺Gr-1⁺ cells derived from HSCs were determined. Experiments were performed 3 times, and similar results were obtained in each. Results shown are the mean ± standard deviation (SD) of triplicates from 1 experiment. (B-C) BM plasma collected from 16-week-old Ptpn11^E76K/+LysM-Cre⁺ mice and Ptpn11^+/+LysM-Cre⁺ control mice (n = 3 mice per genotype) were processed for chemokine-cytokine array analyses. Representative results from 1 pair of the mice are shown (B). Levels of IL-1β in the BM plasma were quantified by enzyme-linked immunosorbent assay (n = 5 mice per genotype) (C). (D) WT HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) were cultured in StemSpan medium supplemented with TPO (100 ng/mL), Flt3 ligand (50 ng/mL), and SCF (100 ng/mL) in the presence or absence of IL-1β (10 ng/mL) and/or IL-1rα (10 ng/mL). Frequencies of Mac-1⁺Gr-1⁺ cells were determined 7 days later. (E-I) Ptpn11^+/+LysM-Cre⁺IL-1R^+/+, Ptpn11^+/+LysM-Cre⁺IL-1R^−/−, Ptpn11^E76K/+LysM-Cre⁺IL-1R^+/+, and Ptpn11^E76K/+LysM-Cre⁺IL-1R^−/− mice were generated and euthanized at the age of 4 months. Spleen weights (normalized against body weights) were documented (n = 8-9 mice per genotype) (E). Percentages of Mac-1⁺Gr-1⁺ cells in the peripheral blood, spleen, and BM were determined (n = 4-7 mice per genotype) (F). Frequencies of LSK cells and HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) in the BM (n = 7-10 mice per genotype) (G) and in the spleen (n = 6-10 mice per genotype) (H) and the cell cycle distribution in HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) in the BM (n = 4 mice per genotype) (I) were determined by multiparameter FACS analyses. (J) Purified HSCs (Lin⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) from WT and IL-1R^−/− mice were cocultured with Mac-1⁺ cells isolated from 4-month-old Ptpn11^+/+LysM-Cre⁺ and Ptpn11^E76K/+LysM-Cre⁺ mice in StemSpan medium supplemented with TPO (100 ng/mL), Flt3 ligand (50 ng/mL), and SCF (100 ng/mL) in a 2-chamber transwell system for 8 days. Frequencies of Mac-1⁺Gr-1⁺ cells differentiated from HSCs in the upper chamber were assayed by FACS analyses. Experiments were performed 3 times, and similar results were obtained in each. Results shown are mean ± SD of triplicates from 1 experiment. (K) Cells from patients with JMML carrying PTPN11 mutations and healthy BM cells were cultured in StemSpan medium (serum free) supplemented with human SCF (hSCF; 50 ng/mL), hTPO (50 ng/mL), and hFlt3 ligand (50 ng/mL) for 3 days (experiment 1 [Exp. 1] and Exp 2). Culture medium was collected and analyzed for IL-1β by FACS with IL-1β antibody–conjugated beads. (L) CD34⁺ cord blood cells were isolated and cocultured with cells from patients with JMML or control cells in transwell systems in StemSpan medium (serum free) supplemented with hSCF (50 ng/mL), hTPO (50 ng/mL), and hFlt3 ligand (50 ng/mL). Five days (Exp. 1) or 12 days (Exp. 2) later, percentages of CD14⁺ cells and CD11c⁺ cells differentiated from CD34⁺ cells were determined by FACS analyses. *P = .05, **P = .01, ***P = .001. IFN-γ, interferon-γ; ns, not significant; TNF-α, tumor necrosis factor α.