Recombinant human il 3

Prior to cell culture, pDCs were purified by sorting to be free of tDCs (AXL⁻), following the gating strategy in Figure S3A. 10,000 sorted bona fide pDCs were cultured in 200 μL R10 complete media consisting of RPMI (Corning) with 10% FBS, 2 mM L-glutamine (Corning), 100 IU Penicillin, 100 mg/mL Streptomycin (Corning), 25 mM HEPES (Corning), 1 mM Sodium Pyruvate (Corning), 100 mM MEM Nonessential Amino Acids (Corning) and 55 mM 2-Mercaptoethanol (GIBCO) in 96 well U-bottom plates at 37°C. All conditions included 10 ng/mL recombinant human IL-3 (R&D Systems; carrier-free) for pDC survival. Stimulation conditions were 100 ng/mL CD40L (R&D Systems; carrier-free) or 5 μg/mL Imiquimod (R837, Invitrogen). For ATAC-seq, 4–6 wells were plated per condition. After 2 days, cells were pooled and sorted as FSC-A, SSC-A, Live, Singlets, CD123⁺ HLA-DR⁺ (see Figure S4A for sorting strategy). For analysis of IFN-I production in Figure 6G, pDCs were re-sorted into “pDC-like,” “tDC-like,” and “cDC-like” after 2 days of stimulation with CD40L, then stimulated for 24h with 5 μg/mL CpG-A (ODN 2216, Invivogen).

Cell lines. OCI-AML1 cells were cultured in Roswell Park Memorial Institute medium (RPMI) 1640 (Sigma-Aldrich) or Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen) supplemented with 10% FBS, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) (Gibco by Life Technologies) supplemented with 10% FBS and 1×PSQ (2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, Gibco by Life Technologies). NK-92 cell line was purchased from ATCC (Manassas, VA) and cells were cultured in α-MEM (Gibco by Life Technologies) supplemented with 0.2 mmol/l myo-inositol (Sigma-Aldrich), 0.02 mmol/l folic acid (Sigma-Aldrich), 0.1 mmol/l 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA), 12.5% horse serum (Invitrogen), 12.5% fetal bovine serum (Gibco, Life Technologies) and 2 ng/ml hIL-2.
Human primary AML cells were cultured in AIM V medium (research and clinical grade, Life technologies) supplemented with 20 ng/ml recombinant human IL-3 (R&D Systems) and 100 ng/ml recombinant human SCF (R&D Systems). All cells were maintained in a humidified incubator at 37°C with 5% CO₂.