Escherichia coli 0111 b4

The following TLR-agonists (InvivoGen; San Diego, CA, USA, if not stated otherwise) were used at following concentrations: (i) 10 ng/mL of the TLR1/2 heterodimer agonist Pam3CSK4, a synthetic triacetylated lipopeptide, (ii) 10 ng/mL of the TLR4 agonist LPS isolated from Escherichia coli 0111:B4 (Sigma Aldrich; St. Louis, MO, USA), (iii) 100 ng/mL of the TLR5 agonist flagellin isolated from Salmonella typhimurium, and (iv) 100 ng/mL of the dual TLR7/8 agonist resiquimod (R848). These compounds were tested on ten unselected AML patients at concentrations of 0.01, 0.1 and 1.0 µg/mL (Pam3CSK4, LPS and flagellin) or respectively of 0.1, 1.0 and 10 µg/mL (R848). At the selected concentrations, the agonists significantly increased the AML blasts’ secretion of the cytokines CCL2, CCL3, CCL4, CXCL8 and IL-6, while the proliferation capacity of the cells was not compromised (Figure S3).

GM-BM were generated from murine bone marrow precursors using recombinant mouse (rm) GM-CSF. Cells were obtained as previously described [65 (link)] with minor modifications. GM-BM were cultured in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 50 μM 2-mercaptoethanol (Sigma-Aldrich) and 20 ng/ml rmGM-CSF (R&D Systems, Minneapolis, MN, USA). After 10 days of culture in 100 mm bacteriological Petri dishes (Corning [Falcon] BD, Bedford, MA, USA) GM-BM were separated using MACS CD11c⁺ labeled magnetic beads (Miltenyi Biotec, Auburn, CA, USA) [26 (link)]. After MACS separation (purity ≥ 95%) CD11c⁺ cells were exposed to: (a) complete RPMI-1640 medium (mock, negative control), (b) live-ECTV [multiplicity of infection, m.o.i. = 5], or (c) uvi-ECTV (m.o.i. = 5, before inactivation). After 60 min virus adsorption at 37°C in a humidified 5% CO₂ atmosphere, mock-, ECTV- or uvi-ECTV-exposed cells were cultured in the absence or the presence of LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St Louis, MO, USA) for 24 h at a final concentration of 1μg/ml, which served as a control of GM-BM maturation.

Whole blood, PBMCs, or PMNs were cultured with human interferon-α (IFNα) (1,000 U/ml, Alpha A/D hybrid, #11200, PBL Interferon Source; Piscataway, NJ), R848 (5 µg/ml, InvivoGen, San Diego, CA), AT-2 HIV-1_MN (3–1,500 ng/ml p24 capsid equivalent), control microvesicles, or lipopolysaccharide (LPS) (100 ng/ml, Escherichia coli 0111:B4; Sigma). Cells were then blocked in PBS with 10% human serum for 20 min at 4°C, resuspended in 100 µl of master mix containing staining buffer (PBS with 2% FBS) and antibodies: CD15-FITC (Biolegend), CD14- PerCP-Cy5.5, and PD-L1-APC (Biolegend). Media for experiments using AT-2_MN was supplemented with FBS instead of human serum to avoid potential blocking effects of human serum [54] (link). Blocking of human IFNα was performed by pre-incubating PBMCs or PMNs with 5 µg/ml anti-IFNAR (clone MMHAR-2; Invitrogen) for 30 min before addition of AT-2 HIV or R848.