Stromal OP9 cells expressing the Notch ligand DLL4 (OP9-DLL4) were provided by Juan Carlos Zúñiga-Pflücker (Sunnybrook Research Institute, Department of Immunology, University of Toronto, Ontario, Canada). Cells were cultured in RPMI1640 (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal calf serum (PAN-Biotech, Aidenbach, Germany), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA), and 50 U/ml penicillin and 50 μg/ml streptomycin (Thermo Fisher Scientific). Cells were confirmed to be mycoplasma-free by Venor GeM Classics Mycoplasma PCR detection kit (BioProducts, Stockerau, Austria) and tested by flow cytometry for surface DLL4. For the generation of moLCs, 5,000 OP9-DLL4 cells per well were seeded in a 96-well round-bottom plate (Corning, Corning, NY). After 24 hours, 20,000 monocytes per well were added with 50 ng/ml GM-CSF (Sanofi, Paris, France) and 10 ng/ml TGF-β1 (PeproTech, Rocky Hill, NJ), followed by incubation for 3 days at 37 °C. Thereafter, cells were either harvested or cultured for additional 24–48 hours in the presence of a DC-cytokine maturation cocktail consisting of 10 ng/ml IL-1β, 10 ng/ml TNF-α, 10 ng/ml IL-6, and 1 μg/ml prostaglandin E2 (all from PeproTech) or for 24 hours in the presence of 5 μg/ml CpG (Miltenyi Biotec, Bergisch Gladbach, Germany), 20 μg/ml PolyI:C (Sigma-Aldrich, St. Louis, MO), or 100 ng/ml lipopolysaccharide (Sigma-Aldrich).

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