Dendritic cells were generated from monocytes in the peripheral blood of healthy individuals, obtained under approval of the Oregon Health and Science University Institutional Review Board, and following the methods described by Lambert et al.
8 Monocytes were isolated from blood (RosetteSep Human Monocyte Enrichment Cocktail; STEMCELL Technologies, Inc., Vancouver, BC, Canada), according to the manufacturer's instructions, and finally suspended in RPMI 1640 medium supplemented with 10% FBS and 200 mM
l-glutamine. Cells were plated in 10-cm-diameter dishes at 20 × 10
6 cells/dish in modified RPMI medium with 10% FBS, interleukin (IL)-4 at 12.5 ng/mL (R&D Systems, Minneapolis, MN), and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 100 ng/mL (PeproTech, Rocky Hill, NJ). The medium was changed after 24 hours, followed by every 72 hours, and dendritic cells were harvested after 7 days. Expression of cell surface molecules was determined by labeling the cells with mouse anti-human monoclonal antibodies diluted in phosphate-buffered saline with 1% FBS and 0.1% sodium azide for 30 minutes on ice: Pacific Blue–tagged anti-CD14 antibody (2 μg/mL, clone M5E2, isotype IgG2aκ); PE-tagged anti-CD11c antibody (25 μg/mL, clone B-ly6, isotype IgG1κ); PE-Cy5.5–tagged anti-CD80 antibody (0.75 μg/mL, clone L307.4, isotype IgG1κ); biotin-tagged anti-CD86 antibody (1.5 μg/mL, clone 2331 [FUN-1], isotype IgG1κ); and FITC-tagged anti-HLA-DR antibody (25 μg/mL, clone G46-6, isotype IgG2aκ) (all obtained from BD Pharmingen, San Jose, CA). After washing with buffer, cells were treated with streptavidin-conjugated APC-Cy7 (2μg/mL, BD Pharmingen) for 15 minutes, washed again, and fixed in 4% paraformaldehyde. Data were acquired on a flow cytometer (BD LSR II; BD Biosciences, San Jose, CA) and analyzed using flow cytometry data analysis software (FCS Express; De Novo Software, Los Angeles, CA). All viable cells, as determined by size, were included in the gates.