Adenovirus-GFP–induced DC immunophenotype in human CD14⁺monocytes.

Fresh monocytes or monocytes cultured for various times in SFM/G were infected with adenovirus-GFP at a multiplicity of infection (MOI) of 200. Cells were harvested 72 to 94 hours later and analyzed by FACS. Monocytes that had never been cultured (“not cultured”) were also examined. Results are representative of 3 experiments with different donors. (A) Demonstration of the purity of starting populations of monocytes. Uncultured elutriated monocytes stained with fluorescein isothiocyanate–conjugated anti-CD33 (x-axis) and phycoerythrin (PE)–conjugated anti-CD11c (y-axis) mAbs. (B) Multipanel histogram analysis of monocytes that were treated in one of the following ways: not cultured; infected with adenovirus-GFP 2 hours after culture; infected 2 hours after culture, washed free of all cytokines at 48 hours, and recultured in fresh SFM (without any added cytokines) for 48 hours; infected with adenovirus-GFP 24 hours after culture; or cultured in SFM/G only. All cultured cell groups were maintained for a total of 96 hours irrespective of time of infection or washout and replacement of ambient culture medium. Open traces represent isotype-matched control antibody staining; filled traces indicate marker-specific antibody. (C) Analysis of CD25 expression after adenovirus infection. Monocytes were cultured 24 hours in SFM/G, infected with adenovirus-GFP, and harvested after an additional 72 hours culture. Light dotted and dashed lines represent anti-CD25–stained uncultured cells and SMF/G-cultured cells, respectively. Heavy solid line represents anti-CD25–stained adenovirus-infected cells.