HSV-2 infection of macaque DCs. Immature macaque and human moDCs were exposed to HSV-2 for 1 hour at 37°C (5 pfu per cell). HSV-2–loaded cells were then washed, counted, and plated for 4 hours up to overnight (ON) reculture in medium containing GM-CSF and IL-4. (A) ICP5, gD, envelope (env) (4 hours), and ICP8 (ON; arrows) were detected in HSV-2–infected cells by immunofluorescence microscopy. HSV-2 protein staining is shown in green and the nuclei in blue (DAPI). A representative example of each staining for macaque moDCs is shown (5 of 5 experiments with ICP5 and gD, 2 of 2 experiments with env, 7 of 8 experiments with ICP8). In the cells from the one animal in which we did not detect ICP8 staining after overnight (ON) reculture, ICP8 expression was detected within 1 to 4 hours of exposure. The relative IgG controls are shown in the lower row for each stain. Uninfected DCs showed no staining with the anti-HSV Abs. Original magnification, × 60. (B) Higher magnification of the middle left ICP8-stained cell from panel A showing the DAPI staining, the ICP8 staining, and their overlay is shown to appreciate the predominant nuclear localization of ICP8. The green ICP8 stain completely masks the blue DAPI-stained nucleus. (C) ICP8 staining was monitored by flow cytometry after 4 hours or ON reculture of medium- (Med) versus HSV-2–exposed (HSV) DCs. At each time point, cells were fixed/permeabilized and bound anti-ICP8 Ab detected with AffiniPure Donkey Anti–mouse IgG and the Qdot 525 streptavidin conjugate. The numbers in each panel indicate the percentage of ICP8⁺ cells (fluorescence versus forward scatter [FSC]) for one representative experiment of macaque moDCs. The quadrants were drawn relative to the medium-treated DCs incubated with anti-ICP8 for each time point. Isotype IgG controls stained less than 1 log (under the horizontal gate) on both infected and uninfected samples. (D) The percentages of ICP8⁺ cells from several experiments (7 for 4 hours and 15 for ON recultured macaque moDCs [filled symbols] and 3 for ON recultured human moDCs [open symbols]; each symbol represents a donor) are summarized for medium-treated versus HSV-infected cells. The horizontal black bars indicate the mean values.