Human Flt3L Expands Dog DCs Figure 1. Morphology of peripheral blood-derived dog dendritic cells cultured with canine IL-4+GM-CSF or Ad.hFlt3L conditioned medium. White cells from dog peripheral blood were purified using Lymphocyte Separation Media and cultured in the presence of recombinant canine IL-4 (50 ng/ml) and GM-CSF (33 ng/ml) or Ad.hsFlt3L conditioned medium (10 ng/ml). The images show the appearance of the culture after 7 days in culture. Low magnification images (206) show typical spheres formed in culture supplemented with canine IL-4+GM-CSF (A) or hsFlt3L (B). Insets show higher magnification images of cells that exhibit typical long cytoplasmic processes characteristic of dendritic cell morphology. Scale bars: 10 mm. C, After 7–9 days in culture non-adherent cells were collected and counted. The graph shows the yield of cells per ml of blood obtained from 27 dogs after culture with hsFlt3L or canine-IL-4/GM-CSF. doi:10.1371/journal.pone.0011074.g001 Phagocytic activity of canine DCs cultured with hsFlt3L or canine IL-4 and GM-CSF Considering that tumor antigen uptake by DCs is a crucial event in the initiation of the anti-tumor immune response induced by the combined Ad-hsFlt3L+Ad-TK therapy [10], we evaluated the phagocytic activity of peripheral blood-derived dendritic cells (PBDCs) generated with hsFlt3L. We compared the phagocytic activity of hsFlt3L- or IL-4/GM-CSF-derived PBDCs to take up yellow green fluorescence beads. After 2h incubation with beads, cells were fixed and stained by immunofluorescence with anti- MHCII or anti-CD11c antibodies. MHCII+ cells and CD11c+ DCs containing beads in their cytoplasm were readily detected in hsFlt3L and IL-4/GM-CSF cultures analyzed by confocal microscopy (Fig. 5A). We quantified the phagocytic activity of PBDCs after activation with CpG or LPS. We found that the basal percentage of CD11c+ DCs with phagocytic activity was higher in cultures derived from hsFlt3L than canine IL-4/GM-CSF (Fig. 5 B). Also, the phagocytic activity of both hsFlt3L- and IL-4/GM- CSF-derived DCs was reduced upon activation with CpG or LPS (Fig. 5 B). PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11074