Human Flt3L Expands Dog DCs Figure 2. Phenotypic characterization of dog peripheral blood DC cultures. Peripheral blood cultures were collected after 7 days of culture with recombinant canine IL-4 and GM-CSF or Ad.hFlt3L conditioned medium. Immune cell populations in the culture were identified using antibodies against CD14 (monocytes), CD11c (dendritic cells) and CD18 (macrophages). Numbers in representative dot plots indicate the percentage of positive cells. Approximately 12,000 events were collected from each sample. Dot plots are representative of 4 dogs. SSC: Side Scatter; FSC: Forward Scatter. doi:10.1371/journal.pone.0011074.g002 Human soluble Flt3L enhances the capacity of DCs to induce proliferation of allogeneic T cells We observed that hsFlt3L can generate DCs from peripheral blood precursors; activation stimulates the release of Th1 cytokines and reduces the phagocytic activity of DCs. Finally, since the ability to prime and stimulate T cell proliferation is an important assessment of the functionality of DCs [25], we performed a mixed leucocyte reaction (MLR) to determine the proliferation of allogeneic lymphocytes induced by mature DCs. DCs were generated from dog peripheral blood, cultured with hsFlt3L or canine IL-4/GM-CSF for 1 week, and activated for additional 24h with CpG (Fig. 6 A). Splenocytes were collected from a second dog and CD4+and CD8a+ T cells were purified by flow cytometry cell sorting after staining with specific antibodies (Fig. 6 B). Dog T cells were co-cultured with increasing numbers of allogeneic mature DCs for 7 days. T cell proliferation was assessed by incorporation of BrdU. As controls, DCs were incubated in the absence of T cells. We found that increasing numbers of hsFlt3L-derived PBDCs exerted a stimulatory effect on the proliferation of allogeneic T cells (Fig. 6 C). Although at a lesser extent, canine IL-4/GM-CSF-derived dog DCs also induced allogeneic T cell proliferation. These results indicate that hsFlt3L exerts a potent trophic effect on dog DCs, which in turn efficiently activate proliferation of allogeneic na¨ıve T cells. Discussion We show that human Flt3L expressed from an adenoviral vector generates dendritic cells from dog peripheral blood precursors. Although previous reports have shown that maturation of DCs can be achieved by culturing peripheral blood leucocytes with T cell conditioned media [29] and canine cytokines [28,30,31], the ability of human cytokines to exert trophic effects on canine immune cells has been a matter of debate. Weber et al. reported no activity of human TNF-a in the growth of canine DCs and little cross-reaction of human Flt3L with canine immune cells [25]. However, Hagglund et al. demonstrated that while the combination of human GM-CSF, human TNF-a and human IL-4 failed to stimulate proliferation of canine DCs, replacement of human IL-4 by human Flt3L in the cytokine cocktail led to generation of DCs in vitro from dog bone marrow progenitors [26]. Moreover, dogs that received daily subcutaneous injections of human recombinant Flt3L for 10 days exhibited increased numbers of leucocytes, mainly monocytes, in peripheral blood and circulating DCs displayed strong allostimulatory potential [27]. Our results show that hsFlt3L exerts a trophic effect on dog DCs that is comparable to the canine cytokine cocktail comprising of IL-4 and GM-CSF. These findings support the rationale of administering an Ad vector encoding hsFlt3L to dog patients bearing spontaneous gliomas to mount an immune response PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11074