Human Flt3L Expands Dog DCs representative MHCII expression histograms (black) and their respective isotype control (red). Approximately 12,000 (A) or 30,000 events (B) were collected from each sample. Dot plots are representative of 3–4 dogs. SSC: Side Scatter; FSC: Forward Scatter. doi:10.1371/journal.pone.0011074.g003 against the tumor. We have previously demonstrated that an immunotherapeutic approach that combines the cytotoxic effect of Ad-TK with the immune stimulation induced by hsFlt3L leads to brain tumor regression and immunological memory in several intracranial rodent tumor models [8,10,32]. After 7 days of incubation with hsFlt3L or canine IL-4/GM- CSF, PBMCs cultures contained ,20–30% CD11c+ DCs. Tallying with previous reports, canine IL-4/GM-CSF led to differentiation of peripheral blood precursors into immature DCs, which produced low levels of cytokines and most of the cells displayed phagocytic activity [28,30,31]. Our findings suggest that the maturation status of hsFlt3L-derived DCs might be slightly different than IL-4-GMCSF-derived DCs. Phagocytic activity was higher when PBMC were cultured in the presence of hsFlt3L, suggesting that these DCs are less mature than IL-4/GM-CSF- derived DCs [25,29,30]. Although activation of both cultures led to increased release of Th1-cytokines, the allostimulatory activity of mature DCs was higher when PBMCs were cultured with hsFlt3L, suggesting an enhanced expression of co-activation molecules. In fact, it has been shown that blocking of CD86 using neutralizing antibodies completely inhibits the stimulatory effect of dog DCs on allogeneic leucocytes [30]. After 7 days of incubation with hsFlt3L or canine IL-4/GM- CSF, PBMCs cultures contained ,30–50% CD14+ monocytes. Although in rodents and humans the expression of CD14 has been shown to be downregulated in DCs upon maturation, it has been proposed that dog DCs derived from PBMC sustain their levels of CD14 [29,30]. The sustained expression of CD14 in our PBMC cultures incubated with both hsFlt3L and IL-4/GM-CSF was in correlation with the robust stimulatory effect of LPS on TNF-a, IL-6 and IFN- c release. Accordingly, LPS strongly inhibited the phagocytic activity of DCs derived from both cultures, suggestive of a more mature phenotype [25,29,30]. While LPS led to maturation of DCs from both cultures, i.e. hsFlt3L and canine IL-4/GM-CSF, we found little or no effect of CpGs on cytokine release from PBMC-derived DCs. This findings are in accordance with previous results that showed no stimulatory effect of CpGs on proliferation of PBMCs [33] and antigen presentation from dog bone marrow-derived DCs [25]. Although expression of TLR9 was detected in dog PBMCs [34], an array of 11 CpGs only induced a minor stimulatory effect on IFN-c and IL-12p40 expression in canine PBMCs, and did not affect the expression of IL-12p35, IL-18 and IL-4 [35]. Weak secretory responses to CpG in canine blood derived monocyte-macrophages were attributed to low expression of TLR9 in these cells [36]. Additionally, it has been proposed that canine immune cells exhibit a weaker response to TLR9 agonists than other species [37]. It is important to note that, in agreement with other authors [30], we observed widespread phenotypic variations between dogs throughout the study, which included over 20 dogs. This variability is an important feature of dog patients for cancer research and the development and testing of novel therapeutics, since they better resemble the variability encountered in the clinical scenario. In conclusion, our results demonstrate that hsFlt3L successfully modulates the function of canine DCs. These findings support the translation of hsFlt3L to be used in combination with Ad-TK for gene therapy applications or in dendritic cell vaccination protocols from rodent glioma models to canine GBM patients to eventually being implemented in human patients. Materials and Methods Ethical Statement All animal work was conducted according to the NIH guidelines and was approved by the Institutional Care and Use Committee (IACUC #2183) at Cedars Sinai Medical Center. Adenoviral vectors Ad-hsFlt3L used for this study is a first-generation replication- defective recombinant adenovirus type 5 vector expressing human soluble Flt3L under the transcriptional control of the human cytomegalovirus intermediate early promoter within the E1 region. Ad-hsFlt3L was generated as previously described [38,39]. Titration was carried out by end-point dilution, cytopathic effect assay, with centrifugation of infected 96-well plates as described in detail by Nyberg-Hoffman et al [40]. The titer for Ad-hsFlt3L was 1.32,2.6461012 iu/ml. Viral prepara- tion was free of replication-competent adenovirus (RCA) and LPS contamination, as assessed by the Limulus amebocyte gel clot assay (Biowhittaker, UK) [41,42]. All relevant adenoviral gener- ation methods and quality control procedures are described in detail in Southgate et al [39]. Generation of human soluble Flt3L conditioned media 56106 COS-7 cells were seeded in a T-75 flask and infected with Ad-hsFlt3L virus at 500 iu/cell in RPMI (CellGro) containing 10% heat-inactivated fetal calf serum, 2 mM L- glutamine, 1% MEM nonessential amminoacids, 100 IU/ml penicillin and 100 mg/ml streptomycin). The supernatant was harvested 72 h later and centrifuged to discard cellular debris. HsFlt3L conditioned media was stored at 280uC. The concen- tration of Flt3L in the conditioned media was determined by ELISA following manufacturer’s instructions (R&D System, Quantikine ELISA kit, Cat # DFK00). Dog blood and splenocytes collection The protocol to collect peripheral blood from dogs was approved by the Institutional Care and Use Committee (IACUC) at Cedars Sinai Medical Center. Peripheral blood was collected by venous puncture under appropriate manual restraint from a total of 27 random-bred dogs housed at Comparative Medicine Department, Cedars Sinai Medical Center. Twenty ml of blood were collected from each dog into BD VacutainerH K2 EDTA Plus Blood Collection Tubes (BD Biosciences, Cat. # 366643). In addition, 1 inch3 blocks of spleen were collected under surgical anesthesia from one dog following an IACUC-approved non- survival surgical procedure. DC generation using hsFlt3L or canine IL-4/GM-CSF Dog peripheral blood leucocytes were obtained by gradient centrifugation using lymphocyte separation medium (LSM, density 1.077–1.080 g/ml, CellgroH Mediatech, Inc. Cat. No 25-072-CV) following the manufacturer’s instructions. Leucocytes were resuspended in RPMI 1640 containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ ml streptomycin. Peripheral blood (PB) leucocytes were incubated PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11074