Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial Weidong Xiong1., Marianela Candolfi1., Chunyan Liu1, A. K. M. Ghulam Muhammad 1 , Kader Yagiz1, Mariana Puntel1, Peter F. Moore2, Julie Avalos3, John D. Young 3 , Dorothy Khan3, Randy Donelson 4 , G. Elizabeth Pluhar5, John R. Ohlfest4,6, Kolja Wawrowsky7, Pedro R. Lowenstein1, Maria G. Castro1* 1 Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Medicine and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 2 Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America, 3 Department of Comparative Medicine, Cedars-Sinai Medical Center, Los Angeles, California, United States of America, 4 Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, United States of America, 5 Department of Small Animal Clinical Sciences, University of Minnesota, St. Paul, Minnesota, United States of America, 6 Department of Neurosurgery, University of Minnesota, Minneapolis, Minnesota, United States of America, 7 Department of Medicine, Cedars-Sinai Medical Center and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America Abstract Background: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads) encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L) and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK). This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs), in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF. Methodology/Principal Findings: Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF- cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-a and IFN-c. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation. Conclusions/Significance: These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells’ vaccination and gene therapeutic approaches from rodent models to canine patients and its future implementation in human clinical trials. Citation: Xiong W, Candolfi M, Liu C, Muhammad AKMG, Yagiz K, et al. (2010) Human Flt3L Generates Dendritic Cells from Canine Peripheral Blood Precursors: Implications for a Dog Glioma Clinical Trial. PLoS ONE 5(6): e11074. doi:10.1371/journal.pone.0011074 Editor: Maciej Lesniak, The University of Chicago, United States of America Received May 12, 2010; Accepted May 19, 2010; Published June 11, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work is supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) Grant 1R21-NS054143, 1UO1 NS052465 and 1RO1-NS057711 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193, and RO1 NS061107 to P.R.L.; The Bram and Elaine Goldsmith and the Medallions Group Endowed Chairs in Gene Therapeutics Research to P.R.L. and M.G.C., respectively, The Linda Tallen & David Paul Kane Foundation Annual Fellowship and the Board of Governors at CSMC. M.C. is supported by NIH/NINDS 1F32NS058156.01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: castromg@cshs.org . These authors contributed equally to this work. Introduction Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. GBM accounts for 35% of all primary brain tumor cases and 80% of malignant brain tumors (www.cbtrus.org). The standard of care for treatment of GBM, which consists of surgical resection, followed by radiation therapy and chemotherapy with temozolomide, leads to a median survival of 6–18 months [1]. Thus, novel approaches are being developed for the treatment of this devastating disease [2–6]. Our previous studies have shown that a combined conditional cytotoxic-immunotherapy approach using adenoviral vectors PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11074