THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2003; 5: 654–667. Published online 23 April 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.400 Lentivirally transduced dendritic cells as a tool for cancer immunotherapy Karine Breckpot 1† Melissa Dullaers 1† Aude Bonehill 1 Sonja Van Meirvenne 1 Carlo Heirman 1 Catherine De Greef 1 Pierre van der Bruggen 2 Kris Thielemans 1 * 1 Laboratory of Molecular and Cellular Therapy, Department of Physiology and Immunology, Medical School of the Vrije Universiteit Brussel (V.U.B.), Laarbeeklaan 103/E, 1090 Brussels, Belgium 2 Ludwig Institute for Cancer Research, Brussels Branch, Avenue Hippocrate 74, UCL 74.59, 1200 Brussels, Belgium *Correspondence to: Dr Kris Thielemans, Medical School of the Vrije Universiteit Brussel (VUB), Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Laarbeeklaan 103/E, 1090 Brussels, Belgium. E-mail: Kris.Thielemans@vub.ac.be † These authors contributed equally to this work. Received: 12 December 2002 Revised: 13 February 2003 Accepted: 24 February 2003 Abstract Background Dendritic cells (DC) are the professional antigen-presenting cells of the immune system, fully equipped to prime naive T cells and thus essential components for cancer immunotherapy. Methods We tested the influence of several elements (cPPT, trip, WPRE, SIN) on the transduction efficiency of human DC. Human and murine DC were transduced with tNGFR-encoding lentiviruses to assess the effect of transduction on phenotype and function. Human DC were transduced with lentiviruses encoding huIi80MAGE-A3 and murine DC with huIi80tOVA to test antigen presentation. Results A self-inactivating (SIN) lentiviral vector containing the trip element was most efficient in transducing human DC. The transduction of DC with trip/SIN tNGFR encoding lentiviral vectors at MOI 15 resulted in stable gene expression in up to 94.6% (murine) and 88.2% (human) of the mature DC, without perturbing viability, phenotype and function. Human huIi80MAGE- A3-transduced DC were able to stimulate MAGE-A3-specific CD4 + and CD8 + T cell clones and could prime both MAGE-A3-specific CD4 + and CD8 + T cells in vitro. Murine huIi80tOVA-transduced DC were able to present OVA peptides in the context of MHC class I and class II in vitro and induced a strong OVA-specific cytotoxic T lymphocyte response in vivo, that was protective against subsequent challenge with OVA-expressing tumor cells. Conclusions We show that, using lentiviral vectors, efficient gene transfer in human and murine DC can be obtained and that these DC can elicit antigen- specific immune responses in vitro and in vivo. The composition of the transfer vector has a major impact on the transduction efficiency. Copyright  2003 John Wiley & Sons, Ltd. Keywords dendritic cell; lentivirus; immunotherapy; T helper cell; cytotoxic T cell Introduction Cancer immunotherapy is based on the fact that tumor cells express antigens which can be recognised by the immune system and lead to tumor rejection. These tumor-associated antigens include tumor-specific shared antigens, differentiation antigens, protein products of mutated genes and rearrangements unique to tumor cells, overexpressed tissue-specific antigens and exogenous viral proteins [1–3]. However, the development of effective therapeutic cancer vaccines has proven difficult, mainly because these tumor Copyright  2003 John Wiley & Sons, Ltd.