title: Activation of monocytes via the CD14 receptor leads to the enhanced lentiviral transduction of immature dendritic cells.
abstract: In this study, we compared dendritic cells (DCs) differentiated from positively selected monocytes (CD14-DCs) to DCs differentiated from adherence-selected monocytes (adh-DCs) with emphasis on lentiviral transduction. Using a second-generation, triple-helix containing, self-inactivating lentiviral vector at a multiplicity of infection (MOI) of 15, we observed enhanced transduction of CD14-DCs (72.8 +/- 5.3%, mean fluorescence intensity [MFI] = 166 +/- 76) compared to adh-DCs (32.3 +/- 13.1%, MFI = 119 +/- 76, n = 5). More importantly, the efficiency to transduce adh-DCs was significantly increased when monocytes were incubated with antiCD14 antibody coupled beads, anti-CD14 antibodies, or lipopolysaccharide (LPS), reaching transduction efficiencies up to 86.6%, 53.3%, and 80.9%, respectively. We showed that this enhanced transduction was correlated to an activation of the monocytes, characterized by the up regulation of the cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and the de novo synthesis of IL-6 and IL-10. However, the enhanced transduction of immature CD14-DCs was not correlated with a progression in the cell cycle from G(0) to G(1). We further showed that CD14-DCs were phenotypically comparable to adh-DCs. Functional analysis revealed that there were no differences in allostimulatory capacity, production of IL-12 p70 on CD40 ligation or expression of IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNF-alpha as evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, we showed that lentivirally transduced CD14-DCs were equally capable as adh-DCs in stimulating MAGE-A3 antigen-specific CD4(+) and CD8(+) T cells in vitro.
authors.affiliation: 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.
authors.forename: Karine
authors.initials: K
authors.lastname: Breckpot
authors.forename: Jurgen
authors.initials: J
authors.lastname: Corthals
authors.forename: Carlo
authors.initials: C
authors.lastname: Heirman
authors.forename: Aude
authors.initials: A
authors.lastname: Bonehill
authors.forename: Annelies
authors.initials: A
authors.lastname: Michiels
authors.forename: Sandra
authors.initials: S
authors.lastname: Tuyaerts
authors.forename: Catherine
authors.initials: C
authors.lastname: De Greef
authors.forename: Kris
authors.initials: K
authors.lastname: Thielemans
mesh_terms: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes; D018414:CD8-Positive T-Lymphocytes; D002453:Cell Cycle; D002454:Cell Differentiation / Q000187:drug effects; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016130:Immunophenotyping; D016086:Lentivirus / Q000235:genetics*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation; D009000:Monocytes / Q000166:cytology / Q000276:immunology*; D009363:Neoplasm Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins