Activation of monocytes via the CD14 receptor leads to the enhanced lentiviral transduction of immature dendritic cells.
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.
Breckpot
Karine
K
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.
Corthals
Jurgen
J
Heirman
Carlo
C
Bonehill
Aude
A
Michiels
Annelies
A
Tuyaerts
Sandra
S
De Greef
Catherine
C
Thielemans
Kris
K
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
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins