title: Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells.
abstract: Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.
authors.affiliation: Laboratory of Experimental Hematology, University of Antwerp, Antwerp University Hospital, Edegem, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Ponsaerts
authors.forename: Glenn
authors.initials: G
authors.lastname: Van den Bosch
authors.forename: Nathalie
authors.initials: N
authors.lastname: Cools
authors.forename: Ann
authors.initials: A
authors.lastname: Van Driessche
authors.forename: Griet
authors.initials: G
authors.lastname: Nijs
authors.forename: Marc
authors.initials: M
authors.lastname: Lenjou
authors.forename: Filip
authors.initials: F
authors.lastname: Lardon
authors.forename: Christine
authors.initials: C
authors.lastname: Van Broeckhoven
authors.forename: Dirk R
authors.initials: DR
authors.lastname: Van Bockstaele
authors.forename: Zwi N
authors.initials: ZN
authors.lastname: Berneman
authors.forename: Viggo F I
authors.initials: VF
authors.lastname: Van Tendeloo
mesh_terms: D000941:Antigens / Q000378:metabolism; D019496:Cancer Vaccines / Q000276:immunology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016895:Culture Media, Serum-Free; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology*; D018274:Electroporation; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D011070:Poly I-C / Q000494:pharmacology; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000941:Antigens; D019496:Cancer Vaccines; D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D011070:Poly I-C