title: An improved protocol for generation of immuno-potent dendritic cells through direct electroporation of CD14+ monocytes.
abstract: In this study we demonstrate a novel protocol showing that electroporation of CD14+ monocytes directly isolated from blood with green fluorescent protein (GFP) RNA results in a 3-fold higher yield of antigen presenting dendritic cells (DCs) when compared to conventional methods employing immature DCs for electroporation. We further show a stable electroporation efficacy resulting in 60% of GFP positive cells. Expression of co-stimulatory molecules and maturation markers such as CD80, CD86, CD83 as well of the chemokine receptor 7 (CCR7) was found in 90% of the mature DCs. Importantly, production of IL-12p70 was 10 times higher in cells electroporated at the monocyte stage compared to cells electroporated at the immature DC stage. Stimulation of autologous naïve lymphocytes by DCs electroporated at monocytes stage elicited proliferation of CD8+ T-cell with 7-fold increase in IFN-gamma release. Blocking of the MHC-Class I molecules significantly inhibited the IFN-gamma release, indicating that antigen presentation was MHC-Class I mediated. In summary, electroporation of CD14+ monocytes with RNA results in a high yield of antigen presenting DCs with high immuno-stimulatory capacity and antigen presentation on MHC-Class I molecules. This improved method may represent an attractive approach for RNA-based DC immunotherapy.
authors.affiliation: Department of Experimental Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands. F.Milano@amc.uva.nl
authors.forename: Francesca
authors.initials: F
authors.lastname: Milano
authors.forename: Jantine W P M
authors.initials: JW
authors.lastname: van Baal
authors.forename: Agnieszka M
authors.initials: AM
authors.lastname: Rygiel
authors.forename: Jacques J G H M
authors.initials: JJ
authors.lastname: Bergman
authors.forename: Sander J H
authors.initials: SJ
authors.lastname: Van Deventer
authors.forename: Martien L
authors.initials: ML
authors.lastname: Kapsenberg
authors.forename: Maikel P
authors.initials: MP
authors.lastname: Peppelenbosch
authors.forename: Kausilia K
authors.initials: KK
authors.lastname: Krishnadath
mesh_terms: D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D016516:CD4-CD8 Ratio; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002450:Cell Communication / Q000276:immunology; D002454:Cell Differentiation; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018274:Electroporation; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D016753:Interleukin-10 / Q000378:metabolism; D018664:Interleukin-12 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008213:Lymphocyte Activation; D008297:Male; D009000:Monocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D054400:Receptors, CCR7; D019707:Receptors, Chemokine / Q000096:biosynthesis; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D015703:Antigens, CD; C515370:CCR7 protein, human; D015395:Histocompatibility Antigens Class I; D018950:Lipopolysaccharide Receptors; D054400:Receptors, CCR7; D019707:Receptors, Chemokine; D016753:Interleukin-10; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D012313:RNA; D007371:Interferon-gamma