A novel plasmid DNA electroporation method allows transfection of murine DC.
Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4(+) T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.
Bros
Matthias
M
Clinical Research Unit Allergology, Department of Dermatology, Johannes Gutenberg-University, Mainz, Germany. mbros@uni-mainz.de
Wiechmann
Nadine
N
Besche
Verena
V
Castor
Timo
T
Sudowe
Stephan
S
Grabbe
Stephan
S
Reske-Kunz
Angelika B
AB
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D018274:Electroporation / Q000379:methods*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D016753:Interleukin-10 / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein / Q000378:metabolism; D063308:Myelin-Oligodendrocyte Glycoprotein; D010957:Plasmids; D014162:Transfection / Q000379:methods*; D019076:Transgenes
C571327:Mog protein, mouse; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D063308:Myelin-Oligodendrocyte Glycoprotein; D016753:Interleukin-10