title: CD83+ human dendritic cells transfected with tumor peptide cDNA by electroporation induce specific T-cell responses: A potential tool for gene immunotherapy.
abstract: Dendritic cells (DC) are the most potent immunostimulatory cells, with the capacity to induce primary T-cell responses. Functional autologous DC can be generated from fetal calf serum-free peripheral blood mononuclear cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor and are stimulated with a defined cytokine cocktail for terminal maturation. We were able to establish a nonviral transfection protocol for these DC by electroporation. Using enhanced green fluorescent protein as a reporter gene, we achieved transfection efficiencies of up to 10%. FACScan analyses revealed a stable phenotype, and the expression of major histocompatibility complex class II and CD83 was not affected by the transfection conditions used. Like their untransfected counterparts, DC that were functionally transfected with green fluorescent protein were potent inducers of allogeneic T cells. To assess whether cDNAs transfected into DC are functionally expressed, human tyrosinase cDNA was transfected into DC. Tyrosinase-transfected DC, but not controls, resulted in antigen-specific tumor necrosis factor-alpha release of the tyrosinase-specific cytolytic T-cell clone IVSB. Taken together, the data show that genuine (CD83+) mature DC can be transfected using a nonviral method, and that the DC retain their functionality. These DC are ideal candidates for immunotherapy (e.g., cancer therapy).
authors.affiliation: Department of Dermatology, Johannes Gutenberg-University, Mainz, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Lohmann
authors.forename: K
authors.initials: K
authors.lastname: Galle
authors.forename: J
authors.initials: J
authors.lastname: Knop
authors.forename: A H
authors.initials: AH
authors.lastname: Enk
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D002478:Cells, Cultured; D018076:DNA, Complementary; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D018274:Electroporation; D005434:Flow Cytometry; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis*; D007167:Immunotherapy / Q000379:methods; D015847:Interleukin-4 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008562:Membrane Glycoproteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection; D000097802:CD83 Antigen
chemical_list: D015703:Antigens, CD; D018076:DNA, Complementary; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D049452:Green Fluorescent Proteins; D007136:Immunoglobulins; D015847:Interleukin-4; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D000097802:CD83 Antigen