title: High-level antigen expression and sustained antigen presentation in dendritic cells nucleofected with wild-type viral mRNA but not DNA.
abstract: Dendritic cells (DC) are potent antigen-presenting cells that hold promise as cell-based therapeutic vaccines for infectious diseases and cancer. Ideally, DC would be engineered to express autologous viral or tumor antigens to ensure the presentation of relevant antigens to host T cells in vivo; however, expression of wild-type viral genes in primary cell lines can be problematic. Nucleofection is an effective means of delivering transgenes to primary cell lines, but its use in transfecting DNA or mRNA into DC has not been widely investigated. We show that nucleofection is a superior means of transfecting human and monkey monocyte-derived DC with DNA and mRNA compared to lipofection and conventional electroporation. However, the delivery of DNA and mRNA had significantly different outcomes in transfected DC. DC nucleofected with DNA encoding green fluorescent protein (GFP) had poor antigen expression and viability and were refractory to maturation with CD40 ligand. In contrast, >90% of DC expressed uniform and high levels of GFP from 3 h to 96 h postnucleofection with mRNA while maintaining a normal maturation response to CD40 ligation. Monkey DC nucleofected with wild-type, non-codon-optimized mRNA encoding simian immunodeficiency virus Gag stimulated robust antigen-specific effector T-cell responses at 24 h and 48 h postnucleofection, reflecting sustained antigen presentation in transfected DC, whereas no detectable T-cell response was noted when DC were nucleofected with DNA encoding the same Gag sequence. These data indicate that mRNA nucleofection may be an optimal means of transfecting DC with autologous tumor or viral antigen for DC-based immunotherapy.
authors.affiliation: Center for Vaccine Research and Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Nada M
authors.initials: NM
authors.lastname: Melhem
authors.forename: Sherrianne M
authors.initials: SM
authors.lastname: Gleason
authors.forename: Xiang Dong
authors.initials: XD
authors.lastname: Liu
authors.forename: Simon M
authors.initials: SM
authors.lastname: Barratt-Boyes
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D000956:Antigens, Viral / Q000096:biosynthesis*; D003713:Dendritic Cells / Q000276:immunology*; D015683:Gene Products, gag / Q000276:immunology; D006801:Humans; D008253:Macaca mulatta; D012333:RNA, Messenger / Q000276:immunology*; D012367:RNA, Viral / Q000276:immunology*; D015302:Simian Immunodeficiency Virus / Q000276:immunology; D014162:Transfection
chemical_list: D000956:Antigens, Viral; D015683:Gene Products, gag; D012333:RNA, Messenger; D012367:RNA, Viral