title: Enhanced dendritic cell antigen presentation in RNA-based immunotherapy.
abstract: BACKGROUND
  Dendritic cells pulsed with mRNA provide a unique approach to tumor immunotherapy. We hypothesized that increased mRNA transfection efficiency and dendritic cell maturation would improve antigen processing and presentation as well as T-cell costimulation, resulting in enhanced induction of antimelanoma immune responses.
  
  
  METHODS
  Immature monocyte-derived dendritic cells were transfected with mRNA by passive pulsing, lipofection, or electroporation. Dendritic cells were either left untreated or matured using the double-stranded RNA poly(I:C). T-Cell cultures were generated by stimulation of naïve T-cells with each set of dendritic cells. Specific antigen presentation and specific effector T-cell generation were analyzed by an IFN-gamma release Elispot assay.
  
  
  RESULTS
  Greatest intracellular green fluorescent protein was observed by flow cytometry following dendritic cell electroporation with green fluorescent protein mRNA. DC presentation of Mart-1/Melan A peptide, as measured by Elispot assay using a specific T-cell clone, was greatest following transfection with Mart-1/Melan A mRNA by electroporation. Maturation of dendritic cells further improved antigen presentation regardless of transfection technique. Specific Mart-1/Melan A effector T cells were produced after culture of naïve T cells with dendritic cells that were electroporated with Mart-1/Melan A mRNA and then matured, but not for dendritic cells that remained immature.
  
  
  CONCLUSIONS
  Efficient mRNA transfection by electroporation as well as dendritic cell maturation results in increased levels of Mart-1/Melan A antigen presentation and enhanced production of antigen-specific effector T cells. This combination of strategies may be used to enhance immune responses to RNA-based dendritic cell vaccines.
authors.affiliation: Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Kalady
authors.forename: Mark W
authors.initials: MW
authors.lastname: Onaitis
authors.forename: Karen M
authors.initials: KM
authors.lastname: Padilla
authors.forename: Sirisha
authors.initials: S
authors.lastname: Emani
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Tyler
authors.forename: Scott K
authors.initials: SK
authors.lastname: Pruitt
mesh_terms: D017951:Antigen Presentation / Q000276:immunology*; D002460:Cell Line; D016922:Cellular Senescence / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018274:Electroporation; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D012333:RNA, Messenger / Q000276:immunology / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection; D014612:Vaccines
chemical_list: D012333:RNA, Messenger; D014612:Vaccines; D007371:Interferon-gamma