title: Transfection of human monocyte-derived dendritic cells with native tumor DNA induces antigen-specific T-cell responses in vitro.
abstract: OBJECTIVE
  Nucleofection of genomic tumor (Tu) DNA into human monocyte-derived dendritic cells (hMoDC) was evaluated for use in producing anti-tumor vaccines able to induce effective T-cell specific immune responses.
  
  
  METHODS
  Cultured hMoDC obtained from HLA-A2+ normal donors were nucleofected with genomic DNA extracted from an HLA-A2+gp100+ Mel 526 cell line and 3' end-labeled with biotinylated TdT nucleotides or from a genetically-modified Mel 526 expressing enhanced green fluorescent protein (EGFP). An Amaxa Nucleofector system was used for electroporation. Nucleofected hMoDC were matured in the presence of cytokines and examined in ELISPOT assays for the ability to present the gp100(209-217) epitope to epitope-specific T cells or to prime autologous naïve T cells in culture.
  
  
  RESULTS
  The nucleofected hMoDC presented gp100 protein to HLA-A2+gp 100-specific T cells as observed in IFN-gamma ELISPOT assays. Spot formation was inhibited by anti-HLA class I and HLA-A2 but not anti-HLA class II antibodies (Abs). Tu DNA-nucleofected hMoDC also primed nasmall yi, Ukrainianve autologous peripheral blood T cells in culture to develop into Tu-reactive effector cells (CTL). These CTL recognized Tu cells which had donated genomic DNA, and these responses were MHC class I- and class II-restricted. The CTL recognized shared Tu antigens encoded in Tu-derived DNA.
  
  
  CONCLUSIONS
  Nucleofection of hMoDC with genomic Tu-derived DNA is a useful strategy for Tu vaccine production: it is feasible, does not require Tu epitope isolation, can be used when few Tu cells are available, and avoids Tu-induced DC suppression.
authors.affiliation: Department of Pathology, University of Pittsburgh School of Medicine and Cancer Institute, Pittsburgh, Pennsylvania 15213-1863, USA.
authors.forename: Elisa
authors.initials: E
authors.lastname: Artusio
authors.forename: Bridget
authors.initials: B
authors.lastname: Hathaway
authors.forename: Joanna
authors.initials: J
authors.lastname: Stanson
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D002294:Carcinoma, Squamous Cell; D045744:Cell Line, Tumor; D002470:Cell Survival; D004273:DNA, Neoplasm / Q000235:genetics*; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D005434:Flow Cytometry; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D015789:HLA-A2 Antigen / Q000276:immunology; D006258:Head and Neck Neoplasms; D006801:Humans; D007378:Interleukins / Q000494:pharmacology; D008545:Melanoma; D010957:Plasmids; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D004273:DNA, Neoplasm; D015789:HLA-A2 Antigen; D007378:Interleukins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor