title: Retroviral-mediated IL-12 gene transduction into human CD34+ cell-derived dendritic cells.
abstract: Genetically modified dendritic cells (DCs) with Th1 type cytokine genes are useful for activating anti-tumor immune response. We made human interleukin (IL)-12 p70 gene-transduced DCs generated from CD34+ progenitor cells using a retrovirus system and investigated the function of IL-12-producing DCs. We used the pMX retroviral vector and made cytokine gene-containing viral vectors referred to as GFP pMX and hIL-12 pMX. Supernatants from BOSC23 cells transfected with GFP pMX and hIL-12 pMX were harvested and used for transfection of DC. Cord blood CD34+ cells were incubated with supernatants containing retrovirus for 48 h with cytokines such as IL-3, IL-6, SCF, Flt3 ligand (FL), bFGF and IGF-I. The cells were cultured for 12 days in the presence of GM-CSF, SCF, FL, IL-4 and TNF-alpha to get mature DC-enriched population. Analysis of surface marker on DCs and allogeneic MLR assay were also performed. After a 14-day culture, 60-70% of cultured CD34+ cells were DC marker (CD1a, DEC205) positive. The IL-12 p70 protein levels in supernatant of DC-GFP and DC-hIL-12 were 0.2 ng/ml and 53 ng/ml/5 x 10(5) DCs for 72 h, respectively. The addition of CH296 fibronectin fragment (FN) increased 3-fold IL-12 gene transduction efficiency into DCs. MLR assay showed that IL-12-producing DC exhibited more potent T cell growth-stimulating activity compared with GFP-DC. These results suggested that genetically modified CD34+ cell-derived DCs with human IL-12 gene are fully efficient in T cell priming, and could be a good tool for effective cancer immunotherapy.
authors.affiliation: Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukuji, Chuo-ku, Tokyo 104-0045, Japan. yaakiyam@gan2.res.ncc.go.jp
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Akiyama
authors.forename: Kouji
authors.initials: K
authors.lastname: Maruyama
authors.forename: Morihiro
authors.initials: M
authors.lastname: Watanabe
authors.forename: Ken
authors.initials: K
authors.lastname: Yamaguchi
mesh_terms: D018949:Antigens, CD1 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018952:Antigens, CD34 / Q000096:biosynthesis / Q000097:blood / Q000276:immunology*; D016207:Cytokines / Q000494:pharmacology; D003713:Dendritic Cells / Q000276:immunology / Q000502:physiology*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007959:Lymphocyte Culture Test, Mixed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000276:immunology / Q000502:physiology; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D018949:Antigens, CD1; D018952:Antigens, CD34; C419393:CD1a antigen; D016207:Cytokines; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018664:Interleukin-12