Preferences help
enabled [disable] Abstract
Number of results
2006 | 53 | 1 | 203-212
Article title

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

Title variants
Languages of publication
Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR+ cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34+ cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor α (TNF-α). Transfection of CD34+ cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.
Physical description
  • Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392: 245-252.
  • Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J (1992) GM-CSF and TNF-α cooperate in the generation of dendritic Langerhans cells. Nature 360: 258-261.
  • Caux C, Massacrier C, Dezutter-Dambuyant C, Vanbervliet B, Jacquet C, Schmitt D, Banchereau J (1995) Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen. J Immunol 155: 5427-5435.
  • Davis TA, Saini AA, Blair PJ, Levine PL, Craighead N, Harlan DM, June CH, Lee KP (1998) Phorbol esters induce differentiation of human CD34+ hemopoietic progenitors to dendritic cells: evidence for protein kinase C-mediated signaling. J Immunol 160: 3689-3697.
  • Hsu FJ, Benike C, Fagnoni F, Liles TM, Czerwinski D, Taidi B, Engleman EG, Levy R (1996) Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med 2: 52-58.
  • Fong L, Engleman EG (2000) Dendritic cells in cancer immunotherapy. Annu Rev Immunol 18: 245-273.
  • Gabrilovich DI, Corak J, Ciernik IF, Kavanaugh D, Carbone DP (1997) Decreased antigen presentation by dendritic cells in patients with breast cancer. Clin Cancer Res 3: 483-490.
  • Irvine AS, Trinder PK, Laughton DL, Ketteringham H, McDermott RH, Reid SC, Haines AMR, Amir A, Husain R, Doshi R, Young LS, Mountain A (2000) Efficient nonviral transfection of dendritic cells and their use for in vivo immunization. Nat Biotechnol 18: 1273-1278.
  • Kim CJ, Prevette T, Cormier J, Overwijk W, Roden M, Restifo NP, Rosenberg SA, Marincola FM (1997) Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro. J Immunother 20: 276-286.
  • Krause SW, Neumann C, Soruri A, Mayer S, Peters JH, Andreesen R (2002) The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells. J Immunother 25: 421-448.
  • Lenz P, Bacot SM, Frazier-Jessen MR, Feldman GM (2003) Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells. FEBS Lett 538: 149-154.
  • Lundqvist A, Noffz G, Pavlenko M, Sæbøe-Larssen S, Fong T, Maitland N, Pisa P (2002) Nonviral and viral gene transfer into different subsets of human dendritic cells yield comparable efficiency of transfection. J Immunother 25: 445-454.
  • Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D (1998) Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4: 328-332.
  • Oki M, Ando K, Hagihara M, Miyatake H, Shimizu T, Miyoshi H, Nakamura Y, Matsuzawa H, Sato T, Ueda Y, Gansuvd B, Kato S, Hotta T (2001) Efficient lentiviral transduction of human cord blood CD34+ cells followed by their expansion and differentiation into dendritic cells. Exp Hematol 29: 1210-1217.
  • Ołdak T, Kruszewski M, Machaj EK, Gajkowska A, Pojda Z (2002) Optimisation of transfection of CD34+ hematopoietic cells derived from human umbilical cord blood. Acta Biochim Polon 49: 625-632.
  • Perez-Diez A, Butterfied LH, Li L, Chakraborty NG, Economou JS, Mukherji B (1998) Generation of CD8+ and CD4+ T-cell response to dendritic cells genetically engineered to express the MART-1/Melan A gene. Cancer Res 63: 2127-2133.
  • Santiago-Schwarz F, Belilos E, Diamond B, Carsons SE (1992) TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages. J Leukoc Biol 52: 274-281.
  • Savary AC, Grazziutti ML, Melichar B, Przepiorka D, Freedman RS, Cowart RE, Cohen DM, Anaissie EJ, Woodside DG, McIntyre BW, Pierson DL, Pellis NR, Rex JH (1998) Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients. Cancer Immunol Immunother 45: 234-240.
  • Sæbøe-Larssen S, Fossberg E, Gaudernack G (2002) mRNA-based electrotransfection of human dendritic cells and induction of cytotoxic T lymphocyte responses against the telomerase catalytic subunit (hTERT). J Immunol Methods 259: 191-203.
  • Schuler G, Schuler-Thurner B, Steinman RM (2003) The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 15: 138-147.
  • Siena S, Di Nicola M, Bregni M, Mortarini R, Anichini A, Lombardi L, Ravagnani F, Parmiani G, Gianni AM (1995) Massive ex vivo generation of functional dendritic cells from mobilized CD34+ blood progenitors for cancer therapy. Exp Hematol 23: 1463-1471.
  • Strobel I, Berchtold S, Götze A, Schulze U, Schuler G, Steinkasserer A (2000) Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes. Gene Ther 7: 2028-2035.
  • Tan PH, Beutelspacher SC, Wang YH, McClure MO, Ritter MA, Lombardi G, George AJT (2005) Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination. Mol Ther 11: 790-800.
  • Temme A, Morgenroth A, Schmitz M, Weigle B, Rohayem J, Lindemann D, Fussel M, Ehninger G, Rieber EP (2002) Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34+ cord blood-derived dendritic cells. Gene Ther 9: 1551-1560.
  • Timmerman JM, Czerwinski DK, Davis TA, Hsu FJ, Benike C, Hao ZM, Taidi B, Rajapaksa R, Caspar CB, Okada CY, van Beckhoven A, Liles TM, Engleman EG, Levy R (2002) Idiotype pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood 99: 1517-1526.
  • Uduehi A, Mailhos C, Truman H, Thrasher AJ, Kinnon C, Hart SL (2003) Enhancement of integrin-mediated transfection of haematopoietic cells with a synthetic vector system. Biotechnol Appl Biochem 38: 201-209.
  • Van Tendeloo VFI, Snoeck H-W, Lardon F, Vanham GLEE, Nijs G, Lenjou M, Hendriks L, Van Broeckhoven C, Moulijn A, Rodrigus I, Verdonk P, Van Bockstaele DR, Berneman ZN (1998) Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells. Gene Ther 5: 700-707.
  • Van Tendeloo VFI, Willems R, Ponsaerts P, Lenjou M, Nijs G, Vanhove M, Muylaert P, Van Cauwelaert P, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2000) High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation mediated gene delivery. Gene Ther 7: 1431-1437.
  • Van Tendeloo VFI, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98: 49-56.
  • Wu MH, Smith SL, Danet GH, Lin AM, Williams SF, Liebowitz DN, Dolan ME (2001a) Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation. Bone Marrow Transplant 27: 1201-1209.
  • Wu MH, Smith SL, Dolan ME (2001b) High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells. Stem Cells 19: 492-499.
  • Wu MH, Liebowitz DN, Smith SL, Williams SF, Dolan ME (2001c) Efficient expression of foreign genes in human CD34+ hematopoietic precursor cells using electroporation. Gene Ther 8: 384-390.
  • Ying H, Zaks TZ, Wang R-F, Irvine KR, Kammula US, Marincola FM, Leitner WW, Restifo NP (1999) Cancer therapy using self-replicating RNA vaccine. Nat Med 5: 823-827.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.