title: Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70.
abstract: BACKGROUND
  For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development.
  
  
  METHODS
  After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C).
  
  
  RESULTS
  Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation.
  
  
  CONCLUSIONS
  Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.
authors: Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, Munich, Germany. AZobywalski@aol.com
authors: Anke
authors: A
authors: Zobywalski
authors: Miran
authors: M
authors: Javorovic
authors: Bernhard
authors: B
authors: Frankenberger
authors: Heike
authors: H
authors: Pohla
authors: Elisabeth
authors: E
authors: Kremmer
authors: Iris
authors: I
authors: Bigalke
authors: Dolores J
authors: DJ
authors: Schendel
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D015925:Cryopreservation; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D018274:Electroporation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015234:HLA-A Antigens / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D018664:Interleukin-12 / Q000096:biosynthesis*; D007959:Lymphocyte Culture Test, Mixed; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D010455:Peptides / Q000276:immunology; D012313:RNA; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors; D014019:Tissue Donors
chemical_list: D015234:HLA-A Antigens; D010455:Peptides; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D016753:Interleukin-10; D049452:Green Fluorescent Proteins; D018664:Interleukin-12; D012313:RNA; D007371:Interferon-gamma