title: Transduction of dendritic cell progenitors with a retroviral vector encoding viral interleukin-10 and enhanced green fluorescent protein allows purification of potentially tolerogenic antigen-presenting cells.
abstract: BACKGROUND
  Dendritic cells (DC) are important antigen-presenting cells that play critical roles in the initiation and modulation of immune responses. Genetic engineering of DC to express immunosuppressive molecules is a novel approach to the inhibition of allograft rejection. Retroviral delivery of viral interleukin (vIL)-10 to replicating myeloid DC progenitors (DCp) impairs their T-cell stimulatory capacity and promotes the induction of antigen-specific T-cell hyporesponsiveness. However, transduction efficiency with retroviral vectors is comparatively low. Enhanced green fluorescent protein (EGFP) is important both as a marker of gene transduction and for the selection of transduced cells. Our aims were to construct a retroviral vector encoding both vIL-10 and EGFP, to positively select transduced DC, and to assess the impact of these highly purified, vIL-10-secreting antigen-presenting cells on allogeneic T-cell responses.
  
  
  METHODS
  DCp propagated from bone marrow of C57BL10 (H2b) mice in granulocyte-macrophage colony-stimulating factor (GM-CSF)+IL-4 were transduced with a retroviral vector encoding both vIL-10 and EGFP by centrifugal enhancement. Gene transfer efficiency was determined by flow cytometry. Transduced cells were flow sorted, and vIL-10 secretion was quantified by ELISA. DC function was assessed by the ability of the cells to induce naive allogeneic (C3H; H2k) T-cell proliferation and cytotoxic T lymphocyte generation.
  
  
  RESULTS
  Retrovirally transduced DC expressed both vIL-10 and EGFP gene products. Approximately 20% of unsorted cells expressed EGFP, as determined by flow cytometry. vIL-10 was produced at a mean rate of 31 ng/40 hr/10(6) cells. After sorting, the incidence of EGFP+ DC was increased dramatically to at least 95%, and the production of vIL-10 was increased approximately three- to fourfold, to a mean of 107 ng/40 hr/10(6) cells. These highly purified, vIL-10-secreting DC exhibited markedly diminished capacity to induce allogeneic T-cell proliferative and cytotoxic responses.
  
  
  CONCLUSIONS
  DCp retrovirally transduced to express both vIL-10 and EGFP can be rapidly identified and sorted to high levels of purity. The availability of highly enriched preparations of vIL-10-transduced DC facilitates studies of their immunoregulatory function and may enhance their therapeutic potential in transplantation or autoimmune disease.
authors.affiliation: Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pennsylvania 15213, USA.
authors.forename: T
authors.initials: T
authors.lastname: Takayama
authors.forename: H
authors.initials: H
authors.lastname: Tahara
authors.forename: A W
authors.initials: AW
authors.lastname: Thomson
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology* / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D003713:Dendritic Cells / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007108:Immune Tolerance; D016753:Interleukin-10 / Q000378:metabolism* / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000502:physiology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D016753:Interleukin-10; D049452:Green Fluorescent Proteins