title: Decline of surface MHC I by adenoviral gene transfer of anti-MHC I intrabodies in human endothelial cells-new perspectives for the generation of universal donor cells for tissue transplantation.
abstract: BACKGROUND
  The seeding of small-calibre vascular polytetrafluoroethylene (PTFE) grafts with endothelial cells provides an increase in biocompatibility of the graft surface. The harvest and ex vivo culture of autologous endothelial cells is highly delicate. Allogeneic human umbilical vein endothelial cells (HUVEC) could be a potential cell source-however, rejection might occur due to major histocompatibility complex (MHC) I mismatches. Lowering cell surface MHC I expression on endothelial cells by gene transfer of an anti-MHC I intrabody might reduce graft failure. The intrabody consists of a single-chain variable fragment (sFv) of an anti-MHC I antibody, carrying a terminal KDEL sequence to retain the molecule together with the MHC I inside the endoplasmic reticulum.
  
  
  METHODS
  Adenoviral gene transfer was used to express the intrabody in HUVEC. The MHC I surface expression was measured 48 h after transduction by flow cytometry. Functional effects of the intrabody expression were analyzed in a calcein release cytotoxicity assay.
  
  
  RESULTS
  A transduction efficiency of more than 95% with EGFP-adenovirus indicates a sufficient gene transfer into HUVEC. Intrabody-adenovirus-transduced HUVEC show a massive reduction in MHC I surface expression creating almost a complete 'knockout' phenotype. Stimulation with inflammatory cytokines could not overcome this effect. The cell lysis of anti-MHC I intrabody-expressing HUVEC in a cytotoxicity assay is reduced when compared with the level of the MHC mismatched control.
  
  
  CONCLUSIONS
  Our data indicate that HUVEC with reduced levels of MHC I might be used as universal donor cells for the seeding of vascular grafts.
authors.affiliation: Institute of Medical Immunology, Campus Charité-Mitte, Schumannstrasse 20/21, 10098 Berlin, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Beyer
authors.forename: Cornelia
authors.initials: C
authors.lastname: Doebis
authors.forename: Annette
authors.initials: A
authors.lastname: Busch
authors.forename: Thomas
authors.initials: T
authors.lastname: Ritter
authors.forename: Abner
authors.initials: A
authors.lastname: Mhashilkar
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Marasco
authors.forename: Horst
authors.initials: H
authors.lastname: Laube
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Volk
authors.forename: Martina
authors.initials: M
authors.lastname: Seifert
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002462:Cell Membrane / Q000276:immunology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000276:immunology*; D004730:Endothelium, Vascular / Q000166:cytology; D018014:Gene Transfer Techniques; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D009842:Oligopeptides / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D016378:Tissue Transplantation / Q000379:methods; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D015395:Histocompatibility Antigens Class I; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D021382:Protein Sorting Signals; C073762:lysyl-aspartyl-glutamyl-leucine