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.
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.
Beyer
Florian
F
Institute of Medical Immunology, Campus Charité-Mitte, Schumannstrasse 20/21, 10098 Berlin, Germany.
Doebis
Cornelia
C
Busch
Annette
A
Ritter
Thomas
T
Mhashilkar
Abner
A
Marasco
Wayne M
WM
Laube
Horst
H
Volk
Hans-Dieter
HD
Seifert
Martina
M
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
D015395:Histocompatibility Antigens Class I; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D021382:Protein Sorting Signals; C073762:lysyl-aspartyl-glutamyl-leucine