Aberrant expression of alpha-Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion.
The contribution of Gal alpha 1,3Gal (alpha-Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine alpha 1,3 galactosyltransferase (alpha 1,3GalT) to obtain alpha-Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post-transduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF-alpha and IFN-gamma stimulation. No differences were detected between non-alpha-Gal- and alpha-Gal-expressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the alpha-Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.
He
Zhong
Z
Division of Clinical Immunology, Karolinska Institutet, Huddinge University Hospital AB, Stockholm, Sweden.
Ehrnfelt
Cecilia
C
Kumagai-Braesch
Makiko
M
Islam
Khalid B
KB
Holgersson
Jan
J
D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D004187:Disaccharides / Q000096:biosynthesis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007371:Interferon-gamma / Q000096:biosynthesis; D007694:Killer Cells, Natural / Q000276:immunology*; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D014161:Transduction, Genetic
D004187:Disaccharides; D008164:Luminescent Proteins; C055075:galactosyl-(1-3)galactose; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma