Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells.
BACKGROUND
The generation of Galalpha1-3Gal (Gal) transferase deficient pigs has increased the interest in non-Gal antigens potentially representing important targets for xenoreactive antibody binding leading to xenograft rejection. The present study addressed the levels and immunoglobulin isotypes of preformed human anti-non-Gal antibodies and their potential to activate porcine endothelial cells.


METHODS
Porcine endothelial cells lacking the Gal epitope (Gal-/-) were used to measure immunoglobulin (Ig) M and IgG subclass anti-non-Gal antibodies, using sera from 80 blood donors and pooled human AB serum. Antibodies specific for the non-Gal Hanganutziu-Deicher (HD) xenoantigen were measured by enzyme-linked immunosorbent assay. Activation of Gal-/- and Gal+/+ endothelial cells by human serum was measured, in the presence or absence of complement inhibitors, by E-selectin cell-surface expression using flow cytometry.


RESULTS
Anti-non-Gal antibody levels varied considerably among individual sera and comprised approximately 10% of total anti-porcine antibodies without sex or age differences. Among the IgG subclasses only IgG1 and IgG2 were detected. Human serum-induced E-selectin expression on Gal-/- cells was less than 20% compared with Gal+/+ cells, correlated with anti-HD IgM and IgG antibody levels (P=0.027 and 0.032, respectively), and was largely complement-independent in accordance with the lack of IgG3 anti-non-Gal antibodies. In contrast, E-selectin upregulation on Gal+/+ cells was reduced in complement blocking experiments.


CONCLUSIONS
Preformed anti-non-Gal antibodies, in particular anti-HD antibodies, were present in all human sera samples, activated porcine endothelial cells, and may therefore play a role in xenograft rejection using organs from GalT-/- pigs.
Institute of Immunology, Rikshospitalet-Radiumhospitalet Medical Center and Faculty of Medicine, University of Oslo, Oslo, Norway. marit.sathre@medisin.uio.no
Marit
M
Saethre
Bettina C
BC
Baumann
Michael
M
Fung
Jörg D
JD
Seebach
Tom Eirik
TE
Mollnes
D000328:Adult; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000910:Antibodies, Heterophile / Q000276:immunology / Q000378:metabolism; D015478:Antigens, Heterophile / Q000276:immunology; D001011:Aorta / Q000166:cytology; D017690:Cell Transplantation; D002478:Cells, Cultured; D003165:Complement System Proteins / Q000096:biosynthesis; D017077:Culture Media, Conditioned; D004187:Disaccharides / Q000276:immunology*; D019040:E-Selectin / Q000096:biosynthesis; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D008297:Male; D008875:Middle Aged; D013552:Swine; D014183:Transplantation, Heterologous
D000888:Antibodies, Anti-Idiotypic; D000910:Antibodies, Heterophile; D015478:Antigens, Heterophile; D017077:Culture Media, Conditioned; D004187:Disaccharides; D019040:E-Selectin; D000939:Epitopes; D007074:Immunoglobulin G; D007075:Immunoglobulin M; C055075:galactosyl-(1-3)galactose; D003165:Complement System Proteins