Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells

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. 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. 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. 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.