Human Immune Reactivity against Liver Sinusoidal Endothelial Cells from GalTα(1,3)GalT-Deficient Pigs

Abstract

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.