Elevated Porcine α-1,6 Fucose On GGTA1/CMAH Knockout Pigs Is a Target of Cytolytic Pre-Formed Human Antibodies.

Abstract

Overcoming human antibody-mediated rejection in pigs has entirely been the result of modifying genes that produce unique carbohydrate epitopes. Pigs deficient in galactose a-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) attract extremely low amounts of human antibody and may be excellent candidates for xenotransplantation. To determine if additional carbohydrate epitopes account for the remaining xenoreactive antibody we identified glycans that were differentially expressed between humans and domestic and genetically modified pigs. The fucosyltransferase gene, FUT8, gives rise to core linked α-1,6 fucose that is more abundant in pigs. To validate the α-1,6 fucose phenotype observed previously we compared lectin affinity of human and pig serum glycoproteins samples by dot blot analysis and confocal microscopy. The expression of fucosylation related genes were assessed by quantitative PCR. Human anti-fucose antibody was isolated by affinity chromatography and tested for specificity to L-fucose by custom macro array. The affinity and cytotoxicity of the isolated human anti-fucose antibody towards human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-1,6 fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-1,6 fucose and may represent focal sites for antibody-mediated rejection. The expression of fucosylation genes in pigs supports the increased addition of α-1,6 fucose while de-emphasizing removal of α-1,6 fucose during synthesis. Isolated human anti-fucose IgA, IgG and IgM are specific for fucose. The isolated human IgG cross-reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-1,6 fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.