Abstract
Analysis of non‐Gal antibody induced after pig‐to‐baboon cardiac xenotransplantation identified the glycan produced by porcine beta‐1,4‐N‐acetyl‐galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti‐SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti‐SDa antibody and increased binding of Dolichos biflorus agglutinin (DBA), a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA, suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2‐KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha‐1,3‐galactosyltransferase deficient (GGTA1‐KO) and cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase deficient (CMAH‐KO), show reduced antibody binding, confirming the presence of B4GALNT2‐dependent antibodies in both humans and non‐human primates. Preclinical xenotransplantation using B4GALNT2‐deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti‐pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.
Highlights
Xenotransplantation (XTx) is limited by recalcitrant antibody- mediated rejection occurring either hyperacutely immediately after transplant (HAR) or at later times, referred to as delayed xenograft rejection.[1,2,3] These rejection mechanisms result from the abundance of human and non-human primate (NHP) antibodies directed to the classic xenogeneic antigen galactose alpha 1,3 galactose (Gal) which leads to chronic or induced antibody-mediated vascular endothelial cell (EC) injury or activation.[4,5] Gal is not expressed in humans or Old World NHPs but is expressed at high levels in porcine tissues
The SDa glycan is commonly expressed in human GI epithelial cells and at widely variant levels in human red blood cell (RBC) and other tissues and fluids
Recent genetic engineering of the porcine B4GALNT2 locus confirms the presence of preformed NHP and human antibody to B4GALNT2-dependent antigens
Summary
Xenotransplantation (XTx) is limited by recalcitrant antibody- mediated rejection occurring either hyperacutely immediately after transplant (HAR) or at later times, referred to as delayed xenograft rejection.[1,2,3] These rejection mechanisms result from the abundance of human and non-human primate (NHP) antibodies directed to the classic xenogeneic antigen galactose alpha 1,3 galactose (Gal) which leads to chronic or induced antibody-mediated vascular endothelial cell (EC) injury or activation.[4,5] Gal is not expressed in humans or Old World NHPs but is expressed at high levels in porcine tissues. Modified pigs, with a mutation in the GGTA-1 locus (GTKO), do not express the Gal antigen.[6] The introduction of GTKO donor organs eliminated anti-Gal-mediated xenograft rejection, but did not eliminate antibody-mediated rejection and instead highlighted the importance of antibody directed to non-Gal pig antigens.[7,8]. Non-Gal antibody in human and NHP serum is reactive to both protein and carbohydrate antigens.[9,10,11,12,13] The currently identified immunogenic EC carbohydrate antigens include Gal, glycans modified with N-glycolylneuraminic acid (Neu5Gc), and a carbohydrate antigen (SDa) produced by the porcine β1,4 N- acetylgalactosaminyltransferase-2 (B4GALNT2). The human blood group A antigen is potentially immunogenic[14]; high rates of A-type blood group polymorphism in the pig permit exclusion of this antigen by selective breeding.[15,16] Collectively, antibody reactivity to the 3 major xenogeneic glycans, Gal, Neu5Gc-modified glycans, and SDa, accounts for the majority of preformed human anti-pig antibody reactivity.[17,18]
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