Antibodies directed at non-gal xenoantigens are responsible for acute humoral xenograft rejection when gal knockout (GalTKO) pig organs are transplanted into non-human primates. We generated IgM and IgG gene libraries using peripheral blood lymphocytes of rhesus monkeys initiating active xenoantibody responses after immunization with GalTKO pig endothelial cells and used these libraries to identify IgVH genes that encode antibody responses to non-gal pig xenoantigens. Immunoglobulin genes derived from the IGHV3-21 germline progenitor encode xenoantibodies directed at non-gal xenoantigens. Transduction of GalTKO cells with lentiviral vectors expressing the porcine α1,3 galactosyltransferase gene responsible for gal carbohydrate expression results in a higher level of binding of “anti-non-gal” xenoantibodies to transduced GalTKO cells expressing the gal carbohydrate, suggesting that anti-non-gal xenoantibodies crossreact with carbohydrate xenoantigens. The galactosyltransferase 2 gene encoding isoglobotriaosylceramide synthase (iGb3 synthase) is not expressed in GalTKO pig cells. Our results demonstrate that anti-non-gal xenoantibodies in primates are encoded by IgVH genes that are restricted to IGHV3-21 and bind to an epitope that is structurally related to but distinct from the Gal carbohydrate.

Purpose of Review-Anti-non-gal xenoantibodies are a major barrier to the survival of genetically modified porcine xenografts. This review summarizes new information on the contribution of antinon-gal xenoantibodies to the activation of porcine endothelial cells and the pathology of rejection and further provides an update on recent advancements in defining the unique features of anti-nongal xenoantibody structure.

Galactoslytransferase‐deficient (GalTKO) pig organs carry great potential as a solution to the shortage of human organ donors. These organs do not initiate hyperacute rejection but undergo acute humoral rejection mediated by xenoantibodies directed at non‐gal xenoantigens. The structure, specificity and germline origin of these antibodies has not been determined.METHODS: Three rhesus monkeys were immunized with sixty million GalTKO pig endothelial cells in order to induce high levels of xenoantibodies directed at GalTKO xenoantigens. The specificity of the immune response to GalTKO xenoantigens was confirmed by flow cytometry. Pre and post transplant peripheral blood B cells were used to prepare IgG and IgM cDNA libraries for identification of the IgVH genes encoding xenoantibodies.RESULTS: IgVH genes encoding anti‐non‐gal xenoantibodies were most closely related to the human germline progenitor V3‐21. The CDR3 region is unique and the J region is most similar to JH4 in 75.8% of the IgG sequences. The germline progenitors encoding anti‐non‐gal xenoantibodies are restricted and are 91% similar to the IGHV3‐11 alleles that encode anti‐carbohydrate xenoantibodies in primates transplanted with wild type and hDAF transgenic pig organs.CONCLUSIONS: The anti‐non‐gal xenoantibody response is restricted to usage of an IgVH repertoire known to encode antibodies to carbohydrates and glycolipids.