Byrne, Guerard W.

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.

Cardiac xenotransplantation (CXTx) is a promising solution to the chronic shortage of donor hearts. Recent advancements in immune suppression have greatly improved the survival of heterotopic CXTx, now extended beyond 2 years, and life-supporting kidney XTx. Advances in donor genetic modification (B4GALNT2 and CMAH mutations) with proven Gal-deficient donors expressing human complement regulatory protein(s) have also accelerated, reducing donor pig organ antigenicity. These advances can now be combined and tested in life-supporting orthotopic preclinical studies in nonhuman primates and immunologically appropriate models confirming their efficacy and safety for a clinical CXTx program. Preclinical studies should also allow for organ rejection to develop xenospecific assays and therapies to reverse rejection. The complexity of future clinical CXTx presents a substantial and unique set of regulatory challenges which must be addressed to avoid delay; however, dependent on these prospective life-supporting preclinical studies in NHPs, it appears that the scientific path forward is well defined and the era of clinical CXTx is approaching.

AbstractBackgroundRejection of Gal‐free (GTKO) donor pig cardiac xenografts is strongly associated with vascular non‐Gal antibody binding, endothelial cell (EC) injury, and activation and microvascular thrombosis. We adopted a pig‐to‐SCID/beige small animal transplant model to compare the pathogenicity of baboon and human anti‐pig antibody.MethodsWild‐type (GT+) or GTKO porcine coronary arteries (PCAs) were transplanted into the infrarenal aorta of SCID/beige mice. Three days after transplant, recipients were infused with anti‐pig antibody (anti‐SLA class I, an isotype control, naive or sensitized baboon serum, or naive human serum). PCAs were recovered 24 h after antibody infusion and examined using histology, immunohistochemistry, and in situ hybridization.ResultsDose‐dependent intragraft thrombosis occurred after infusion of anti‐SLA I antibody (but not isotype control) in GT+ and GTKO PCA recipients. Naive baboon serum induced thrombosis in GT+ grafts. Thrombosis was significantly reduced by pre‐treating naive baboon serum with Gal polymer and not observed when this serum was infused to GTKO PCA recipients. Naive human serum caused dose‐dependent intragraft thrombosis of GTKO PCAs. In all cases, thrombosis involved graft‐specific vascular antibody and complement deposition, macrophage adherence, EC delamination, and subendothelial thrombus formation.ConclusionsThis study provides the first direct in vivo comparison of the pathogenicity of naive human and baboon serum. The results suggest that human preformed non‐Gal antibody may have increased pathogenicity compared to baboon. This model, which showed a rejected graft histopathology similar to antibody‐mediated rejection in cardiac xenotransplantation, may be useful to assess the pathogenicity of individual protein or carbohydrate specific non‐Gal reactive antibodies.

Abstract Background Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non‐ G alα(1,3) G al (non‐ G al) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized Gal TKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Methods Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non‐ G al pig antigens using primary porcine aortic endothelial cells ( pAEC s) and cell culture‐adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (Gal TKO ) swine. Results The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture‐adapted Gal TKO pAEC KO :15502 cells as a routine method to determine the reactivity of anti‐non‐ G al antibodies in human and baboon serum. Conclusions We have developed an assay that allows the detection of natural and induced non‐Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non‐ G al antibody responses.

Abstract Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA‐1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non‐Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNA s which encode pig antigens that bind to IgG induced after pig‐to‐primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N‐acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK‐B4T) was produced. Expression of porcine B4GALNT2 in HEK‐B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK‐B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK‐B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non‐Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4 GALNT 2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4 GALNT 2 genes, respectively. The B4 GALNT 2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4 GALNT 2 in human HEK cells ( HEK ‐B4T) results in increased binding of antibody to the B4 GALNT 2 enzyme, and increased reactivity with anti‐Sd a and DBA . HEK ‐B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO : CD 55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non‐Gal antibody, measured using GTKO PAEC s, and the induction of antibodies which preferentially bind to HEK ‐B4T cells. Conclusion The functional isolation of the porcine B4 GALNT 2 gene from a PAEC expression library, the pattern of B4 GALNT 2 gene expression and its sensitization of HEK ‐B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4 GALNT 2 produces a new immunogenic non‐Gal glycan which contributes in part to the non‐Gal immune response detected after pig‐to‐baboon cardiac xenotransplantation.

AbstractThe histopathology of cardiac xenograft rejection has evolved over the last 20 yr with the development of new modalities for limiting antibody‐mediated injury, advancing regimens for immune suppression, and an ever‐widening variety of new donor genetics. These new technologies have helped us progress from what was once an overwhelming anti‐Gal‐mediated hyperacute rejection to a more protracted anti‐Gal‐mediated vascular rejection to what is now a more complex manifestation of non‐Gal humoral rejection and coagulation dysregulation. This review summarizes the changing histopathology of Gal‐ and non‐Gal‐mediated cardiac xenograft rejection and discusses the contributions of immune‐mediated injury, species‐specific immune‐independent factors, transplant and therapeutic procedures, and donor genetics to the overall mechanism(s) of cardiac xenograft rejection.

Abstract Classic hyperacute rejection is dependent on the activation of the terminal components of complement. Recently, xenoantibodies with limited abilities to activate the classical pathway of complement in vitro have been implicated in the acute vascular rejection of xenografts. It is unclear how these Abs affect their pathogenic activities in vivo. In this study, we demonstrate the ability of an anti-Gal-α1,3Gal (Gal) IgG1, with modest complement-activating abilities in vitro, to induce xenograft rejection. This rejection was dependent on the activation of complement, on FcγR-mediated interactions, and on the presence of NK cells. Inhibition of any one of these factors resulted in the abrogation of IgG1-mediated rejection. In contrast, an anti-Gal IgG3 mAb induced classic, hyperacute rejection that was solely dependent on complement activation. Our observations implicate two types of IgG-mediated rejection; one that is dependent on complement activation, and a second that is uniquely dependent on complement, FcγR, and NK cells.

Abstract As immune responses likely contribute to degeneration of bioprosthetic heart valves (BHVs), this nonhuman primate study compares the anti-Gal antibody response to BHVs from wild type (WT) pigs (current BHVs) and those from α-galactosyltransferase knockout (GTKO) pigs. Stented glutaraldehyde fixed BHVs from WT (n=4) and GTKO (n=3) pigs were commercially manufactured and implanted in the mitral position in nonhuman primates using standard surgical techniques. Recipients were treated with Lovenox (1mg/kg BID) for 5 weeks reducing to 1mg/kg daily for one week and then discontinued. Recipients were sensitized to the αGal antigen by immunization to match IgG levels found in humans. Serum antibody responses were monitored by ELISA. WT BHVs were explanted at 3 hours, 1 year, 2 years and the fourth is currently ongoing at 3 years. One GTKO BHV was explanted at 218 days. The remaining two GTKO BHVs are currently ongoing at 3 years. After immunization, circulating anti-Gal IgG levels were comparable in both groups at the time of implantation and were equal or greater than human levels. Anti-Gal antibody levels decreased in both WT and GTKO recipients after implantation. WT recipients, however, retained elevated levels (greater than 20% preimplant values) of anti-Gal IgG for at least the first year post implant. Conversely anti-Gal IgG levels in all GTKO recipients fell within one month and remained at less than 20% preimplant values. Analysis of the area under the curve showed a significant increase of anti-Gal IgG in the WT BHV group compared to GTKO BHV recipients (p<0.01). In this nonhuman primate model, the persistently and significantly (p<0.01) elevated levels of anti-Gal IgG antibody observed in WT but not GTKO BHV recipients, indicate continuing immune stimulation to the αGal antigen. Current commercially available BHVs are similarly known to contain αGal antigen. Anti-Gal antibody has been shown to increase calcification of processed pericardium in the rat implant model. These data support the hypothesis that preformed and induced anti-Gal antibody in BHV recipients may initiate an early immune response, which promotes subsequent calcification. BHVs made from GTKO pigs would eliminate this major xenoreactive antigen and provide a prosthesis with reduced immunogenicity. Such BHVs made from GTKO pigs may have greater durability and be potentially used in younger patients with more active immune systems.