Late graft failure of pig-to-rhesus renal xenografts has features of glomerulopathy and recipients have anti-swine leukocyte antigen class I and class II antibodies.

Prolonged survival in preclinical renal xenotransplantation demonstrates that early antibody mediated rejection (AMR) can be overcome. It is now critical to evaluate and understand the pathobiology of late graft failure and devise new means to improve post xenograft outcomes. In renal allotransplantation the most common cause of late renal graft failure is transplant glomerulopathy-largely due to anti-donor MHC antibodies, particularly anti-HLA DQ antibodies. We evaluated the pig renal xenograft pathology of four long-surviving (>300 days) rhesus monkeys. We also evaluated the terminal serum for the presence of anti-SLA class I and specifically anti-SLA DQ antibodies. All four recipients had transplant glomerulopathy and expressed anti-SLA DQ antibodies. In one recipient tested for anti-SLA I antibodies, the recipient had antibodies specifically reacting with two of three SLA I alleles tested. These results suggest that similar to allotransplantation, anti-MHC antibodies, particularly anti-SLA DQ, may be a barrier to improved long-term xenograft outcomes.

Patients on the Transplant Waiting List Have Anti-Swine Leukocyte Antigen Class I Antibodies.

Organ supply remains inadequate to meet the needs of many patients who could benefit from allotransplantation. Xenotransplantation, the use of animals as organ donors, provides an opportunity to alleviate this challenge. Pigs are widely accepted as the ideal organ donor, but humans and nonhuman primates have strong humoral immune responses to porcine tissue. Although carbohydrate xenoantigens have been studied intensively, the primate Ab response also targets class I and class II swine leukocyte Ags (SLAs). Human Abs that recognize HLAs can cross-react with SLA molecules because epitopes can be shared across species. However, ∼15% of people may also exhibit Abs toward class II SLAs despite lacking Abs that also recognize class II HLAs. Here, we extend these studies to better understand human Ab responses toward class I SLAs. When tested against a panel of 18 unique class I SLA proteins, 14 of 52 sera samples collected from patients in need of an organ transplant contained Abs that bound class I SLAs. Class I SLA-reactive sera may contain IgM only, IgG, only, or IgM and IgG capable of recognizing the pig proteins. The presence of class I HLA-reactive Abs was not essential to generating anti-class I SLA Ig. Last, anti-class I SLA reactivity varied by serum; some recognized a single SLA allele, whereas others recognized multiple class I SLA proteins.

Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs.

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, 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-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.

Importance of the Major Histocompatibility Complex (Swine Leukocyte Antigen) in Swine Health and Biomedical Research.

In pigs, the major histocompatibility complex (MHC), or swine leukocyte antigen (SLA) complex, maps to Sus scrofa chromosome 7. It consists of three regions, the class I and class III regions mapping to 7p1.1 and the class II region mapping to 7q1.1. The swine MHC is divided by the centromere, which is unique among mammals studied to date. The SLA complexspans between 2.4 and 2.7 Mb, depending on haplotype, and encodes approximately 150 loci, with at least 120 genes predicted to be functional. Here we update the whole SLA complex based on the Sscrofa11.1 build and annotate the organization for all recognized SLA genes and their allelic sequences. We present SLA nomenclature and typing methods and discuss the expression of SLA proteins, as well as their role in antigen presentation and immune, disease, and vaccine responses. Finally, we explore the role of SLA genes in transplantation and xenotransplantation and their importance in swine biomedical models.

Life-supporting Kidney Xenotransplantation From Genetically Engineered Pigs in Baboons: A Comparison of Two Immunosuppressive Regimens.

BACKGROUND: The aims of this study were to evaluate the efficacy of US Food and Drug Administration-approved drugs in genetically engineered pig-to-baboon kidney xenotransplantation and compare the results with those using an anti-CD40 monoclonal antibody (mAb)-based regimen. METHODS: Ten life-supporting kidney transplants were carried out in baboons using α1,3-galactosyltransferase gene-knockout/CD46 pigs with various other genetic manipulations aimed at controlling coagulation dysregulation. Eight transplants resulted in informative data. Immunosuppressive therapy consisted of induction with antithymocyte globulin and anti-CD20mAb, and maintenance based on either (1) CTLA4-Ig and/or tacrolimus (+rapamycin or mycophenolate mofetil) (GroupA [US Food and Drug Administration-approved regimens], n = 4) or (2) anti-CD40mAb + rapamycin (GroupB, n = 4). All baboons received corticosteroids, interleukin-6R blockade, and tumor necrosis factor-α blockade. Baboons were followed by clinical and laboratory monitoring of kidney function, coagulation, and immune parameters. At euthanasia, morphological and immunohistochemical studies were performed on the kidney grafts. RESULTS: The median survival in GroupB was 186 days (range 90-260), which was significantly longer than in GroupA; median 14 days (range 12-32) (P < 0.01). Only GroupA baboons developed consumptive coagulopathy and the histopathological features of thrombotic microangiopathic glomerulopathy and interstitial arterial vasculitis. CONCLUSIONS: Recognizing that the pig donors in each group differed in some genetic modifications, these data indicate that maintenance immunosuppression including anti-CD40mAb may be important to prevent pig kidney graft failure.

Data on B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy.

This report is related to the research article entitled "B cell phenotypes in baboons with pig artery patch grafts receiving conventional immunosuppressive therapy" (Yamamoto et al., in press). Herein we provide the data regarding pig artery patch xenotransplantation into the baboon׳s aorta, trough levels of tacrolimus and rapamycin in the blood after transplantation, analysis of B cell phenotype on the basis of IgD and CD27 expression in the blood, and analysis of T cell phenotype on the basis of CD28 and CD95 expression in the blood.

Xenotransplantation panel for the detection of infectious agents in pigs.

BACKGROUND: Recent advances in xenotransplantation have produced organs from pigs that are well tolerated in primate models because of genetic changes engineered to delete major antigens from donor animals. To ensure the safety of human transplant recipients, it will be essential to understand both the spectrum of infectious agents in donor pigs and their potential to be transmitted to immunocompromised transplant recipients. Equally important will be the development of new highly sensitive diagnostic methods for use in the detection of these agents in donor animals and for the monitoring of transplant recipients. METHODS: Herein, we report the development of a panel of 30 quantitative polymerase chain reaction (qPCR) assays for infectious agents with the potential to be transmitted to the human host. The reproducibility, sensitivity and specificity of each assay were evaluated and were found to exhibit analytic sensitivity that was similar to that of quantitative assays used to perform viral load testing of human viruses in clinical laboratories. RESULTS: This analytical approach was used to detect nucleic acids of infectious agents present in specimens from 9 sows and 22 piglets derived by caesarean section. The most commonly detected targets in adult animals were Mycoplasma species and two distinct herpesviruses, porcine lymphotrophic herpesvirus 2 and 3. A total of 14 piglets were derived from three sows infected with either or both herpesviruses, yet none tested positive for the viruses indicating that vertical transmission of these viruses is inefficient. CONCLUSIONS: The data presented demonstrate that procedures in place are highly sensitive and can specifically detect nucleic acids from target organisms in the panel, thus ensuring the safety of organs for transplantation as well as the monitoring of patients potentially receiving them.

CRISPR/Cas and recombinase-based human-to-pig orthotopic gene exchange for xenotransplantation.

BACKGROUND: Tools for genome editing in pigs are improving rapidly so that making precise cuts in DNA for the purposes of deleting genes is straightforward. Development of means to replace pig genes with human genes with precision is very desirable for the future development of donor pigs for xenotransplantation. MATERIALS AND METHODS: We used Cas9 to cut pig thrombomodulin (pTHBD) and replace it with a plasmid containing a promoterless antibiotic selection marker and the exon for human thrombomodulin. PhiC31 recombinase was used to remove the antibiotic selection marker to create porcine aortic endothelial cells expressing human instead of pTHBD, driven by the endogenous pig promoter. RESULTS: The promoterless selection cassette permitted efficient enrichment of cells containing correctly inserted transgene. Recombinase treatment of selected cells excised the resistance marker permitting expression of the human transgene by the endogenous pTHBD promoter. Gene regulation was maintained after gene replacement because pig endogenous promoter was kept intact in the correct position. CONCLUSIONS: Cas9 and recombinase technology make orthotopic human for pig gene exchange feasible and pave the way for creation of pigs with human genes that can be expressed in the appropriate tissues preserving gene regulation.

Examining the Biosynthesis and Xenoantigenicity of Class II Swine Leukocyte Antigen Proteins.

Genetically engineered pig organs could provide transplants to all patients with end-stage organ failure, but Ab-mediated rejection remains an issue. This study examines the class II swine leukocyte Ag (SLA) as a target of epitope-restricted Ab binding. Transfection of individual α- and ß-chains into human embryonic kidney cells resulted in both traditional and hybrid class II SLA molecules. Sera from individuals on the solid organ transplant waiting list were tested for Ab binding and cytotoxicity to this panel of class II SLA single-Ag cells. A series of elution studies from an SLA-DQ cell line were performed. Our results indicate that human sera contain Abs specific for and cytotoxic against class II SLA. Our elution studies revealed that sera bind the SLA-DQ molecule in an epitope-restricted pattern. Site-specific mutation of one of these epitopes resulted in statistically decreased Ab binding. Humans possess preformed, specific, and cytotoxic Abs to class II SLA that bind in an epitope-restricted fashion. Site-specific epitope mutagenesis may decrease the Ab binding of highly sensitized individuals to pig cells.

Swine Leukocyte Antigen Class II Is a Xenoantigen.

BACKGROUND: Over 130 000 patients in the United States alone need a lifesaving organ transplant. Genetically modified porcine organs could resolve the donor organ shortage, but human xenoreactive antibodies destroy pig cells and are the major barrier to clinical application of xenotransplantation. The objective of this study was to determine whether waitlisted patients possess preformed antibodies to swine leukocyte antigen (SLA) class II, homologs of the class II HLA. METHODS: Sera from people currently awaiting solid organ transplant were tested for IgG binding to class II SLA proteins when expressed on mammalian cells. Pig fibroblasts were made positive by transfection with the class II transactivator. As a second expression system, transgenes encoding the alpha and beta chains of class II SLA were transfected into human embryonic kidney cells. RESULTS: Human sera containing IgG specific for class II HLA molecules exhibited greater binding to class II SLA positive cells than to SLA negative cells. Sera lacking antibodies against class II HLA showed no change in binding regardless of the presence of class II SLA. These antibodies could recognize either SLA-DR or SLA-DQ complexes. CONCLUSIONS: Class II SLA proteins may behave as xenoantigens for people with humoral immunity toward class II HLA molecules.

Efficient generation of targeted and controlled mutational events in porcine cells using nuclease-directed homologous recombination.

BACKGROUND: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. METHODS: Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by Sanger sequence, restriction enzyme digest and flow cytometry. RESULTS: Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. CONCLUSIONS: A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.

A Novel Approach in Combined Liver and Kidney Transplantation With Long-term Outcomes.

OBJECTIVE: The aim of this study was to compare the outcomes of simultaneous and delayed implantation of kidney grafts in combined liver-kidney transplantation (CLKT). BACKGROUND DATA: Delayed function of the renal graft (DGF), which can result from hypotension and pressor use related to the liver transplantation (LT), may cause worse outcomes in CLKT. METHODS: A total of 130 CLKTs were performed at Indiana University between 2002 and 2015 and studied in an observational cohort study. All kidneys underwent continuous hypothermic pulsatile machine perfusion until transplant: 69 with simultaneous kidney transplantation (KT) (at time of LT, group 1) and 61 with delayed KT (performed at a later time as a second operation, group 2). All patients received continuous veno-venous hemodialysis during the LT. Propensity score match analysis in a 1:1 case-match was performed. RESULTS: Mean kidney cold ischemia time was 10 ± 3 and 50 ± 15 hours, for groups 1 and 2 (P < 0.0001), respectively. The rate of DGF was 7.3% in group 1, but no DGF was seen in group 2 (P = 0.0600). Kidney function was significantly better in group 2, if the implantation of kidneys was delayed >48 hours (P < 0.01). Patient survival was greater in group 2 at 1 year (91%), and 5 year (87%) post-transplantation (P = 0.0019). On multivariate analysis, DGF [hazard ratio (HR), 165.7; 95% confidence interval (CI), 9.4-2926], extended criteria donor kidneys (HR, 15.9; 95% CI 1.8-145.2), and recipient hepatitis C (HR, 5.5; 95% CI 1.7-17.8) were significant independent risk factors for patient survival. CONCLUSIONS: Delayed KT in CLKT (especially if delayed >48 h) is associated with improved kidney function with no DGF post-KT, and improved patient and graft survival.

Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs.

BACKGROUND: Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA). METHODS: Peripheral blood mononuclear cells from SLA identical wild type (WT), α1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads. RESULTS: Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns. CONCLUSIONS: Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.

Eliminating Xenoantigen Expression on Swine RBC.

BACKGROUND: The rapidly improving tools of genetic engineering may make it possible to overcome the humoral immune barrier that prevents xenotransplantation. We hypothesize that levels of human antibody binding to donor tissues from swine must approximate the antibody binding occurring in allotransplantation. It is uncertain if this is an attainable goal. Here we perform an initial analysis of this issue by comparing human antibody binding to red blood cells (RBC) isolated from knockout swine and to allogeneic or autologous human RBC. METHODS: Human sera were incubated with RBC isolated from various genetically engineered swine or from humans. The level of IgG and IgM binding to these cells were compared using either flow cytometry or a novel mass spectrometric assay. RESULTS: Mass spectroscopic quantitation of human antibody binding demonstrated that as few as 3 gene inactivations can reduce the levels human antibody binding to swine RBC that is as low as autologous human RBC. Flow cytometry showed that RBC from 2-gene knockout swine exhibited less human antibody binding than human blood group O allogeneic RBC in 22% of tested sera. Deletion of a third gene from pigs resulted in 30% of human samples having less IgG and IgM RBC xenoreactivity than alloreactivity. CONCLUSIONS: Xenoantigenicity of swine RBC can be eliminated via gene disruption. These results suggest that the gene knockout approach may be able reduce antigenicity in other pig tissues to levels that enable the xenotransplantation humoral barrier to be overcome.

Intestinal Graft Failure: Should We Perform the Allograft Enterectomy Before or With Retransplantation?

BACKGROUND: Intestinal graft dysfunction is sometimes irreversible and requires allograft enterectomy with or without retransplantation. There is no comprehensive assessment of allograft enterectomy regarding indications and outcomes. The aim of this study was to evaluate management of patients with intestinal graft failure with special reference to indications and outcomes of allograft enterectomy and the procedure's validity as a bridge to retransplantation. METHODS: Graft and patient survivals, reason for graft failure, and rejection episodes were evaluated in 221 intestinal recipients (primary transplantation [n = 201], retransplantation [n = 20]). Indications, surgical factors, and outcomes of allograft enterectomy were investigated. RESULTS: Reasons for isolated enterectomy included systemic infection in 11, gastrointestinal bleeding in 1, and severe electrolyte imbalance in 1, all of which were associated with rejection. One isolated intestinal transplantation patient underwent isolated enterectomy due to cytomegalovirus enteritis. One multivisceral transplantation patient underwent isolated allograft enterectomy due to bowel necrosis. Of these 15 patients, 3 died from persistent infection postoperatively, whereas 8 underwent retransplantation with median interval of 74 days (42-252 days). Allosensitization occurred between isolated enterectomy and retransplantation in 2, one of whom lost the second graft due to rejection. Simultaneous allograft enterectomy and retransplantation was performed in 3 isolated intestinal transplantation and 9 multivisceral transplantation patients. Patient survival after retransplantation was similar between patients who underwent isolated allograft enterectomy and those who did simultaneous enterectomy with retransplantation (P = 0.82). CONCLUSIONS: In cases of irreversible intestinal graft dysfunction, isolated allograft enterectomy successfully provides recovery from comorbidities as a lifesaving procedure and does not compromise outcomes of retransplantation.

Pig Liver Xenotransplantation: A Review of Progress Toward the Clinic.

Experience with clinical liver xenotransplantation has largely involved the transplantation of livers from nonhuman primates. Experience with pig livers has been scarce. This brief review will be restricted to assessing the potential therapeutic impact of pig liver xenotransplantation in acute liver failure and the remaining barriers that currently do not justify clinical trials. A relatively new surgical technique of heterotopic pig liver xenotransplantation is described that might play a role in bridging a patient with acute liver failure until either the native liver recovers or a suitable liver allograft is obtained. Other topics discussed include the possible mechanisms for the development of the thrombocytopenis that rapidly occurs after pig liver xenotransplantation in a primate, the impact of pig complement on graft injury, the potential infectious risks, and potential physiologic incompatibilities between pig and human. There is cautious optimism that all of these problems can be overcome by judicious genetic manipulation of the pig. If liver graft survival could be achieved in the absence of thrombocytopenia or rejection for a period of even a few days, there may be a role for pig liver transplantation as a bridge to allotransplantation in carefully selected patients.

Silencing the porcine iGb3s gene does not affect Galα3Gal levels or measures of anticipated pig-to-human and pig-to-primate acute rejection.

BACKGROUND: The Galα(1,3)Gal epitope (α-GAL), created by α-1,3-glycosyltransferase-1 (GGTA1), is a major xenoantigen causing hyperacute rejection in pig-to-primate and pig-to-human xenotransplantation. In response, GGTA1 gene-deleted pigs have been generated. However, it is unclear whether there is a residual small amount of α-Gal epitope expressed in GGTA1(-/-) pigs. Isoglobotrihexosylceramide synthase (iGb3s), another member of the glycosyltransferase family, catalyzes the synthesis of isoglobo-series glycosphingolipids with an α-GAL-terminal disaccharide (iGb3), creating the possibility that iGb3s may be a source of α-GAL epitopes in GGTA1(-/-) animals. The objective of this study was to examine the impact of silencing the iGb3s gene (A3GalT2) on pig-to-primate and pig-to-human immune cross-reactivity by creating and comparing GGTA1(-/-) pigs to GGTA1(-/-) - and A3GalT2(-/-) -double-knockout pigs. METHODS: We used the CRISPR/Cas 9 system to target the GGTA1 and A3GalT2 genes in pigs. Both GGTA1 and A3GalT2 genes are functionally inactive in humans and baboons. CRISPR-treated cells used directly for somatic cell nuclear transfer produced single- and double-gene-knockout piglets in a single pregnancy. Once grown to maturity, the glycosphingolipid profile (including iGb3) was assayed in renal tissue by normal-phase liquid chromatography. In addition, peripheral blood mononuclear cells (PBMCs) were subjected to (i) comparative cross-match cytotoxicity analysis against human and baboon serum and (ii) IB4 staining for α-GAL/iGb3. RESULTS: Silencing of the iGb3s gene significantly modulated the renal glycosphingolipid profile and iGb3 was not detected. Moreover, the human and baboon serum PBMC cytotoxicity and α-GAL/iGb3 staining were unchanged by iGb3s silencing. CONCLUSIONS: Our data suggest that iGb3s is not a contributor to antibody-mediated rejection in pig-to-primate or pig-to-human xenotransplantation. Although iGb3s gene silencing significantly changed the renal glycosphingolipid profile, the effect on Galα3Gal levels, antibody binding, and cytotoxic profiles of baboon and human sera on porcine PBMCs was neutral.

Immunogenicity of Renal Microvascular Endothelial Cells From Genetically Modified Pigs.

BACKGROUND: Disrupting the porcine GGTA1 and CMAH genes [double knockout (DKO)] that produce the gal-α(1,3)-gal and N-glycolylneuraminic acid xenoantigens reduces human antibody binding to porcine peripheral blood mononuclear cells. It is important to examine rejection pathways at an organ-specific level. The object of this study is to evaluate the human preformed antibody reactivity against DKO renal microvascular endothelial cells (RMEC) in vitro. METHODS: Characteristics of DKO RMEC were analyzed using flow cytometry. Human IgG/M binding to primary RMEC, immortalized RMEC (iRMEC), and iRMEC-deficient in B4GALNT2 genes were examined using flow cytometric crossmatch assay. RESULTS: Porcine RMEC expressed gal-α(1,3)-gal, N-glycolylneuraminic acid, and Dolichos biflorus agglutinin glycans recognized by human preexisting antibodies in humans. Antigenicity of DKO RMEC was lower than GGTA1 KO RMEC. The disruption of B4GALNT2 gene in DKO iRMEC further reduced human IgG/IgM binding. CONCLUSIONS: Silencing the porcine GGTA1, CMAH, and B4GALNT2 genes is an effective strategy to reduce human preformed antibody binding to RMEC. Porcine RMEC will be a useful reagent for the further study of xenoimmunology.