Background.The most common cause of late graft failure in renal allotransplantation is chronic antibody-mediated rejection caused by donor-specific antibodies against class II human leukocyte antigen (HLA), particularly HLA-DQ. In preclinical renal xenotransplantation, graft failure 1-mo posttransplant is characterized by glomerulopathy and immunoglobulin G (IgG) staining in the glomerulus. Rhesus renal xenograft recipients with late graft failure also have anti-swine leukocyte antigen (SLA)-DQ antibodies present in their serum suggesting that, like allotransplantation, late xenograft failure may be driven by antidonor major histocompatibility complex class II antibodies, particularly SLA-DQ. Some patients have anti-SLA-DQ antibodies, but the magnitude of this problem is unclear.Methods.We evaluated patient sera for the presence of anti-SLA-DQ antibodies in engineered immortalized cells, to determine patients' reactivity toward 7 different SLA-DQ molecules. Next, we created glycoprotein, alpha-galactosyltransferase 1/beta-1,4-N-acetyl-galactosaminyltransferase 2/SLA-DQ knockout (KO) pigs so that we could evaluate the impact of SLA-DQ on the level of antipig antibodies by performing crossmatches with serum from na & iuml;ve and HLA class II-sensitized patients and SLA-DQ KO peripheral blood mononuclear cells.Results.Na & iuml;ve and HLA class II-sensitized patients had anti-SLA-DQ immunoglobulin M and IgG that were pan-specific rather than SLA-DQ allele-specific. Crossmatching patient sera with peripheral blood mononuclear cells from the SLA-DQ KO pigs revealed that many patients had anti-SLA-DQ antibodies. Eliminating SLA-DQ reduced human immunoglobulin M and IgG binding to primary pig cells.Conclusions.SLA-DQ is a xenoantigen for most patients. SLA-DQ KO pigs may help address this problem.

Attack of donor tissues by pre-formed anti-pig antibodies is well known to cause graft failure in xenotransplantation. Genetic engineering of porcine donors to eliminate targets of these pre-formed antibodies coupled with advances in immunosuppressive medicines have now made it possible to achieve extended survival in the pre-clinical pig-to-non-human primate model. Despite these improvements, antibodies remain a risk over the lifetime of the transplant, and many patients continue to have pre-formed donor-specific antibodies even to highly engineered pigs. While therapeutics exist that can help mitigate the detrimental effects of antibodies, they act broadly potentially dampening beneficial immunity. Identifying additional xenoantigens may enable more targeted approaches, such as gene editing, to overcome these challenges by further eliminating antibody targets on donor tissue. Because we have found that classical class I swine leukocyte antigens are targets of human antibodies, we now examine whether related pig proteins may also be targeted by human antibodies. We show here that non-classical class I swine leukocyte proteins (SLA-6, -7, -8) can be expressed at the surface of mammalian cells and act as antibody targets.

ABSTRACTObjectiveTo evaluate the clinically relevant anti‐CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig‐to‐rhesus renal xenograft model.Summary Background DataCD40/CD40L co‐stimulation blockade‐based immunosuppression has been more successful than calcineurin‐based protocols in prolonging xenograft survival in preclinical models.MethodsGGTA1 knockout/CD55 transgenic pig kidneys were transplanted into rhesus monkeys (n = 6) receiving an iscalimab‐based immunosuppressive regimen.ResultsTwo grafts were lost early (22 and 26 days) because of ectatic donor ureters with otherwise normal histology. The other recipients survived 171, 315, 422, and 439 days with good renal function throughout the posttransplant course. None of the recipients experienced serious infectious morbidity.ConclusionsIt may be reasonable to evaluate an iscalimab‐based immunosuppressive regimen in clinical renal xenotransplantation.

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.

Renal transplantation is the preferred treatment of ESKD, but the shortage of suitable donor kidneys from the cadaver pool means that many patients with ESKD will not receive a kidney transplant. Xenotransplantation has long represented a solution to the kidney shortage, but the occurrence of antibody-mediated rejection has precluded its clinical development. Developments in somatic cell nuclear transfer in pigs and gene editing tools have led to the creation of new donor pigs with greatly improved crossmatches to patients. In addition, improvements in preclinical kidney xenotransplant survival using new anti-CD40/CD154-based immunosuppression have pushed xenotransplantation to the point where it is reasonable to consider initiating a clinical trial to evaluate this potential therapy in patients.

Abstract 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.

Pig liver xenotransplantation is limited by a thrombocytopenic coagulopathy that occurs immediately following graft reperfusion. In vitro and ex vivo studies from our lab suggested that the thrombocytopenia may be the result of a species incompatibility in platelet glycosylation. Realization that platelet α-granules contain antibodies caused us to reevaluate whether the thrombocytopenia in liver xenotransplantation could occur because IgM and IgG from inside platelet α-granules bound to pig liver sinusoidal endothelial cells (LSECs). Our in vitro analysis of IgM and IgG from inside α-granules showed that platelets do carry xenoreactive antibodies that can bind to known xenoantigens. This study suggests that thrombocytopenia occurring following liver xenotransplantation could occur because of xenoreactive antibodies tethering human platelets to the pig LSEC enabling the platelet to be phagocytosed. These results suggest genetic engineering strategies aimed at reducing xenoantigens on the surface of pig LSEC will be effective in eliminating the thrombocytopenia that limits survival in liver xenotransplantation.

Objective: Pig-to-primate renal xenotransplantation is plagued by early antibody-mediated graft loss which precludes clinical application of renal xenotransplantation. We evaluated whether temporary complement inhibition with anti-C5 antibody Tesidolumab could minimize the impact of early antibody-mediated rejection in rhesus monkeys receiving pig kidneys receiving costimulatory blockade-based immunosuppression. Methods: Double (Gal and Sd a ) and triple xenoantigen (Gal, Sd a , and SLA I) pigs were created using CRISPR/Cas. Kidneys from DKO and TKO pigs were transplanted into rhesus monkeys that had the least reactive crossmatches. Recipients received anti-C5 antibody weekly for 70 days, and T cell depletion, anti-CD154, mycophenolic acid, and steroids as baseline immunosuppression (n = 7). Control recipients did not receive anti-C5 therapy (n = 10). Results: Temporary anti-C5 therapy reduced early graft loss secondary to antibody-mediated rejection and improved graft survival ( P < 0.01). Deleting class I MHC (SLA I) in donor pigs did not ameliorate early antibody-mediated rejection (table). Anti-C5 therapy did not allow for the use of tacrolimus instead of anti-CD154 (table), prolonging survival to a maximum of 62 days. Conclusion: Inhibition of the C5 complement subunit prolongs renal xenotransplant survival in a pig to non-human primate model.

Seventy to ninety percentage of preformed xenoreactive antibodies in human serum bind to the galactose-alpha(1,3)-galactose Gal epitope, and the creation of Gal knockout (KO) pigs has eliminated hyperacute rejection as a barrier to xenotransplantation. Now other glycan antigens are barriers to move ahead with xenotransplantation, and the N-glycolyl neuraminic acid, Neu5Gc (or Hanganutziu-Deicher antigen), is also a major pig xenoantigen. Humans have anti-Neu5Gc antibodies. Several data indicate a strong immunogenicity of Neu5Gc in humans that may contribute to an important part in antibody-dependent injury to pig xenografts. Pig islets express Neu5Gc, which reacted with diet-derived human antibodies and mice deleted for Neu5Gc reject pancreatic islets from wild-type counterpart. However, Neu5Gc positive heart were not rejected in Neu5Gc KO mice indicating that the role of Neu5Gc-specific antibodies has to be nuanced and depend of the graft situation parameters (organ/tissue, recipient, implication of other glycan antigens). Recently generated Gal/Neu5Gc KO pigs eliminate the expression of Gal and Neu5Gc, and improve the crossmatch of humans with the pig. This review summarizes the current and recent experimental and (pre)clinical data on the Neu5Gc immunogenicity and emphasize of the potential impact of anti-Neu5Gc antibodies in limiting xenotransplantation in humans.

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti-pig antibodies significantly improved survival in a pig-to-rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan-T cell depletion and an anti-CD154-based maintenance regimen. Selective depletion of CD4+ T cells but not CD8+ T cells resulted in long-term survival (median survival time >400 days vs 6 days). These studies suggested that CD4+ T cells may have a more prominent role in xenograft rejection compared with CD8+ T cells. Although animals that received selective depletion of CD8+ T cells showed signs of early cellular rejection (marked CD4+ infiltrates), animals receiving selective CD4+ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4+ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti-pig antibody and CD4 depletion resulted in consistent, long-term xenograft survival.

The humoral barrier has been the limiting factor in moving xenotransplantation towards the clinic. Improvements in somatic cell nuclear transfer and genome editing, particularly CRISPR-Cas9, have made it possible to create pigs with multiple glycan xenoantigen deletions for the purposes of reducing xenoreactive antibody binding to the xenografted organ. Recent studies have also considered the aetiology and existence of antibodies directed at the swine leucocyte antigen (SLA) complex, and potential genetic engineering strategies to avoid these antibodies. Evaluation of xenoreactive antibody binding is very important for the advancement of xenotransplantation, because if patients do not have any detectable xenoreactive antibody, then it is reasonable to expect that cellular rejection and not antibody-mediated rejection (AMR) will be the next hurdle to clinical application.

Background. Highly sensitized patients are difficult to match with suitable renal allograft donors and may benefit from xenotransplant trials. We evaluate antibody binding from sensitized patients to pig cells and engineered single allele cells to identify anti-human leukocyte antigen (HLA) antibody cross-species reactivity with swine leukocyte antigen (SLA). These novel testing strategies assess HLA/SLA epitopes and antibody-binding patterns and introduce genetic engineering of SLA epitopes. Methods. Sensitized patient sera were grouped by calculated panel reactive antibody and luminex single antigen reactivity profile and were tested with cloned GGTA1/CMAH/B4GalNT2 glycan knockout porcine cells. Pig reactivity was assessed by direct flow cytometric crossmatch and studied following elution from pig cells. To study the antigenicity of individual class I HLA and SLA alleles in cells, irrelevant sera binding to lymphoblastoid cells were minimized by CRISPR/Cas9 elimination of endogenous class I and class II HLA, B-cell receptor, and Fc receptor genes. Native HLA, SLA, and mutants of these proteins after mutating 144K to Q were assessed for antibody binding. Results. Those with predominately anti-HLA-B&C antibodies, including Bw6 and Bw4 sensitization, frequently have low pig reactivity. Conversely, antibodies eluted from porcine cells are more commonly anti-HLA-A. Single HLA/SLA expressing engineered cells shows variable antigenicity and mutation of 144K to Q reduces antibody binding for some sensitized patients. Conclusions. Anti-HLA antibodies cross-react with SLA class I in predictable patterns, which can be identified with histocompatibility strategies, and SLA class I is a possible target of genetic engineering.

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.

Xenotransplantation of pig organs into people may help alleviate the critical shortage of donors which faces organ transplantation. Unfortunately, human antibodies vigorously attack pig tissues preventing the clinical application of xenotransplantation. The swine leukocyte antigens (SLA), homologs of human HLA molecules, can be xenoantigens. SLA molecules, encoded by genes in the pig major histocompatibility complex, contribute to protective immune responses in pig. Therefore, simply inactivating them through genome engineering could reduce the ability of the human immune system to surveil transplanted pig organs for infectious disease or the development of neoplasms. A potential solution to this problem is to identify and modify epitopes in SLA proteins to eliminate their contribution to humoral xenoantigenicity while retaining their biosynthetic competence and ability to contribute to protective immunity. We previously showed that class II SLA proteins were recognized as xenoantigens and mutating arginine at position 55 to proline, in an SLA-DQ beta chain, could reduce human antibody binding. Here, we extend these observations by creating several additional point mutants at position 55. Using a panel of monoclonal antibodies specific for class II SLA proteins, we show that these mutants remain biosynthetically competent. Examining antibody binding to these variants shows that point mutagenesis can reduce, eliminate, or increase antibody binding to class II SLA proteins. Individual mutations can have opposite effects on antibody binding when comparing samples from different people. We also performed a preliminary analysis of creating point mutants near to position 55 to demonstrate that manipulating additional residues also affects antibody reactivity.

Objective: Xenotransplantation using pig organs could end the donor organ shortage for transplantation, but humans have xenoreactive antibodies that cause early graft rejection. Genome editing can eliminate xenoantigens in donor pigs to minimize the impact of these xenoantibodies. Here we determine whether an improved cross-match and chemical immunosuppression could result in prolonged kidney xenograft survival in a pig-to-rhesus preclinical model. Methods: Double xenoantigen (Gal and Sda) knockout (DKO) pigs were created using CRISPR/Cas. Serum from rhesus monkeys (n = 43) was cross-matched with cells from the DKO pigs. Kidneys from the DKO pigs were transplanted into rhesus monkeys (n = 6) that had the least reactive cross-matches. The rhesus recipients were immunosuppressed with anti-CD4 and anti-CD8 T-cell depletion, anti-CD154, mycophenolic acid, and steroids. Results: Rhesus antibody binding to DKO cells is reduced, but all still have positive CDC and flow cross-match. Three grafts were rejected early at 5, 6, and 6 days. Longer survival was achieved in recipients with survival to 35, 100, and 435 days. Each of the 3 early graft losses was secondary to IgM antibody-mediated rejection. The 435-day graft loss occurred secondary to IgG antibody-mediated rejection. Conclusions: Reducing xenoantigens in donor pigs and chemical immunosuppression can be used to achieve prolonged renal xenograft survival in a preclinical model, suggesting that if a negative cross-match can be obtained for humans then prolonged survival could be achieved.

Martens, Gregory MD; Ladowski, Joseph M. PhD; Reyes, Luz M. MSc, PhD; Easlick, Juliet; Tector, Matt PhD; Tector, A Joseph MD, PhD, FACS Author Information