Non-Classical Swine Leukocyte Antigens SLA-6, -7, and -8, Are Xenoantigens for Some Waitlisted Patients.

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.

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.

Xenoreactive antibodies in α-granules of human platelets bind pig liver endothelial cells.

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.

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.

Current Status of Renal Xenotransplantation and Next Steps.

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.

Xenoantigen Deletion and Chemical Immunosuppression Can Prolong Renal Xenograft Survival.

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.

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.

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.

The fate of human platelets exposed to porcine renal endothelium: a single-pass model of platelet uptake in domestic and genetically modified porcine organs.

BACKGROUND: Thrombocytopenia may represent a significant challenge to the clinical application of solid-organ xenotransplantation. When studied in a pig-to-primate model, consumptive coagulopathy has challenged renal xenografts. New strategies of genetic manipulation have altered porcine carbohydrate profiles to significantly reduce human antibody binding to pig cells. As this process continues to eliminate immunologic barriers to clinical xenotransplantation, the relationship between human platelets and pig organs must be considered. METHODS: Genetically modified pigs that were created by the CRISPR/Cas9 system with α-1,3-galactosyltransferase (GGTA1)(-/-) or GGTA1(-/-) cytidine monophosphate-N-acetylneuraminic acid hydroxylase(-/-) phenotype, as well as domestic pigs, were used in this study. Autologous porcine platelets were isolated from donor animal blood collection, and human platelets were obtained from a blood bank. Platelets were fluorescently labeled and in a single-pass model, human, or autologous platelets were perfused through porcine organs at a constant concentration and controlled temperature. Platelet uptake was measured by sampling venous output and measuring sample florescence against input florescence. In vitro study of the interaction between human platelets and porcine endothelial cells was accomplished by immunohistochemical stain and confocal microscopy. RESULTS: Differences between human and autologous platelet loss through the porcine kidney were not significant in any genetic background tested (WT P = 0.15, GGTA1(-/-)P = 0.12, GGTA1(-/-) cytidine monophosphate-N-acetylneuraminic acid hydroxylase(-/-)P = 0.25). The unmodified porcine liver consumed human platelets in a single-pass model of platelet perfusion in fewer than 10 min. WT suprahepatic inferior vena cava fluoresce reached a maximum of 76% of input fluoresce within the human platelet cohort and was significantly lower than the autologous platelet control cohort (P = 0.001). Confocal microscopic analysis did not demonstrate a significant association between human platelets and porcine renal endothelial cells compared with porcine liver endothelial positive controls. CONCLUSIONS: Our results suggest that in the absence of immunologic injury, human platelets respond in a variable fashion to organ-specific porcine endothelial surfaces. Human platelets are not removed from circulation by exposure to porcine renal endothelium but are removed by unmodified porcine hepatic endothelium. Kidneys possessing genetic modifications currently relevant to clinical xenotransplantation failed to consume human platelets in an isolated single-pass model. Human platelets did not exhibit significant binding to renal endothelial cells by in vitro assay.

Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/ß4GalNT2 genes.

BACKGROUND: Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The ß4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes. METHODS: The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/ß4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. RESULTS: Cells from GGTA1/CMAH/ß4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/ß4GalNT2. CONCLUSIONS: Inactivation of the ß4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/ß4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/ß4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

Biochemical analysis of CTLA-4 immunoreactive material from human blood.

BACKGROUND: CTLA-4 was initially described as a membrane-bound molecule that inhibited lymphocyte activation by interacting with B7.1 and B7.2 molecules on antigen presenting cells. Alternative splicing of mRNA encoding the CTLA-4 receptor leads to the production of a molecule (sCTLA-4) that lacks a membrane anchor and is therefore secreted into the extracellular space. Despite studies finding that people with autoimmune disease more frequently express high levels of sCTLA-4 in their blood than apparently healthy people, the significance of these findings is unclear. METHODS: Molecules isolated from blood using CTLA-4 specific antibodies were analyzed with ligand binding assays, mass spectroscopy, and biochemical fractionation in an effort to increase our understanding of CTLA-4 immunoreactive material. RESULTS: Mass spectroscopy analysis of the molecules recognized by multiple CTLA-4-specific antibodies failed to identify any CTLA-4 protein. Even though these molecules bind to the CTLA-4 receptors B7.1 and B7.2, they also exhibit properties common to immunoglobulins. CONCLUSION: We have identified molecules in blood that are recognized by CTLA-4 specific antibodies but also exhibit properties of immunoglobulins. Our data indicates that what has been called sCTLA-4 is not a direct product of the CTLA-4 gene, and that the CTLA-4 protein is not part of this molecule. These results may explain why the relationship of sCTLA-4 to immune system activity has been difficult to elucidate.

Lack of association between sCTLA-4 levels in human plasma and common CTLA-4 polymorphisms.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an important downregulatory molecule expressed on both T and B lymphocytes. Numerous population genetics studies have documented significant associations between autoimmune diseases and single nucleotide polymorphisms (SNPs) within and around the CTLA-4 region of chromosome 2 in man. Furthermore, circulating levels of a soluble form of CTLA-4 (sCTLA-4) have been reported in a variety of autoimmune mediated diseases. Despite these findings, the relationship between levels of sCTLA-4 protein, mRNA transcript levels, and SNPs within the CTLA-4 region have not been clearly defined. In order to further clarify this relationship, we have tested four different SNPs within the CTLA-4 region among subjects whom are negative (n = 53) versus positive (n = 28) for sCTLA-4. RESULTS: Our data do not support a clear association between sCTLA-4 levels and any of the four SNPs tested. CONCLUSION: The variation in the SNPs tested does not appear to effect sCTLA-4 protein levels, despite reports that they affect sCTLA-4 mRNA.

A mechanistic study of immune system activation by fusion of antigens with the ligand-binding domain of CTLA4.

Fusion proteins consisting of the ligand-binding domain of CTLA4 covalently attached to an antigen (Ag) are potent immunogens. This fusion strategy effectively induces Ag-specific immunity both when introduced as a DNA-based vaccine and as a recombinant protein. CTLA4 is a ligand for B7 molecules expressed on the surface of antigen-presenting cells (APCs), and this interaction is critical for the fusion protein to stimulate Ag-specific immunity. We show that interaction of the fusion protein with either B7-1 or B7-2 is sufficient to stimulate immune activity, and that T cells are essential for the development of IgG responses. In addition, we demonstrate that human dendritic cells (DCs) pulsed with CTLA4-Ag fusion proteins can efficiently present Ag to T cells and induce an Ag-specific immune response in vitro. These studies provide further mechanistic understanding of the process by which CTLA4-Ag fusion proteins stimulate the immune system, and represent an efficient means of generating Ag-specific T cells for immunotherapy.