Xenotransplantation is finally moving toward clinical application. Despite the progress in survival in preclinical models and the limited attempts at clinical heart transplantation, the initial problem of xenoreactive antibodies in human serum remains a proximal barrier to widespread success in preclinical and clinical settings. Detailed understanding of the pretransplant status of recipients in the preclinical as well as clinical setting is critical to moving the field forward. The differences in the pretransplant donor-specific antibody (DSA) levels in the preclinical and clinical models are outlined so that we can begin to think about the two scenarios in a complementary fashion and facilitate decision making for research priorities and movement into the clinical realm.

Transplantation has become a preferred therapy for the treatment of end stage organ failure, improving the quality and duration of recipients lives. The major limitation of organ transplantation is the shortage of suitable donor organs available for clinical use. Xenotransplantation using genetically modified pig organs could provide an unlimited source of organs, allowing all patients in need to receive a transplant in a timely fashion. Xenotransplantation was limited to the experimental realm because of the presence of anti-pig antibodies that are present in the blood of every human (1, 2).The development of genetic engineering tools, especially CRISPR/Cas9 and somatic cell nuclear transfer made it possible to create pigs missing key glycan pig antigens so that the antibodies did not bind to the new pig (3-5). Preclinical results using kidneys from new donor pigs has improved to the point where nonhuman primate recipients are living for more than 4 years (Andrew Adams personal communication). The improvements in survival seen in preclinical models has led to clinical attempts at heart and kidney xenotransplantation (6, 7). Thus far in the first 5 clinical xenotransplant cases success has been modest, with only one graft (kidney) functioning past 60 days to date. The other patients receiving pig xenografts (2 hearts and 2 kidneys) succumbed to early antibody mediated rejection (AMR) (7-9). Nevertheless, the developments in preclinical and compassionate use xenotransplantation have resulted in the first FDA approved clinical trial with renal xenotransplantation. This article will deal with the issues that are likely to be the focus of the next 25 years with regards to development of clinical xenotransplantation.

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.

INTRODUCTION:"Delayed" antibody-mediated xenograft rejection is one of the most important obstacles to clinical application of pig organ xenografts. The aim of this study was to assess the impact of a structured desensitization regimen including proteasome inhibition and next-generation costimulation blockade on xenoreactive antibodies. METHODS:Rhesus macaques with moderate-high pre-treatment xenoreactive antibody titers (N = 2) were selected. Recipients received twice-weekly carfilzomib (20 mg/m2), anti-CD154 (20 mg/kg) every other week, and CD4 and CD20 lymphocyte cell depletion. Bone marrow was acquired to assess plasma cell depletion in response to proteasome inhibition. A flow cytometry-based xenoreactive crossmatch assay was performed to assess levels of circulating xenoreactive antibodies. RESULTS:The desensitization regimen resulted in a >50% depletion of CD38+CD27+ bone marrow plasma cells; these changes were progressive over the duration of the desensitization treatment period. The desensitization strategy and plasma cell depletion resulted in a progressive reduction in anti-pig IgG antibodies. Following xenotransplantation, both desensitized recipients demonstrated superior graft survival to a highly xenoreactive recipient (MST 30 days vs. 6 days), but neither desensitized recipient experienced prolonged graft survival. CONCLUSIONS:A structured desensitization regimen including proteasome inhibition and costimulation blockade results in plasma cell depletion and resultant reduction in circulating xenoreactive anti-pig IgG antibodies, with a modest improvement in xenograft survival. This desensitization regimen has promise for pig-to-NHP xenotransplant models.

INTRODUCTION:Xenotransplantation has emerged as a promising solution to organ shortage, generating numerous publications. However, no studies have analyzed the research dynamics of xenotransplantation research. We aimed to systematically assess xenotransplantation publication activity. METHODS:A systematic literature search was conducted up to November 22, 2024. Studies on xenotransplantation of solid organs and islets of Langerhans from animals to humans, or perfusion with human blood or its derivatives were included. Publication information, publishing journal, publication type, organ, donor species, and topics studied were extracted. RESULTS:Of 2944 publications, 706 met inclusion criteria: 41.2% original articles, 41.1% reviews, 14.2% publications without original data, 1.6% case reports, 1.3% research letters, 0.6% systematic reviews/meta-analyses. Publication activity displayed two peaks: in the 1990s, driven by the gene editing advancements, and in the early 2020s, following the first pig-to-human transplantation. The top five publishing countries were the USA with (48.2%), Germany (10.2%), UK (5.4%), Sweden (4.8%), and China (4.2%). Xenotransplantation journal accounted for 19.7% of publications, transplantation journals for 27.6%, and general medical journals for 5.4%. Islets of Langerhans were studied in 23.1% of studies, and the most studied organs were heart (21.2%), followed by kidney (17.1%), liver (12.2%), and lung (6.2%). The most represented thematic groups were rejection, immune mechanisms, overall challenges, gene editing, current research, and prospects. CONCLUSION:This first systematic assessment of xenotransplantation research highlights its growing global interest and evolving focus areas. The low proportion of publications with original data underscores the need for more original research. Limited representation in general medical journals highlights the importance of engaging a broader audience as clinical trials approach.

This report summarizes the content of a debate sponsored by eGenesis Bio, organized by the International Xenotransplantation Association (IXA), and attended by more than 150 delegates in the context of the IPITA-IXA-CTRMS Joint Congress held in San Diego in October 2023. The debate centered around two important immunological topics relating to xenotransplantation. The first was a debate relating to the statement that "HLA-sensitized patients are at higher risk for rejecting a pig xenograft." Stuart Knechtle provided evidence to support this statement and Massimo Mangiola opposed it. Before the debate, a majority (>80%) of the audience agreed with this statement. After listening to the debate, this percentage was reduced to approximately 60%. The second debated statement was "Recipients of pig xenografts who develop anti-pig antibodies are at higher risk for rejecting a subsequent allograft." This was proposed by A. Joseph Tector and opposed by L & eacute;o H. B & uuml;hler. Before the debate, once again a majority of the audience (approximately 60%) believed that prior sensitization to a pig xenograft would be detrimental to the survival of a subsequent allograft. However, after listening to the debate, only about 40% believed this statement to be correct. The topics discussed remain complex and answers are not yet conclusive. However, the present evidence suggests that allosensitization may prove detrimental to subsequent xenotransplantation, whilst sensitization to pig antigens may not be detrimental to subsequent allotransplantation.

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.

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.

Phillipe Abreu, Joao Manzi, Vighnesh Venkatasamy, Rafael Miyashiro, Akin Tekin, Gennaro Selvaggi, Alfred Joseph Tector, Rodrigo Vianna. Korean J Transplant 2023;37:242. https://doi.org/10.4285/ATW2023.F-8356

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.

Background: The use of deceased donors after circulatory death (DCD) for liver transplantation (LT) is still underutilized. It provides an acceptable solution to increase the organ donor pool without affecting outcomes. We analyzed data of DCD donors for LT in order to identify risk factors for graft and patient survival. Methods: This was a retrospective cohort study using data reviewed by screening SRTR/UNOS of patients that underwent liver transplantation with DCD donor at the University of Miami, Jackson Memorial Hospital in Miami, FL from 2017 to 2019. Different surrogates were analyzed, including donor, recipient, procurement and transplant operation characteristics. The primary outcome was patient and graft survival. Continuous variables were analyzed using a 2-sided student t-test. Categorical variables were presented as frequencies and proportion and compared using chi-squared or Fisher’s exact test as appropriate. Univariable Cox proportional hazards models were fit in order to assess the impact of individual covariables on the instantaneous rate of events, with time-to-event analysis also being ascertained through Kaplan-Meier estimates. Results: 54 cases were analyzed with a median MELD score of 18.5. Alcoholic cirrhosis was the most prevalent etiology for liver disease. There was 1 case with intra-hepatic biliary stricture, and 7 patients with anastomotic strictures that required stenting. The 1-year survival rate following liver transplant using DCD donors was 90.7%. The total cold ischemic time (CIT) (HR 1.9, 95% CI 1.1-3.2) and diagnosis of rejection (HR 9, 95% CI 1.1-78.8) were statistically significant affecting graft failure. Estimated blood loss (EBL) with HR of 1.1 (95% CI 1.1-1.2) was found to be statistically impacting survival rate. Conclusion: DCD donors are excellent options to increase the availability of organs for patients waiting for a LT. CIT and EBL are the main predictors of poor outcomes and thus should be carefully taken into account when matching organs to recipients.