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Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts
Abstract
Background:
Genetically engineered pigs could provide a source of kidneys for clinical transplantation. The two longest kidney graft survivals reported to date have been 136 and 310 days, but graft survival >30 days has been unusual until recently.
Methods:
Donor pigs (n=4) were on an α1,3-galactosyltransferase gene-knockout (GTKO)/human complement regulatory protein (CD46) background (GTKO/CD46). In addition, the pigs were transgenic for at least one human coagulation regulatory protein. Two baboons received a kidney from a six-gene pig (GroupA) and two from a three-gene pig (GroupB). Immunosuppressive therapy was identical in all four cases and consisted of anti-thymoglobulin (ATG)+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R mAbs were administered to reduce the inflammatory response. Baboons were followed by clinical/laboratory monitoring of immune/coagulation/inflammatory/physiological parameters. At biopsy or euthanasia, the grafts were examined by microscopy.
Results:
The two GroupA baboons remained healthy with normal renal function >7 and >8 months, respectively, but then developed infectious complications. However, no features of a consumptive coagulopathy, eg, thrombocytopenia and reduction of fibrinogen, or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response, and histology of biopsies taken at approximately 4, 6, and 7 months and at necropsy showed no significant abnormalities. In contrast, both GroupB baboons developed features of a consumptive coagulopathy and required euthanasia on day 12.
Conclusions:
The combination of (i) a graft from a specific six-gene genetically modified pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory therapy prevented immune injury, a protein-losing nephropathy, and coagulation dysfunction for >7 months. Although the number of experiments is very limited, our impression is that expression of human endothelial protein C receptor (±CD55) in the graft is important if coagulation dysregulation is to be avoided.
... This proved a major step forward. Since then, almost all groups have employed an anti-CD154 or anti-CD40mAb as the basis of their immunosuppressive regimen [53,[66][67][68][69][70][71][72][73][74][75][76][77]. ...
... The withdrawal of the original anti-CD154mAbs because of their thrombogenic effect [92][93][94] necessitated the use of anti-CD40mAbs, first introduced into xenotransplantation by Mohiuddin et al [68][69][70]. However, increasing data indicate that anti-CD154 agents are superior to anti-CD40 agents in preventing both the adaptive immune response and some aspects of the innate response [75,95]. ...
... When the problems relating to coagulation dysfunction between pig and human were confirmed, the introduction of GTKO. hCD46 pigs that additionally expressed human thrombomodulin reduced the incidence of thrombotic microangiopathy in the pig graft and of consumptive coagulopathy in the recipient NHPs (Table 5; Figure 5) [70]. This prolonged life-supporting kidney graft survival to 7-8 months, with termination of the experiments from infectious complications rather than from rejection [70]. ...
An overview is provided of the evolution of strategies towards xenotransplantation during the past almost 40 years, focusing on advances in gene-editing of the organ-source pigs, pre-transplant treatment of the recipient, immunosuppressive protocols, and adjunctive therapy. Despite initial challenges, including hyperacute rejection resulting from natural (preformed) antibody binding and complement activation, significant progress has been made through gene editing of the organ-source pigs and refinement of immunosuppressive regimens. Major steps were the identification and deletion of expression of the three known glycan xenoantigens on pig vascular endothelial cells, the transgenic expression of human “protective” proteins, e.g., complement-regulatory, coagulation-regulatory, and anti-inflammatory proteins, and the administration of an immunosuppressive regimen based on blockade of the CD40/CD154 T cell co-stimulation pathway. Efforts to address systemic inflammation followed. The synergy between gene editing and judicious immunomodulation appears to largely prevent graft rejection and is associated with a relatively good safety profile. Though there remains an incidence of severe or persistent proteinuria (nephrotic syndrome) in a minority of cases. This progress offers renewed hope for patients in need of life-saving organ transplants.
... Unlike porcine TBM, porcine EPCR appears compatible with human thrombin and protein C activation mechanisms in vitro [38]. Additionally, EPCR provides some protective effects even in the absence of human TBM expression [41]. Transgenic human EPCR in a pig-to-baboon renal transplant model-in the setting of GGTA1KO, transgenic CD46, transgenic CD55, transgenic CD47, and transgenic tissue factor pathway inhibitor-enabled survival up to 9 months [41]. ...
... Additionally, EPCR provides some protective effects even in the absence of human TBM expression [41]. Transgenic human EPCR in a pig-to-baboon renal transplant model-in the setting of GGTA1KO, transgenic CD46, transgenic CD55, transgenic CD47, and transgenic tissue factor pathway inhibitor-enabled survival up to 9 months [41]. The EPCR group median survival exceeded the median survival of the human TBM expressing xenografts in the same series, albeit with lower-than-expected TBM expression [41]. ...
... Transgenic human EPCR in a pig-to-baboon renal transplant model-in the setting of GGTA1KO, transgenic CD46, transgenic CD55, transgenic CD47, and transgenic tissue factor pathway inhibitor-enabled survival up to 9 months [41]. The EPCR group median survival exceeded the median survival of the human TBM expressing xenografts in the same series, albeit with lower-than-expected TBM expression [41]. ...
Purpose of Review
The first successful pig to human cardiac xenotransplantation in January 2022 represented a major step forward in the fields of heart failure, immunology, and applied genetic engineering, using a 10-gene edited (GE) pig. This review summarizes the evolution of preclinical modelling data which informed the use of each of the 10 genes modified in the 10-GE pig: GGTA1, Β4GalNT2, CMAH, CD46, CD55, TBM, EPCR, CD47, HO-1, and growth hormone receptor.
Recent Findings
The translation of the 10-GE pig from preclinical modelling to clinical compassionate xenotransplant use was the culmination of decades of research combating rejection, coagulopathy, inflammation, and excessive xenograft growth.
Summary
Understanding these 10 genes with a view to their combinatorial effects will be useful in anticipated xenotransplant clinical trials.
... 32 When hEPCR was not included in genetic modifications of xenografts, graft survival significantly decreased and CC was observed. 54 This is in comparison with survival of >7 months with the inclusion of hEPCR, suggesting that hEPCR in the graft is critically important to avoid coagulation dysregulation. 54 However, different effects on complement activation and subsequent coagulation dysregulation have been observed in xenografts expressing hCD55 or hEPCR. ...
... 54 This is in comparison with survival of >7 months with the inclusion of hEPCR, suggesting that hEPCR in the graft is critically important to avoid coagulation dysregulation. 54 However, different effects on complement activation and subsequent coagulation dysregulation have been observed in xenografts expressing hCD55 or hEPCR. 55 Expression of hCD47 on pig cells not only suppresses the activation of human macrophages and inflammatory cytokine production 8 but also is implicated in the inhibition of platelets. ...
The recent report of the first pig kidney transplant in a living human brings hope to thousands of people with end‐stage kidney failure. The scientific community views this early success with caution as kidney xenotransplantation exhibits many challenges and barriers. One of these is coagulation dysregulation. This includes (i) pig von Willebrand Factor (vWF) interaction with human platelets, which can induce abnormal clotting responses, heightening the risk of graft failure, (ii) the inefficiency of pig thrombomodulin in activating human protein C, which emphasizes the species‐specific variations that aggravate coagulation challenges, and (iii) the development of thrombotic microangiopathy in the pig grafts and the occurrence of systemic consumptive coagulopathy in the recipients. Indeed, coagulation dysregulation largely results from differences in endothelial cell response and incompatibilities between pig and human coagulation–anticoagulation pathways. These barriers can be resolved by modifications to pig vWF and the expression of human thrombomodulin and endothelial protein C receptors in pig cells, serving as strategic interventions to align the coagulation systems of the two species more closely. These coagulation challenges have clinical implications in how they affect graft survival and patient outcome. Genetic engineering of the organ‐source pig and the administration of various drugs have assisted in correcting this coagulation dysregulation. Hence, comprehending and controlling coagulation dysregulation is crucial for progress in xenotransplantation as a viable option for treating patients with terminal kidney disease.
... One approach to cope with these inflammatory and coagulatory challenges is the genetic engineering of the organ-source pig, for example via the introduction of transgenes for human thrombomodulin (hTBM), human heme oxygenase-1, human A20, or human CD39 [1,16]. Besides the genetic modification of the organ-source pig, there are several therapeutic strategies to prevent or reduce inflammatory responses following xenotransplantation, for example, corticosteroids [19,20], white blood cell filtration [16], anti-complement agents like C1-inhibitors [15] or cobra venom factor [19], IL-6 receptor blockade agents [21,22], anti-histone antibodies [22], TNF-α inhibitors [23], NF-κBinhibitors [22], alpha 1-antitrypsin [15], platelet inhibitors [15], or triiodothyronine [15]. ...
... Increased levels of several cytokines and chemokines (for example IL-6, IL-8, IL-12, INF-γ, and MCP-1) have been observed in different xenotransplantation models [16,20,30]. In pig-tobaboon orthotopic cardiac xenotransplantation experiments, the increase of IL-6, IL1β, TNF, INF-γ, and CXCL9 was seen as an indicator of a "cytokine storm" following CPB [16]. ...
Introduction
Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti‐inflammatory and anti‐coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ‐source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting.
To elucidate this, we present data from pig‐to‐baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti‐inflammatory drugs.
Methods
Genetically modified piglets (GGTA1‐KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive‐bred baboons ( n = 14). All animals received an anti‐inflammatory drug therapy including a C1 esterase inhibitor, an IL‐6 receptor antagonist, a TNF‐α inhibitor, and an IL‐1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co‐stimulation blockade. During the experiments, leukocyte counts, levels of C‐reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections ( n = 2) or technical failures ( n = 2).
Results
Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL‐8, increased about 2‐fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL‐12/IL‐23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach.
Conclusion
Our preclinical experience with the anti‐inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well‐practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti‐inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.
... Xenografts often suffer from endothelial injury and dysfunction. This can result in increased blood clot formation, platelet activation, and vascular constriction [13]. chronic endothelial activation and inflammation can lead to the narrowing of blood vessels, reducing blood flow to the transplanted organ. ...
... One approach is to genetically modify donor animals to knock out or replace certain genes that are responsible for the expression of antigens recognized by the recipient's immune system. For example, by inactivating or replacing genes associated with the production of galactose-alpha-1,3-galactose (Gal), which is a major target for xenograft rejection, it's possible to reduce the risk of hyperacute rejection [13]. Genetic engineering can be used to introduce human genes into the donor animals. ...
... Steps were taken to express at least one human coagulation-regulatory protein, e.g., thrombomodulin (TBM), endothelial protein C receptor [EPCR], tissue factor pathway inhibitor (TFPI), and/or CD39, on the pig vascular endothelial cells. Survival of pig kidneys and hearts transplanted into NHPs was extended to months rather than weeks 78 . ...
... II). Based on (i) the innovative biotechnology for pig gene modification aimed at reducing the effect of the primate immune response to the xenograft, and (ii) the administration of novel immunosuppressive agents that block the CD40/CD154 costimulation pathway, significant progress has been made in pig-to-NHP organ xenotransplantation models 51,78,[108][109][110] . These advances have led to prolonged survival of pig kidney grafts in NHPs, and today survival is being recorded in months or years. ...
In suitable patients with end-stage organ failure, the transplantation of organs from living or deceased human donors offers a much-improved quality and length of life. However, the availability of deceased human donor organs is grossly inadequate. Gene-edited pigs might provide an alternative source of organs for clinical transplantation (xenotransplantation). However, there are major immunobiological barriers to successful pig organ transplantation in human or nonhuman primate recipients. These barriers include antibody binding, activation of complement, the innate cellular response, coagulation dysregulation between pig and primate, and a systemic inflammatory response, in addition to the T cell response. These have steadily been overcome by a combination of (i) genetic engineering of the organ-source pig (aimed mainly at the innate immune response), and (ii) the administration of novel immunosuppressive agents (directed towards the adaptive immune response). The immunological barriers that remain relate to both the innate and adaptive immune responses. Pig kidney transplants have now supported immunosuppressed (anephric) nonhuman primates for periods in excess of a year and pig heart transplants for up to 9 months, although these encouraging results cannot yet be achieved consistently.
... Based on (i) the innovative biotechnology for pig gene modification aimed at reducing the effect of the primate immune response to the xenograft, and (ii) the administration of novel immunosuppressive agents that block the CD40/CD154 co-stimulation pathway, significant progress has been made in the pig-to-NHP kidney xenotransplantation model [22][23][24][25][26] . These advances have led to prolonged survival of pig kidney grafts in NHPs, and today survival is being recorded in months or years. ...
... As long ago as 2000, it was observed that a pig kidney grew rapidly in the first few weeks after transplantation into a NHP (as if it were still in a rapidly-growing pig) 36 . The cause was uncertain, but this phenomenon has been more recently confirmed by others 23,37,38 . After approximately 3 months, its rate of growth may reduce to equate with that of the recipient baboon. ...
In suitable patients with end-stage renal disease, the transplantation of kidneys from living or deceased human donors offers a much-improved quality and length of life. However, the availability of donor kidneys is grossly inadequate. Gene-edited pigs might provide an alternative source of kidneys for clinical transplantation (xenotransplantation). However, there are major pathobiological barriers to successful pig kidney transplantation in human or nonhuman primate (NHP) recipients. These have steadily been overcome by a combination of (i) genetic engineering of the organ-source pig, and (ii) the administration of novel immunosuppressive agents. Pig kidney transplants have now supported immunosuppressed (anephric) NHPs for periods in excess of a year, although this cannot yet be achieved consistently. Studies of pig kidney function after transplantation indicate that the pig kidney can likely fulfill all of the requirements of a human kidney. Potential infectious complications are likely to be similar to those seen in any immunosuppressed patient, and the potential risks of infection with a pig microorganism will be minimized by the breeding and housing of the organsource pigs in biosecure ‘clean’ environments. The attitudes of patients, their family members, healthcare providers, and the public appear to be positive towards xenotransplantation if it will be lifesaving. For the first clinical trial, we suggest that patients on the wait-list aged 55-65 years in good physiological condition with blood group O or B (and who are possibly diabetic) who are unlikely ever to receive a deceased human donor kidney (because of death or the development of comorbidities that result in their removal from the waitlist) might accept a pig kidney if it will negate the need for dialysis for one or more years.
... (maximal survival 193 days) 16 . Finally, a group at the University of Alabama published kidney growth data for a series of six baboons, using varying genotype porcine donors from United Therapeutics 17,18 . Kidney length increased up to~100% over 20 weeks post-transplant. ...
... While complete chemistry data after kidney xenotransplantation in NHPs is rarely published, Soin et al. reported borderline hypercalcemia as well as hypophosphatemia in recipients of hDAF porcine kidneys 14 . High normal calcium and hypophosphatemia was also reported in three baboon recipients of more advanced United Therapeutics porcine donors by 3-4 weeks PTT 17 . However, this study is the first to our knowledge to have provided urinalysis, bone investigation, and molecular data to address these phenotypes. ...
Porcine kidney xenotransplantation is accelerating towards clinical translation. However, despite the demonstrated ability of porcine kidneys to remove metabolic waste products, questions remain about their ability to faithfully recapitulate renal endocrine functions after transplantation. Here we analyze xenograft growth and function of two kidney dependent endocrine pathways in seventeen cynomolgus macaques after kidney xenotransplantation from gene edited Yucatan minipigs. Xenograft growth, the renin-angiotensinogen aldosterone-system, and the calcium-vitamin D-parathyroid hormone axis are assessed using clinical chemistries data, renin activity and beta-C-terminal-telopeptide assays, kidney graft RNA-sequencing and serial ultrasonography. We demonstrate that xenografts transplanted from minipigs show only modest growth and do not substantially contribute to recipient RAAS pathway activity. However, parathyroid hormone-independent hypercalcemia and hypophosphatemia are observed, suggesting a need for close monitoring and timely intervention during human testing. Further study of these phenotypes is warranted in designing prospective clinical trials.
... The accumulation of platelets and fibrin in the pig graft results in a thrombotic microangiopathy (Fig. 12), resulting in a consumptive coagulopathy in the recipient [50,51]. Importantly, this was still seen in regimens where anti-CD154mAb therapy was substituted with anti-CD40mAb therapy [52]. ...
... Therefore, one of the goals of genetic manipulation has been to regulate the coagulation system by creating source animals that express at least one human coagulation pathway regulatory protein, e.g., thrombomodulin (hTBM), tissue factor inhibitor (hT-FPI), and/or endothelial protein C receptor (hEPCR) [53]. When such pigs were available, pig kidneys transplanted into NHPs showed an increased survival to approximately 8 months [52]. ...
... [1][2][3] As clinical trials draw closer, it becomes important to measure the function of pig organ xenografts in the "foreign" environment of the NHP host, and consider whether there are abnormalities in the function that need to be addressed. [4][5][6][7][8][9] The common global clinical measurement of renal function is the glomerular filtration rate (GFR). 10 In progressive renal injury, compensation from remaining nephrons will maintain homeostatic electrolyte balances, but the overall GFR diminishes. ...
... Details of transplantation and immunosuppressive drug therapy used have been reported previously 6,17 and will only be briefly summarized here. Pig kidney transplantation involved end-to-side anastomoses of the pig renal artery to the baboon abdominal aorta and of the pig renal vein to the baboon inferior vena cava. ...
With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculated were similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.
... For example, pig heart transplantation to baboons allowed survival for up to 200-900 days (Mohiuddin et al., 2014). Other pig organs like kidneys or skin were also xenotransplanted to baboons with a similar success rate (Tena et al., 2017;Iwase et al., 2017). Moreover, GM pig organs were xenotransplanted into humans in a few clinical trials. ...
Somatic cell nuclear transfer (SCNT), or cloning, is used to reprogram cells and generate genetically identical embryos and animals. However, the cloning process is inefficient, limiting its application to producing valuable animals. In swine, cloning is mainly utilized to produce genetically modified animals. Indeed, recombinant DNA technologies have evolved considerably in recent years, with homologous recombination and gene editing technologies becoming more efficient and capable of recombining both alleles in a single cell. The selection of appropriate cells and their use as nuclear donors for SCNT is the most common method for generating edited and genetically modified animals for commercial and research purposes. This article reviews current applications of swine cloning and shares our personal experiences with the procedure in this species.
... In addition, the expression of human complement and coagulation regulatory genes in donor pigs has been used to regain control over the activation of the plasma cascade systems [23][24][25][26]. While impressive progress has been achieved, complement and coagulation dysregulations are still presented as key features of short-or long-term xenograft rejection in preclinical and clinical models [26][27][28][29][30]. This indicates that the currently available strategies to preserve the regulatory functions of xenograft ECs are not sufficient to achieve longlasting control and graft survival. ...
In xenotransplantation, the vascular endothelium serves as the first point of contact between the recipient’s blood and the transplanted donor organ. The loss of the endothelium’s ability to control the plasma cascades plays a critical role in the dysregulation of the complement and coagulation systems, which greatly contribute to graft rejection and hinder long-term xenograft survival. Although it is known that an intact glycocalyx is a key feature of a resting endothelium that exhibits optimal anticoagulant and anti-inflammatory properties, the role of the endothelial glycocalyx in xenotransplantation is barely investigated so far. Here, we discuss the central role of endothelial cells and the sugar-rich endothelial glycocalyx in regulating the plasma cascades, and how the loss of these functions contributes to graft damage and rejection. We highlight the importance of preserving the regulatory functions of both endothelial cells and the glycocalyx as strategies to improve xenotransplantation outcomes.
... In this study we evaluated results associated with using 3-, 9-, or 10-GE pig hearts, tested in a baboon heterotopic cardiac transplant model in the context of a cold-perfused ischemia minimization technique and a previously validated, clinically applicable co-stimulation-based immunosuppression regimen targeting CD40/CD154. 5,10,17,[22][23][24] ...
... Early research suggested that kidney graft size has been increased [12]. But in recent studies, development of partial stricture of ureter has been observed. ...
Book series on Medical Science gives the opportunity to students and doctors from all over the world to publish their research work in a set of Preclinical sciences, Internal medicine, Surgery and Public Health. This book series aim to inspire innovation and promote academic quality through outstanding publications of scientists and doctors. It also provides a premier interdisciplinary platform for researchers, practitioners, and educators to publish the most recent innovations, trends, and concerns as well as practical challenges encountered and solutions adopted in the fields of Medical Science. It also provides a remarkable opportunity for the academic, research and doctors communities to address new challenges and share solutions and discuss future research directions.
... Graft failure is primarily caused by an antibody-mediated rejection [34]. In contrast, for baboons receiving anti-CD154 antibody-based [46] or anti-CD40 protocols, the experiments were terminated mainly because of infection or other complications [47]. ...
Background
Xenograft kidney transplantation has been receiving increasing attention. The purpose of this study is to use bibliometric analysis to identify papers in this research field and explore their current status and development trends.
Methods
Using the data in the Web of Science core database from Clarivate Analytics as the object of study, we used ‘TS = Kidney OR Renal AND xenotransplantation’ as the search term to find all literature from 1980 to 2 November 2022.
Results
In total, 1005 articles were included. The United States has the highest number of publications and has made significant contributions in this field. Harvard University was at the forefront of this study. Professor Cooper has published 114 articles in this field. Xenotransplantation has the largest number of relevant articles. Transplantation was the most cited journal. High-frequency keywords illustrated the current state of development and future trends in xenotransplantation. The use of transgenic pigs and the development of coordinated co-stimulatory blockers have greatly facilitated progress in xenotransplantation research. We found that ‘co-stimulation blockade’, ‘xenograft survival’, ‘pluripotent stem cell’, ‘translational research’, and ‘genetic engineering’ were likely to be the focus of attention in the coming years.
Conclusions
This study screened global publications related to xenogeneic kidney transplantation; analyzed their literature metrology characteristics; identified the most cited articles in the research field; understood the current situation, hot spots, and trends of global research; and provided future development directions for researchers and practitioners.
... Recently, to combat hyperacute xenograft rejection, α1,3-galactosyltransferase genetic knockout pigs were generated, and renal xenograft survival was drastically improved. [141][142][143] Another pathway to address known difficulties surrounding hyperacute xenograft rejection is through modification of host endothelial cells, as they are involved in graft rejection for every organ. ETV2, a master regulator of hematoendothelial lineages, has become a major target for the generation of human-pig chimeras with human-derived endothelial cells to minimize the likelihood of hyperacute graft rejection. ...
Orthotopic liver transplantation (OLT) currently serves as the sole definitive treatment for thousands of patients suffering from end-stage liver disease; and the existing supply of donor livers for OLT is drastically outpaced by the increasing demand. To alleviate this significant gap in treatment, several experimental approaches have been devised with the aim of either offering interim support to patients waiting on the transplant list or bioengineering complete livers for OLT by infusing them with fresh hepatic cells. Recently, interspecies blastocyst complementation has emerged as a promising method for generating complete organs in utero over a short timeframe. When coupled with gene editing technology, it has brought about a potentially revolutionary transformation in regenerative medicine. Blastocyst complementation harbors notable potential for generating complete human livers in large animals, which could be used for xenotransplantation in humans, addressing the scarcity of livers for OLT. Nevertheless, substantial experimental and ethical challenges still need to be overcome to produce human livers in larger domestic animals like pigs. This review compiles the current understanding of interspecies blastocyst complementation and outlines future possibilities for liver xenotransplantation in humans.
... The immune barriers have largely been overcome by two approaches -i) the breeding of pigs genetically engineered to protect their organs and cells from the human innate (antibody) immune response, and ii) the administration of novel immunosuppressive agents that suppress the human adaptive (cellular) immune response. The combination of the transplantation of a heart or kidney from a pig with multiple genetic modifications with treatment of the recipient with one of the novel immunosuppressive drugs now available has resulted in pig organs supporting the lives of NHPs for months or even years [8][9][10][11][12][13] . The transplantation of pig livers and lungs has to date been much less successful, in part because of the more complex nature of the immune response to these organs 14,15 . ...
Surgeons working in the field of organ transplantation are increasingly directing attention to resolving the shortage of deceased human donor organs by the transplantation of organs from genetically-engineered pigs, a field known as xenotransplantation (cross-species transplantation). If the remaining immunologic hurdles can be overcome, the ready availability of pig organs would enable all suitable patients to receive an organ transplant. Furthermore, the organ graft would be available whenever needed, thus reducing the need for prolonged renal dialysis or other supportive therapies. In addition, with regard to protecting the graft from the human immune response, xenotransplantation provides us the first opportunity of modifying the donor rather than just suppressing the response in the recipient, which may have detrimental effects on the heath of the patient. However, xenotransplantation offers a much greater potential than just organ transplantation. Pig islets could prove to be a cure for diabetes mellitus, corneas for corneal blindness, and pig dopamine-producing cells may reduce the disabilities of Parkinson’s disease. Pig red blood cells offer the prospect of a limitless supply for transfusion, and the implantation of gene-edited pig heart valves may increase the viability of these valves far longer than at present. There would be unlimited skin and tissues for the treatment of burns and many other conditions. Importantly, living human donors would no longer be put at risk, and surgeons would no longer have to resort to the transplantation of organs of suboptimal status. Indeed, xenotransplantation could well be the next great medical revolution.
... The editing of pig genes to fill the intra-species incompatibility gaps have further contributed to advanced interest in xenotransplantation. Using recent gene-editing technologies, xenotransplantation from multi-transgenic alpha-1,3-galactosyltransferase knockout pigs has demonstrated a marked prolongation of renal xenograft survival, ranging from days to greater than 6 months for islets, 1 year for life-supporting kidneys, and >2 years in a heterotopic non-life-supporting cardiac xenograft model (7)(8)(9)(10)(11). However, it is not clear which gene manipulations are essential for successful xenogeneic islet transplantation. ...
... 16 This has been developed using a number of preclinical porcine to non-human primate models. [21][22][23][24] In two studies porcine kidneys (in one case following removal of the gene encoding α 1.3-galactosyl transferase, responsible for the galactose α-1,3 galactose epitope and in the other 10 genetic modifications as described below for clinical cardiac xenotransplantation) have been grafted into brain dead human recipients with no signs of hyperacute rejection during the (inevitably) short (up to 72 hours) duration of the experiment. 25,26 The first gene modified pig to human cardiac transplant was carried out in January 2022. ...
Early in 2022 the first pig to human cardiac xenotransplant was performed. The graft initially performed well, and rejection was well controlled. However, the graft failed, and the patient died 60 days after the procedure. The ethical issues relating to xenotransplantation include the risk/benefit to the individual, the risk of porcine-derived infectious agents crossing into humans, animal welfare and rights, issues of human and animal identity and concerns relating to fair allocation of organs and appropriate use of resources.
These ethical issues are often addressed using emotional arguments, or through consequentialist or deontological lens. An alternative is to use approaches based on virtue ethics to understand the moral purpose (telos) of the research and the virtues (character traits) needed to be a good research clinician. In this review we will consider the virtues of justice, courage, temperance and practical wisdom, as well as the role of clinical curiosity, and their application to xenotransplantation. This provides an alternative approach for the clinical academic and others involved in the research to reflect on their practice.
The escalating incidence of heart failure globally, and in the United States, necessitates innovative solutions beyond conventional human cardiac transplantation due to donor heart shortage. Recent measures to overcome this shortage include the novel idea of cardiac xenotransplantation, with the first procedure done in January 2022 at the University of Maryland. However, the patient did not survive in the postoperative phase, highlighting potential challenges in cardiac xenotransplantation. Trace amounts of research exist on the physiological impacts subsequent to innate anatomical differences of porcine hearts, regardless of genetic modifications in growth rates. As such, this review aims to explore and address the critical implications of utilizing genetically modified porcine hearts for cardiac xenotransplantation as it pertains to postoperative physiological function. An analysis of literature discussing multiple anatomical and physiological factors, such as differences in organ dimensions, vasculature, and cardiac conduction, was carried out. Although xenotransplantation offers a promising solution, the present analysis of relevant literature points out potentially important considerations relating to long‐term survivability.
Organ transplantation remains the foremost effective intervention for end‐stage organ failure. Nevertheless, the scarcity of donors has resulted in prolonged waiting times for countless patients globally. The advent of xenografts presents a promising solution to the organ shortage crisis. Although the utilization of xenografts has a long history, it is only in recent years that breakthroughs in genetically modified pigs have rendered successful xenotransplantation a feasible option. In the past 4 years, numerous subclinical and clinical trials have involved xenotransplantation from genetically modified pigs to humans. However, the outcomes have been disappointing, necessitating a reassessment of basic and preclinical research to address the emerging challenges. Furthermore, immunosuppressive therapies remain essential in xenotransplantation. The range of immunosuppressive agents, encompassing traditional immunosuppressants and monoclonal antibodies such as anti‐CD154/CD40 monoclonal antibodies, exhibits considerable diversity. However, the most effective drug combination for achieving optimal efficacy remains elusive. This review will offer a succinct overview of the results from recent clinical and subclinical xenotransplantation trials. Moreover, it will highlight recent advancements in immunosuppressive strategies and discuss potential future research directions in this field.
Background
Long‐term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig‐to‐human xenotransplantation .
Methods
A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/β4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti‐CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated.
Results
PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T‐cell proliferation stimulated by pig PBMCs. CsA inhibited human T‐cell proliferation in a typical dose‐dependent manner. When Tac and CsA concentrations reached 5 and 200 ng/mL, respectively, the proliferation rates of CD3 ⁺ /CD4 ⁺ /CD8 ⁺ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T‐cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL‐2, IL‐6, IFN‐γ, TNF‐α, IL‐4, IL‐10, and IL‐17). Although anti‐CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T‐cell proliferation, low‐dose anti‐CD154 mAb combined with low‐dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects.
Conclusion
Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T‐cell responses in vitro. If used in combination with anti‐CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.
Background:
Significant progress has been made in kidney xenotransplantation in the past few years, and this field is accelerating towards clinical translation. Therefore, surveillance of the xenograft with appropriate tools is of great importance. Ultrasonography has been widely used in kidney allotransplantation and served as an economical and non-invasive method to monitor the allograft. However, questions remain whether the ultrasonographic criteria established for human kidney allograft could also be applied in xenotransplantation.
Methods:
In the current study, we established a porcine-rhesus life sustaining kidney xenotransplantation model. The xenograft underwent intensive surveillance using gray-scale, colorful Doppler ultrasound as well as 2D shear wave elastography. The kidney growth, blood perfusion, and cortical stiffness were measured twice a day. These parameters were compared with the clinical data including urine output, chemistry, and pathological findings.
Results:
The observation continued for 16 days after transplantation. Decline of urine output and elevated serum creatinine were observed on POD9 and biopsy proven antibody-mediated rejection was seen on the same day. The xenograft underwent substantial growth, with the long axis length increased by 32% and the volume increased by threefold at the end of observation. The resistive index of the xenograft arteries elevated in response to rejection, together with impaired cortical perfusion, while the peak systolic velocity (PSV) was not compromised. The cortical stiffness also increased along with rejection.
Conclusion:
In summary, the ultrasound findings of kidney xenograft shared similarities with those in allograft but possessed some unique features. A modified criteria needs to be established for further application of ultrasound in kidney xenotransplantation.
Background
Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation.
Methods
We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays.
Results
High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR.
Conclusions
Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.
Disruption of specific target genes is a first step to uncover the gene function. Large-sized animals are difficult to be targeted in embryo level, causing damages in early embryo developments. The recent development of genome editing techniques is being evolved to the easier ZFN, TALEN, and CRISPR/Cas9 technologies than the conventional laborious one in various animals, giving generation of gene knock-out pigs. The pig genome is about 2.7 Gb size in length, which is packaged in 18 autosomes with sex chromosomes X and Y [1, 2]. Genetic manipulation in pigs was conventionally performed by breeding selection to keep specific segments of the genome. The first genetic modification in the pig was the transgenic case [3]. Then, homologous recombination-based gene knocking out [4, 5] and knocking in were made in pigs and knock-ins [6], respectively. Gene KO disrupts specific genetic sequence to inactivate the genes that encode proteins, while knock-in gene is reversed with the gene’s encoded protein.
Besides the immunological damages, the xenotransplantation also induces several blood and vascular damages through fatal hemorrhagic coagulation, phagocytotic cell death, and activation of platelets as thrombocytopenic consequence. Several biochemical events such as inflammatory response and thrombotic failure occur at xenotransplantation of solid organs. Therefore, to preventively suppress such events, several regulating factors including anti-coagulant factor, anti-inflammatory factor, and cytoprotection factor-expressing pig transgenic strains are strategically considered. From the mice experiments, human EPCR-expressing transgenic mice protect themselves from reperfusion injury of warm renal ischemia. Heart organs of these mice showed the prolonged survival time when the organs were heterotrophic-transplanted in C6-deleted rats and showed less hemorrhage and edema levels [1].
As an alternative prevention of xenograft rejection, induction of immunological tolerance status is another strategy in which the host immune system of human recipients is specifically not responsive to the xenografted organs of pigs [1–3], but the human immune system is normally with the responding ability to infectious pathogens. There are huge progresses in strategies to induce tolerance across xenoantigenic hurdles and rejection barriers. Also, immune tolerance induction is potentially beneficial in xenotransplantation. By advanced progress in recent technologies of gene editing, survival time of xenotransplanted grafts of multiple-transgenic α1,3Gal-T KO pigs has greatly been prolonged, which is ranged from several days to months for transplanted organ grafts, allowing life-supporting healthy. For example, 2 years more survivals have been observed in non-life-supporting heterotopic model of xenotransplantation of cardiac xenografts. To achieve such success, continuous treatment with immunosuppressive drugs is prerequisite. In fact, to obtain much improved outcome, immunosuppression is required in host recipients and hence, anti-CD40 or anti-CD154 monoclonal Abs as immunosuppression agents are continuously administered. The status eventually causes death of recipient host from infectious diseases involved in chronically administered immunosuppression or from immune rejection of xenografts [4, 5], because immunosuppression is unsuccessful [6]. Human T-cell responses against xenoantigenic barriers of pigs are much strong rather than those of allogeneic antigens [7]. This invites a new strategic option for the tolerance induction in human host. Because two different immune responses including low T-cell-dependent Abs levels and innate immune response activation are directly associated with responsible damages of xenografts, immune tolerance against xenografts has been achieved using by approaches of mixed chimerism and thymus transplantation in pig-to-mouse xenotransplantation model. Such successful application has also been applied to pig-to-baboon xenotransplantation model as an experimental extension. To achieve such immune tolerance, alternative studies are cooperatively required for macro-chimerism persistence, prolonged survival of pig xenografts, and donor unresponsiveness [8]. Successful tolerance induction reflects success in xenotransplantation.
Hyper acute rejection (HAR) occurs 48 h immediately after transplantation. HAR is caused by cytotoxic antibody against donor’s HLA or ABO antigen. Its symptoms include high fever, edema, complement activation, and blood coagulation. Blood coagulation is also caused by bleeding from endothelial cells, and its symptoms are heart attack, stroke, pulmonary embolism, and thrombosis. In blood vessel, blood clot (thrombus) formation induces endothelial cell injury and abnormal blood stream. During HAR, antibody-mediated rejection (AMR) occurs by donor antigen-induced Abs, which were directed against ABO antigenic carbohydrates, donor-specific molecule HLA proteins, antigens of endothelial cells, or surfaced antigens on pig cells. Despite technology development, there are still obstacles and hurdles of HAR and cellular xenogeneic rejection (CXR). First, as a representative xenoantigen, GGTA1 (α1,3-galactosyltransferase) generates a major α1,3-Gal epitope as xenoantigen. Its synthesized α1,3-Gal residue-linked epitopes are present on surfaces of most mammal cells in synthetic mechanism of the donor substrates α1,3-Gal+Galβ1,4GlcNAc (N-acetyllactosamine; LacNAc) to yield Galα1,3Galβ1,4GlcNAc-R carbohydrate structures, known as α1,3-Gal antigenic epitope. For specific aspects of α1,3-Gal epitope, first, the α1,3-Gal antigenic epitope is not existed in humans and several species including Old World monkeys and apes, indicating that α1,3-Gal epitope is a major xenoantigen. Second, cytidine monophosphate (CMP)-N-acetylneuraminic acid (NeuAc) hydroxylase (CMAH) hydroxylates sialic acid Z(Sia), Neu5Ac. The CMAH gene is widely expressed in endothelial cells of mammals, except highly evolved species, humans. Neu5Gc is one of non-Gal xenoantigens. Third, β4GalNT2 (β1,4N-acetylgalactosaminyl transferase) synthesizes the Sia-α2,3-(GalNAc-β1,4)Gal-β1,4-GlcNAc structure known as Sd(a) antigen, transferring a donor substrate of β1,4-GalNAc residue to the Gal residue of an α2,3-sialylglycan structure. Sd(a) is one of non-α1,3Gal xenoantigens (Fig. 10.1). Fourth, human HLA-compatible swine leukocyte antigen (SLA) is a swine type MHC protein, which is highly polymorphic. SLA of pigs, like HLA of humans, is directly associated with the immune responses of pigs to microbial and viral infections as well as vaccinations.
The pig has long been used as a research animal and has now gained importance as a potential source of organs for clinical xenotransplantation. When an organ from a wild-type (i.e., genetically unmodified) pig is transplanted into an immunosuppressed nonhuman primate, a vigorous host immune response causes hyperacute rejection (within minutes or hours). This response has been largely overcome by 1) extensive gene editing of the organ-source pig and 2) the administration to the recipient of novel immunosuppressive therapy based on blockade of the CD40/CD154 T cell costimulation pathway. Gene editing has consisted of 1) deletion of expression of the 3 known carbohydrate xenoantigens against which humans have natural (preformed) antibodies and 2) the introduction of human ‘protective’ genes. The combination of gene editing and novel immunosuppressive therapy has extended life-supporting pig kidney graft survival to greater than 1 y and of pig heart survival to up to 9 mo. This review briefly describes the techniques of gene editing, the potential risks of transfer of porcine endogenous retroviruses with the organ, and the need for breeding and housing of donor pigs under biosecure conditions.
End-stage organ failure can result from various preexisting conditions and occurs in patients of all ages, and organ transplantation remains its only treatment. In recent years, extensive research has been done to explore the possibility of transplanting animal organs into humans, a process referred to as xenotransplantation. Due to their matching organ sizes and other anatomical and physiological similarities with humans, pigs are the preferred organ donor species. Organ rejection due to host immune response and possible interspecies infectious pathogen transmission have been the biggest hurdles to xenotransplantation's success. Use of genetically engineered pigs as tissue and organ donors for xenotransplantation has helped to address these hurdles. Although several preclinical trials have been conducted in nonhuman primates, some barriers still exist and demand further efforts. This review focuses on the recent advances and remaining challenges in organ and tissue xenotransplantation.
Expected final online publication date for the Annual Review of Animal Biosciences, Volume 12 is February 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Half a million patients in the USA alone require treatment for burns annually. Following an extensive burn, it may not be possible to provide sufficient autografts in a single setting. Genetic manipulations (GM) of pigs offer the possibility of reducing primate humoral and cellular rejection of pig skin xenografts and thus extending graft survival. We compared the survival of skin grafts from pigs with 9‐GM with that of autografts and allografts in squirrel monkeys. Monitoring for rejection was by (1) macroscopic examination, (2) histopathological examination of skin biopsies, and (3) measurement of anti‐monkey and anti‐pig IgM and IgG antibodies. Autografts ( n = 5) survived throughout the 28 days of follow‐up without histopathological features of rejection. Median survival of allografts ( n = 6) was 14 days and of pig xenografts ( n = 12) 21 days. Allotransplantation was associated with an increase in anti‐monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti‐pig IgM and IgG. In all cases, histopathologic features of rejection were similar. 9‐GM pig skin xenografts survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti‐pig IgM and IgG antibodies, these did not cross‐react with monkey antigens, indicating that a primary 9‐GM pig skin graft would not be detrimental to a subsequent monkey skin allograft.
Recent human decedent model studies1,2 and compassionate xenograft use³ have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.
Heart transplantation (HTx) has proved itself to be the definite therapy for patients with end- stage cardiac failure. While HTx remains the cornerstone of the surgical armamentarium, it,s long- term success has resulted in an unbalanced supply- demand equation due to severe scarcity of valid organ donors. Therefore, further developments in the field of transplant therapy remain curtailed, unless other resources are encouraged. The present report is a review on the concept evolution, present status and reasonable outlook of three different, but confluent, emerging therapies, namely, a) xenotransplantation, b) 3D additive bioprinting and c) gene therapy.
Background:
Blood vessels that contain endothelial cells (ECs) on the surface are in direct contact with host blood and are the first target of xenograft rejection. Currently, our understanding of human anti-pig vessel immune responses is primarily based on in vitro assays using pig ECs. Therefore, it is necessary to develop an animal model that permits in vivo study of human immunological rejection of pig vessels.
Methods:
Pig artery tissues (PAT) were transplanted into human immune system (HIS) mice or immunodeficient NSG mice (as controls). Intragraft human immune cell infiltration and antibody deposition were quantified using histology and immunohistochemistry. Donor antigen-specific immune responses were quantified using a mixed lymphocyte reaction and a complement-dependent killing assay.
Results:
Pig CD31+ ECs were detected and increased 2-fold from weeks 3 to 5 in PAT xenografts from immunodeficient NSG mice. However, compared with NSG mice, PAT xenografts in HIS mice had significantly lower numbers of porcine CD31+ ECs and showed a marked reduction from week 3 to week 5. PAT xenograft rejection in HIS mice is associated with intensive infiltration of human immune cells, deposition of human IgM and IgG antibodies, and the formation of a tertiary lymphoid structure. Robust donor pig antigen-specific human T cells and antibody responses were detected in PAT-transplanted HIS mice.
Conclusion:
We have developed a humanized mouse model to evaluate human anti-pig xenoimmune responses by PAT transplantation in vivo. This model is expected to facilitate the refinement of pig gene-editing strategies (the expression on EC surface) and the testing of local immunosuppressive strategies for clinical pig organ xenotransplantation.
Antibody-mediated rejection (AMR) is the commonest cause of failure of a pig graft after transplantation into an immunosuppressed nonhuman primate (NHP). The incidence of AMR compared to acute cellular rejection is much higher in xenotransplantation (46% vs. 7%) than in allotransplantation (3% vs. 63%) in NHPs. Although AMR in an allograft can often be reversed, to our knowledge there is no report of its successful reversal in a pig xenograft. As there is less experience in preventing or reversing AMR in models of xenotransplantation, the results of studies in patients with allografts provide more information. These include (i) depletion or neutralization of serum anti-donor antibodies, (ii) inhibition of complement activation, (iii) therapies targeting B or plasma cells, and (iv) anti-inflammatory therapy. Depletion or neutralization of anti-pig antibody, for example, by plasmapheresis, is effective in depleting antibodies, but they recover within days. IgG-degrading enzymes do not deplete IgM. Despite the expression of human complement-regulatory proteins on the pig graft, inhibition of systemic complement activation may be necessary, particularly if AMR is to be reversed. Potential therapies include (i) inhibition of complement activation (e.g., by IVIg, C1 INH, or an anti-C5 antibody), but some complement inhibitors are not effective in NHPs, for example, eculizumab. Possible B cell-targeted therapies include (i) B cell depletion, (ii) plasma cell depletion, (iii) modulation of B cell activation, and (iv) enhancing the generation of regulatory B and/or T cells. Among anti-inflammatory agents, anti-IL6R mAb and TNF blockers are increasingly being tested in xenotransplantation models, but with no definitive evidence that they reverse AMR. Increasing attention should be directed toward testing combinations of the above therapies. We suggest that treatment with a systemic complement inhibitor is likely to be most effective, possibly combined with anti-inflammatory agents (if these are not already being administered). Ultimately, it may require further genetic engineering of the organ-source pig to resolve the problem entirely, for example, knockout or knockdown of SLA, and/or expression of PD-L1, HLA E, and/or HLA-G.
Background:
Xenoantigens other than Gal, Neu5Gc, and Sda may be playing a role in pig graft rejection. We investigated the incidence of antibodies to unknown pig xenoantigen in different human groups.
Methods:
We collected blood from TKO/hCD55 pigs (n = 3), and isolated PBMCs and RBCs. Serum samples were collected from (i) healthy human volunteers (n = 43), (ii) patients with end-stage renal disease (ESRD) (n = 87), (iii) the same patients after kidney allotransplantation (n = 50), and (iv) renal allotransplant recipients experiencing T cell-mediated rejection (allo-TCMR, n = 10). The sera were initially incubated with TKO/hCD55 pRBCs (1 × 108 cells) for 1 h to absorb anti-pig antibodies (except against SLA and possibly other antigens not expressed on pRBCs) and then the serum (absorbed or unabsorbed) was tested for antibody binding and complement-dependent cytotoxicity (CDC) to TKO/hCD55 pig PBMCs.
Results:
A significant reduction in IgM/IgG binding and CDC was observed in the absorbed sera. Serum obtained before and after renal allotransplantation showed no significant difference in IgM or IgG binding to, or in CDC of, TKO/hCD55 pig cells. IgM antibodies (but rarely IgG) against unknown xenoantigens expressed on TKO/hCD55 PBMCs, possibly against swine leukocyte antigens, were documented in healthy humans, patients with ESRD, and those with renal allografts undergoing acute T cell rejection. IgM (but not CDC) was higher in patients experiencing allo-TCMR.
Conclusion:
Human sera contain IgM antibodies against unknown pig xenoantigens expressed on TKO/hCD55 pPBMCs. Although not confirmed in the present study, the targets for these antibodies may include swine leukocyte antigens.
Background:
A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome.
Methods:
Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells.
Findings:
After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes.
Interpretation:
Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future.
Funding:
The University of Maryland School of Medicine, and the University of Maryland Medical Center.
Solid organ transplant (SOT) recipients require meticulously tailored immunosuppressive regimens to minimize graft loss and mortality. Traditional approaches focus on inhibiting effector T cells, while the intricate and dynamic immune responses mediated by other components remain unsolved. Emerging advances in synthetic biology and material science have provided novel treatment modalities with increased diversity and precision to the transplantation community. This review investigates the active interface between these two fields, highlights how living and non-living structures can be engineered and integrated for immunomodulation, and discusses their potential application in addressing the challenges in SOT clinical practice.
In June 2022, the US Food and Drug Administration Center for Biologics Evaluation and Research held the 73rd meeting of the Cellular, Tissue, and Gene Therapies Advisory Committee for public discussion of regulatory expectations for xenotransplantation products. The members of a joint American Society of Transplant Surgeons/American Society of Transplantation committee on xenotransplantation compiled a meeting summary focusing on 7 topics believed to be key by the committee: (1) preclinical evidence supporting progression to a clinical trial, (2) porcine kidney function, (3) ethical aspects, (4) design of initial clinical trials, (5) infectious disease issues, (6) industry perspectives, and (7) regulatory oversight.
The mechanistic/mammalian target of rapamycin (mTOR) is one of the systems that are necessary to maintain cell homeostasis, such as survival, proliferation, and differentiation. mTOR inhibitors (mTOR-Is) are utilized as immunosuppressants and anti-cancer drugs. In organ allotransplantation, current regimens infrequently include an mTOR-I, which are positioned more commonly as alternative immunosuppressants. In clinical allotransplantation, long-term efficacy has been established, but there is a significant incidence of adverse events, for example, inhibition of wound healing, buccal ulceration, anemia, hyperglycemia, dyslipidemia, and thrombocytopenia, some of which are dose-dependent. mTOR-Is have properties that may be especially beneficial in xenotransplantation. These include suppression of T cell proliferation, increases in the number of T regulatory cells, inhibition of pig graft growth, and anti-inflammatory, anti-viral, and anti-cancer effects. We here review the potential benefits and risks of mTOR-Is in xenotransplantation and suggest that the benefits exceed the adverse effects.
Progress in pig organ xenotransplantation has been made largely through (1) genetic engineering of the organ-source pig to protect its tissues from the human innate immune response, and (2) development of an immunosuppressive regimen based on blockade of the CD40/CD154 costimulation pathway to prevent the adaptive immune response. In the 1980s, after transplantation into nonhuman primates (NHPs), wild-type (genetically unmodified) pig organs were rejected within minutes or hours. In the 1990s, organs from pigs expressing a human complement-regulatory protein (CD55) transplanted into NHPs receiving intensive conventional immunosuppressive therapy functioned for days or weeks. When costimulation blockade was introduced in 2000, the adaptive immune response was suppressed more readily. The identification of galactose-α1,3-galactose as the major antigen target for human and NHP anti-pig antibodies in 1991 allowed for deletion of expression of galactose-α1,3-galactose in 2003, extending pig graft survival for up to 6 months. Subsequent gene editing to overcome molecular incompatibilities between the pig and primate coagulation systems proved additionally beneficial. The identification of 2 further pig carbohydrate xenoantigens allowed the production of 'triple-knockout' pigs that are preferred for clinical organ transplantation. These combined advances enabled the first clinical pig heart transplant to be performed and opened the door to formal clinical trials.
After pig-to-baboon kidney transplantation, episodes of hypovolemia and hypotension from an unexplained mechanism have been reported. This study evaluated the renin-angiotensin-aldosterone system post-kidney xenotransplantation. Kidneys from genetically-engineered pigs were transplanted into 5 immunosuppressed baboons after the excision of the native kidneys. Immunosuppressive therapy was based on the blockade of the CD40/CD154 costimulation pathway. Plasma renin, angiotensinogen (AGT), angiotensin II (Ang II), aldosterone levels, and urine osmolality and electrolytes were measured in healthy pigs, healthy nonimmunosuppressed baboons, and immunosuppressed baboons with life-supporting pig kidney grafts. After pig kidney transplantation, plasma renin and Ang II levels were not significantly different, although Ang II trended lower, even though plasma AGT and potassium were increased. Plasma aldosterone levels were unchanged. Urine osmolality and sodium concentration were decreased. Even in the presence of increasing AGT and potassium levels, lower plasma Ang II concentrations may be because of reduced, albeit not absent, the reactivity of pig renin to cleave baboon AGT, suggesting an impaired response of the renin-angiotensin-aldosterone system to hypovolemic and hypotensive episodes. The maintenance of aldosterone may be protective. The reduced urine osmolality and sodium concentration reflect the decreased ability of the pig kidney to concentrate urine. These considerations should not prohibit successful clinical pig kidney xenotransplantation.
Natural preformed and de novo antibodies against pig antigens are a major cause of pig xenograft rejection in nonhuman primates (NHPs). In vivo studies in pig-to-NHP models are time consuming. In vitro assays, for example, antibody binding to pig cells, complement-dependent cytotoxicity assays, provide valuable information quickly and inexpensively. Using in vitro assays for several years, it has been documented that (1) during the first year of life, humans and NHPs develop anti-wild-type pig antibodies, but humans develop no or minimal antibody to triple-knockout (TKO) pig cells. (2) Some adult humans have no or minimal antibodies to TKO pig cells and are therefore unlikely to rapidly reject a TKO organ, particularly if the organ also expresses human "protective" proteins. (3) There is good correlation between immunoglobulin (Ig)M (but not IgG) binding and complement injury. (4) All Old World NHPs develop antibodies to TKO pig cells and are not optimal recipients of TKO organs. (5) galactosyltransferase gene-knockout/β4GalNT2KO pigs are preferred for Old World NHPs. (6) Humans develop anti-pig IgE and IgA antibodies against pig cells, but their role remains uncertain. (7) In a small percentage of allosensitized humans, antibodies that cross-react with swine leukocyte antigens may be detrimental to a pig organ xenograft. (8) Prior sensitization to pig antigens is unlikely to be detrimental to a subsequent allograft. (9) Deletion of expression of Gal and Neu5Gc is associated with a reduction in the T-cell response to pig cells. All of these valuable observations have largely predicted the results of in vivo studies.
Zusammenfassung
Unter „Xenotransplantation“ wird die Übertragung von funktionsfähigen Zellen, Geweben oder Organen zwischen verschiedenen Spezies verstanden, insbesondere von Schweinen auf den Menschen. In den meisten Industrieländern klafft eine große Lücke zwischen der Anzahl geeigneter Spenderorgane und der Anzahl benötigter Transplantate. Weltweit können nur etwa 10% des Organbedarfs durch Spenden gedeckt werden. Eine erfolgreiche Xenotransplantation könnte diesen Mangel mildern oder sogar weitgehend vermeiden. Das Schwein wird aus verschiedenen Erwägungen heraus als am besten geeignete Spenderspezies angesehen. Bei einer Übertragung porziner Organe auf Primaten treten verschiedene immunologisch bedingte Abstoßungsreaktionen auf, die das übertragene Organ innerhalb kurzer Zeit zerstören können, wie die HAR (hyperakute Abstoßung), die AVR (akute vaskuläre Abstoßung) und die spätere zelluläre Abstoßung. Diese Abstoßungsreaktionen müssen durch genetische Modifikationen im Schwein und eine geeignete immunsuppressive Behandlung des Empfängers kontrolliert werden. Dazu müssen Tiere mit mehrfachen genetischen Veränderungen produziert und im Hinblick auf ihre Eignung für eine erfolgreiche Xenotransplantation geprüft werden. Inzwischen können die HAR und auch die AVR durch Knockouts von antigenen Oberflächenepitopen (z. B. αGal [Galaktose-α1,3-Galaktose]) und transgene Expression humaner Gene mit antiinflammatorischer, antiapoptotischer oder antikoagulativer Wirkung zuverlässig kontrolliert werden. Nach orthotopen Transplantationen in nicht humane Primaten konnten inzwischen mit Schweineherzen Überlebensraten von bis zu 264 Tagen und mit porzinen Nieren von 435 Tagen erzielt werden. Eine Übertragung pathogener Erreger auf den Empfänger kann bei Einhaltung einschlägiger Hygienemaßnahmen ausgeschlossen werden. PERV (porzine endogene Retroviren) können durch RNA-(Ribonukleinsäure-)Interferenz oder Gen-Knockout ausgeschaltet werden. Sie stellen damit kein Übertragungsrisiko für den Empfänger mehr dar. Anfang 2022 wurde in Baltimore (USA) ein Schweineherz mit 10 genetischen Modifikationen auf einen Patienten mit schwerem Herzleiden übertragen, mit dem der Empfänger 2 Monate offenbar ohne größere Probleme lebte. Es wird erwartet, dass Xenotransplantate vom Schwein in absehbarer Zeit zur klinischen Anwendungsreife kommen werden. Dazu werden klinische Versuche zur systematischen Erfassung aller Auswirkungen solcher Transplantate auf den Patienten sowie geeignete rechtliche und finanzielle Rahmenbedingungen benötigt.
Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.
Graft inflammation impairs the induction of solid organ transplant tolerance and enhances acute and chronic rejection. Elucidating the mechanisms by which inflammation is induced after organ transplantation could lead to novel therapeutics to improve transplant outcomes. In this Review we describe endogenous substances - damage-associated molecular patterns (DAMPs) - that are released after allograft reperfusion and induce inflammation. We also describe innate immune signalling pathways that are activated after solid organ transplantation, with a focus on Toll-like receptors (TLRs) and their signal adaptor, MYD88. Experimental and clinical studies have yielded a large body of evidence that TLRs and MYD88 are instrumental in initiating allograft inflammation and promoting the development of acute and chronic rejection. Ongoing clinical studies are testing TLR inhibition strategies in solid organ transplantation, although avoiding compromising host defence to pathogens is a key challenge. Further elucidation of the mechanisms by which sterile inflammation is induced, maintained and amplified within the allograft has the potential to lead to novel anti-inflammatory treatments that could improve outcomes for solid organ transplant recipients.
Immunosuppressed patients and experimental nonhuman primates are at risk of opportunistic infection. We report a Myroides spp infection in an immunosuppressed baboon that had received a life-supporting kidney from a genetically-engineered pig.
The baboon received a costimulation blockade-based immunosuppressive regimen as well as two anti-inflammatory agents (tocilizumab and etanercept). Although the pig kidney functioned well, approximately four months after the transplant the baboon became less active and ate and drank poorly. On day 136, it collapsed and died despite inotropic and fluid support. A blood culture drawn before death grew Myroides spp.
To our knowledge, Myroides spp has not been reported as a cause of opportunistic infection in either patients with organ allotransplants or experimental animals. We summarize what is known about this rare organism, and suggest it should be considered in any immunocompromised patient or animal. In the present case, we suggest the baboon died of circulatory shock following infection through an indwelling intravenous catheter.
Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
The use of animals as donors of tissues and organs for xenotransplantations may help in meeting the increasing demand for organs for human transplantations. Clinical studies indicate that the domestic pig best satisfies the criteria of organ suitability for xenotransplantation. However, the considerable phylogenetic distance between humans and the pig causes tremendous immunological problems after transplantation, thus genetic modifications need to be introduced to the porcine genome, with the aim of reducing xenotransplant immunogenicity. Advances in genetic engineering have facilitated the incorporation of human genes regulating the complement into the porcine genome, knockout of the gene encoding the formation of the Gal antigen (α1,3-galactosyltransferase) or modification of surface proteins in donor cells. The next step is two-fold. Firstly, to inhibit processes of cell-mediated xenograft rejection, involving natural killer cells and macrophages. Secondly, to inhibit rejection caused by the incompatibility of proteins participating in the regulation of the coagulation system, which leads to a disruption of the equilibrium in pro- and anti-coagulant activity. Only a simultaneous incorporation of several gene constructs will make it possible to produce multitransgenic animals whose organs, when transplanted to human recipients, would be resistant to hyperacute and delayed xenograft rejection.
Background:
Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our α1,3-galactosyltransferase gene knockout swine herd.
Methods:
Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed.
Results:
Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days.
Conclusions:
Decreased survivals of α1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.
Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence, predominantly from animal models, indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. We examined the effects of EPO on human T-cell alloimmunity by first documenting that CD4(+) and CD8(+) T cells express EPO receptor (EPO-R) on their surfaces. In mixed lymphocyte reactions, EPO induced a dose-dependent decrease in allogeneic CD4(+) T-cell proliferation (EPO 1000 U/ml: 44.6%±22.9% of vehicle, P<0.05; 2000 U/ml: 11.1%±4% of vehicle, P<0.001) without inducing cell death. The effects required signals transmitted directly through the EPO-R expressed on T cells, resulting in diminished Th1 differentiation without effects on regulatory T-cell induction. Mechanistic studies revealed that EPO prevented IL-2-induced proliferation by uncoupling IL-2 receptor signaling, inhibiting phosphorylation of the intracellular intermediaries AKT and extracellular signal-regulated kinase that are known to mediate T-cell expansion. EPO treatment reduced expansion of human naïve CD4(+) T cells after adoptive transfer into NOD scid γc(null) mouse recipients, verifying the effects in vivo. Although activated T cells expressed CD131, an alternative EPO receptor, addition of a specific CD131 agonist peptide, ARA290, did not alter T-cell proliferation or cytokine production. Our findings link EPO-R signaling on T cells to inhibition of T-cell immunity, providing one mechanism that could explain the observed protective effects of EPO in kidney transplant recipients.
Solid organ transplantation is a vital therapy for end stage diseases. Decades of research have established that components of the adaptive immune system are critical for transplant rejection, but the role of the innate immune system in organ transplantation is just emerging. Accumulating evidence indicates that the innate immune system is activated at the time of organ implantation by the release of endogenous inflammatory triggers. This review discusses the nature of these triggers in organ transplantation and also potential mediators that may enhance inflammation resolution after organ implantation.
Consumptive coagulopathy (CC) remains a challenge in pig-to-primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild-type (n = 2), alpha1,3-galactosyltransferase gene-knockout (GT-KO; n = 1) or GT-KO and transgenic for the complement-regulatory protein, CD46 (GT-KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post-Tx day 1. Subsequently, platelet-leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig-to-primate kidney Tx.
We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.
The importance of CD40/CD154 costimulatory pathway blockade in immunosuppression strategies is well-documented. Efforts are currently focused on monoclonal antibodies specific for CD40 because of thromboembolic complications associated with monoclonal antibodies directed towards CD154. Here we present the rational development and characterization of a novel antagonistic monoclonal antibody to CD40. Rhesus macaques were treated with the recombinant anti-CD40 mAb, 2C10, or vehicle before immunization with keyhole limpet hemocyanin (KLH). Treatment with 2C10 successfully inhibited T cell-dependent antibody responses to KLH without significant peripheral B cell depletion. Subsequently, MHC-mismatched macaques underwent intraportal allogeneic islet transplantation and received basiliximab and sirolimus with or without 2C10. Islet graft survival was significantly prolonged in recipients receiving 2C10 (graft survival time 304, 296, 265, 163 days) compared to recipients receiving basiliximab and sirolimus alone (graft survival time 8, 8, 10 days). The survival advantage conferred by treatment with 2C10 provides further evidence for the importance of blockade of the CD40/CD154 pathway in preventing alloimmune responses. 2C10 is a particularly attractive candidate for translation given its favorable clinical profile.
Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4-7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4-7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.
In the past, ABO blood group incompatibility was considered an absolute contraindication for kidney transplantation. Progress in defined desensitization practice and immunologic understanding has allowed increasingly successful ABO incompatible transplantation during recent years. This paper focused on the history, disserted outcomes, desensitization modalities and protocols, posttransplant immunologic surveillance, and antibody-mediated rejection in transplantation with an ABO incompatible kidney allograft. The mechanism underlying accommodation and antibody-mediated injury was also described.
Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Rejection in Gal knockout (GTKO) pigs due to elicited non-Gal antibody response required further genetic modifications of donor pigs and better control of the B-cell response to xenoantigens. We report significant prolongation of heterotopic alpha Galactosyl transferase "knock-out" and human CD46 transgenic (GTKO.hCD46Tg) pig cardiac xenografts survival in specific pathogen free baboons. Peritransplant B-cell depletion using 4 weekly doses of anti-CD20 antibody in the context of an established ATG, anti-CD154 and MMF-based immunosuppressive regimen prolonged GTKO.hCD46Tg graft survival for up to 236 days (n = 9, median survival 71 days and mean survival 94 days). B-cell depletion persisted for over 2 months, and elicited anti-non-Gal antibody production remained suppressed for the duration of graft follow-up. This result identifies a critical role for B cells in the mechanisms of elicited anti-non-Gal antibody and delayed xenograft rejection. Model-related morbidity due to variety of causes was seen in these experiments, suggesting that further therapeutic interventions, including candidate genetic modifications of donor pigs, may be necessary to reduce late morbidity in this model to a clinically manageable level.
Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.
Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p
Xenograft outcomes are dictated by xenoantigen expression, for example, Gal α 1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal-transferase gene-knockout (GalT-KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis-related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF-transgenic and GalT- KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal-expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT-KO grafts (1 of 14). Protective-type genes (heme oxygenase-I, superoxide dismutases and CD39) together with von Willebrand factor and P-selectin were upregulated in all renal grafts. Transcriptional responses in Gal-expressing xenografts were comparable to those seen in the infrequent GalT-KO rejection.
In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor-1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.
Inflammation is known to preclude tolerance after transplantation. We have previously shown that systemic inflammation in xenograft recipients (SIXR) precedes activation of coagulation in the absence of T cell responses. Accordingly, SIXR may amplify innate and adaptive immune responses against xenografts after pig-to-primate xenotransplantation, even with efficient immunosuppressive therapy. We evaluated the impact of anti-inflammatory agents on pro-inflammatory cytokines and chemokines in pig artery patch and heart xenograft recipients. Baboons received an artery patch (Group1, n=8) or heart (Group2, n=4) from genetically engineered pigs. All baboons received lymphodepletion with thymoglobulin (ATG) and costimulation blockade-based immunosuppression (anti-CD40 and/or CTLA4Ig). In Group1, baboons received either (i) no anti-inflammatory agents (n=2), (ii) cobra venom factor (CVF, n=2), (iii) α1-antitrypsin (AAT, n=2), or (iv) interleukin (IL)-6 receptor antagonist (IL-6RA, n=2). In Group2, all baboon received corticosteroids, either without (n=2) or with (n=2) IL-6RA. Serum IFN-γ, TNF-α, IL-1β, IL-17, IL-6, IL-8, MCP-1, and sCD40L levels were measured by Luminex. Fibrinogen, D-dimers, and C-reactive protein (C-RP) were also measured. Recipient baboon T cell proliferation was evaluated by mixed lymphocyte reaction (MLR) before and after transplantation. Pig and baboon tissue factor (TF) mRNA levels in heart xenografts were measured by RT-PCR. In no recipient was a marked increase in T cell response to pig cells observed after transplantation. In Groups 1 and 2, post-transplantation levels of IFN-γ, TNF-α, IL-1β, and IL-17 remained comparable to or lower than pre-transplant levels, except in one heart recipient that succumbed to CMV infection. In Group1, when no anti-inflammatory agent was administered, post-transplant levels of IL-6, IL-8, and MCP-1 were elevated. After CVF, IL-6, IL-8, and MCP-1 remained low. After IL-6RA, IL-6 and MCP-1 were elevated. After AAT, IL-8 was elevated. sCD40L became elevated intermittently in most recipients irrespective of the administered anti-inflammatory agent. In Group2, IL-6 was transiently elevated, particularly after IL-6RA administration. MCP-1 gradually increased by 2 months in Group2 recipients. sCD40L generally remained low except in one recipient. In Group1 and Group2 recipients, C-RP levels were elevated except after IL-6RA administration, while D-dimers were elevated regardless of administration of anti-inflammatory agent. In Group2, pig TF mRNA levels were increased in heart xenografts compared to naive pig hearts, irrespective of IL-6 receptor antagonist administration. Additionally, baboon TF mRNA levels were detectable in heart xenografts, but not in naive pig hearts. Some pro-inflammatory cytokines and chemokines are elevated in xenograft recipients, even with efficient T cell-directed immunosuppressive therapy. Persistent elevation of D-dimers, and individual cytokines and chemokines suggest a continuous inflammatory response, despite administration of anti-inflammatory agents. Systemic administration of combined anti-inflammatory agents as well as complement regulation may be essential to prevent SIXR after xenotransplantation.
Background:
It has been well documented that the level of serum/plasma free triiodothyronine (fT3) falls rapidly following brain death or during certain surgical procedures, for example, heart surgery carried out on cardiopulmonary bypass. The level in patients following cardiopulmonary bypass usually recovers within 2 days.
Methods:
We have measured serum fT3 in healthy naïve baboons (n = 31), healthy naïve monkeys (n = 5), and after pig-to-baboon heterotopic heart xenotransplantation (xenoTx) (Group 1, n = 9), orthotopic liver xenoTx (Group 2, n = 10), artery patch xenoTx (Group 3, n = 9), and in monkey-to-monkey heterotopic heart alloTx (Group 4, n = 5).
Results:
The mean level of fT3 in healthy naïve baboons was 3.1 ± 0.9 pg/ml and in healthy naïve monkeys was 2.6 ± 0.3 pg/ml. Following pig heart, liver, and artery patch xenoTx and monkey heart alloTx, there was an immediate rapid fall in fT3 level. Recovery of fT3 was more rapid in Groups 3 and 4 than in Groups 1 and 2. In Group 1, within 4 days fT3 had recovered, but only to the lower limit of normal range, where it remained throughout follow-up (for up to 42 days). In Group 2, no recovery was seen during the 7 days of follow-up. In immunosuppressed baboons with pig patch grafts that received IL-6R blockade (n = 2), the fT3 tended to rise higher than in those that received no IL-6R blockade (n = 6).
Conclusions:
Following operative procedures, there is a dramatic fall in serum fT3 levels. The persistent low level of fT3 after pig heart and liver xenoTx may be associated with a continuing inflammatory state. We suggest that consideration should be given to the replacement of T3 therapy to maintain normal fT3 levels, particularly in nonhuman primates undergoing orthotopic pig heart or liver xenoTx.
Background:
Renal ischemia-reperfusion (I/R) injury is associated with delayed graft function and results in poor long-term graft survival. We previously showed that splenectomy (SPLN) protects the kidney from I/R injury and reduces serum TNF-α levels. Herein, we further investigated the effects of SPLN on inflammatory responses and tissue injury in renal I/R by examining the expression of major inflammatory cytokines and heat shock protein 70 (HSP70). Because it was shown previously that the anti-TNF-α agent infliximab (IFX) attenuated renal I/R injury, we also investigated whether IFX administration mimics the effects of SPLN.
Methods:
The left renal pedicles of adult male Wistar rats were clamped for 45 minutes and then reperfused for 24 hours; right nephrectomy and SPLN were performed immediately. A separate cohort was administered IFX 1 hour before surgery in lieu of SPLN.
Results:
Serum creatinine and blood urea nitrogen levels were markedly elevated by I/R injury; these increases were significantly reversed by IFX. Furthermore, IFX inhibited the induction of inflammatory cytokines and HSP70 during renal I/R injury. Time-dependent profiles revealed that the expression of inflammatory cytokines was elevated immediately after I/R, whereas levels of HSP70, serum creatinine, and blood urea nitrogen began to rise 3 hours postreperfusion. Macrophages/monocytes were significantly increased in I/R-injured kidneys, but not in those administered IFX. The outcomes of SPLN mirrored those of IFX administration.
Conclusions:
Splenectomy and TNF-α inhibition both protect the kidney from I/R injury by reducing the accumulation of renal macrophages/monocytes and induction of major inflammatory cytokines.
Background:
Erythropoietin exerts anti-inflammatory, antiapoptotic, and cytoprotective effects in addition to its hematopoietic action. A nonhematopoietic erythropoietin analogue, ARA 290, has similar properties. The efficacy of pancreatic islet transplantation (PITx) is reduced due to islet damage that occurs during isolation and from the severe inflammatory reactions caused by the transplantation procedure. We investigated whether ARA 290 protects islets and ameliorates inflammatory responses following PITx thus improving engraftment.
Methods:
The effects of ARA 290 on pancreatic islets of C57BL/6J (H-2) mice and on murine macrophages were investigated using an in vitro culture model. As a marginal PITx, 185 islets were transplanted into the liver of streptozotocin-induced diabetic mice (H-2) via the portal vein. Recipients were given ARA 290 (120 μg/kg) intraperitoneally just before and at 0, 6, and 24 hours after PITx. Liver samples were obtained at 12 hours after PITx, and expression levels of proinflammatory cytokines were assessed.
Results:
ARA 290 protected islets from cytokine-induced damage and apoptosis. Secretion of pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) from macrophages was significantly inhibited by ARA 290. After the marginal PITx, ARA 290 treatment significantly improved the blood glucose levels when compared to those of control animals (P < 0.001). Upregulation of monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, IL-1β, and IL-6 messenger RNA expression within the liver was suppressed by ARA 290 treatment.
Conclusions:
ARA 290 protected pancreatic islets from cytokine-induced damage and apoptosis and ameliorated the inflammatory response after PITx. ARA 290 appears to be a promising candidate for improvement of PITx.
Significant progress in life-supporting kidney xenograft survival in nonhuman primates (NHPs) has been associated largely with the increasing availability of pigs with genetic modifications that protect the pig tissues from the primate immune response and/or correct molecular incompatibilities between pig and primate. Blockade of the CD40/CD154 costimulation pathway with anti-CD154 mAb therapy has contributed to prolongation of kidney xenograft survival, although this agent may not be clinically available. An anti-CD40 mAb-based regimen is proving equally successful, but blockade of the CD28/B7 pathway is inadequate. Severe proteinuria were uniformly documented in the early studies of pig kidney xenotransplantation, but whether this resulted from immune injury or from physiological incompatibilities between the species, or both, remained uncertain. Recent experiments suggest it was related to a continuing immune response. Before 2014, the longest survival of a pig kidney graft in a NHP was 90 days, though graft survival >30 days was unusual. Recently this has been extended to >125 days, without features of a consumptive coagulopathy or a protein-losing nephropathy. In conclusion, overcoming the immune, coagulation, and inflammatory responses by the development of precise genetic modifications in donor pigs, along with effective immunosuppressive and anticoagulant/anti-inflammatory therapy is advancing the field towards clinical trials.
Copyright © 2015. Published by Elsevier Ltd.
In pig-to-baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T-cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D-dimer) and inflammation (increased C-reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation.
Baboons received heart (n = 8) or artery patch (n = 16) transplants from genetically engineered pigs and a costimulation blockade-based regimen. Heart grafts functioned for 15-130 days. Artery recipients were euthanized after 28-84 days. Platelet counts, fibrinogen, D-dimer, and CRP were measured.
Thrombocytopenia and reduced fibrinogen developed only in recipients of hearts not expressing a coagulation-regulatory protein (n = 4), but not in other heart or patch recipients. However, in heart recipients (n = 8), there were sustained increases in D-dimer (<0.5 to 1.9 ug/ml [P < 0.01]) and CRP (0.26-2.2 mg/dl [P < 0.01]). In recipients of artery patches, there were also sustained increases in D-dimer (<0.5 to 1.4 ug/ml [P < 0.01]) and CRP (0.26 to 1.5 mg/dl [P < 0.001]). An IL-6R antagonist suppressed the increase in CRP, but not D-dimer.
The pig artery patch model has proved valuable for determining immunosuppressive regimens that prevent sensitization to pig antigens. This model also provides information on the sustained systemic inflammation in xenograft recipients (SIXR). An IL-6R antagonist may help suppress this response.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Inflammation is a complex response that involves interactions between multiple proteins in the human body. The interaction between inflammation and coagulation is well-recognized, but its role in the dysregulation of coagulation in xenograft recipients is not well-understood. Additionally, inflammation is known to prevent the development of T cell tolerance after transplantation. Recent evidence indicates that systemic inflammation precedes and may be promoting activation of coagulation after pig-to-primate xenotransplantation. Activated recipient innate immune cells expressing tissue factor are increased after xenotransplantation, irrespective of immunosuppressive therapy. With immunosuppression, C-reactive protein (C-RP), fibrinogen, and interleukin-6 levels are significantly increased in pig artery patch recipients. In pig organ recipients, increased C-RP levels are observed prior to the development of features of consumptive coagulopathy. Systemic inflammation in xenograft recipients (SIXR) may be a key factor in the development of dysregulation of coagulation, as well as in resistance to immunosuppressive therapy. While genetic modification of the donor pigs provides protection against humoral responses and the development of thrombotic microangiopathy, therapeutic prevention of SIXR may be essential in order to prevent systemic dysregulation of coagulation in xenograft recipients without the use of intensive immunosuppression.
Copyright © 2015. Published by Elsevier Ltd.
The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Background
Three costimulation blockade-based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4-Ig (abatacept) or anti-CD40mAb+CTLA4-Ig (belatacept) can successfully replace anti-CD154mAb.Methods
All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n = 4) expressed additional CD55, and those into Group B (n = 3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n = 2) or abatacept (Regimen 2: n = 2) or anti-CD40mAb+belatacept (Regimen 3: n = 2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy.ResultsGroup A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2.Conclusions
The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.
Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series.
Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype.
Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome.
This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Background
Dysregulation of coagulation is considered a major barrier against successful pig organ xenotransplantation in non-human primates. Inflammation is known to promote activation of coagulation. The role of pro-inflammatory factors as well as the relationship between inflammation and activation of coagulation in xenograft recipients is poorly understood.Methods
Baboons received kidney (n = 3), heart (n = 4), or artery patch (n = 8) xenografts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human complement-regulatory protein CD46 (GTKO/CD46). Immunosuppression (IS) was based on either CTLA4Ig or anti-CD154 costimulation blockade. Three artery patch recipients did not receive IS. Pro-inflammatory cytokines, chemokines, and coagulation parameters were evaluated in the circulation after transplantation. In artery patch recipients, monocytes and dendritic cells (DC) were monitored in peripheral blood. Expression of tissue factor (TF) and CD40 on monocytes and DC were assessed by flow cytometry. C-reactive protein (C-RP) levels in the blood and C-RP deposition in xenografts as well as native organs were evaluated. Baboon and pig C-RP mRNA in heart and kidney xenografts were evaluated.ResultsIn heart and kidney xenograft recipients, the levels of INFγ, TNF-α, IL-12, and IL-8 were not significantly higher after transplantation. However, MCP-1 and IL-6 levels were significantly higher after transplantation, particularly in kidney recipients. Elevated C-RP levels preceded activation of coagulation in heart and kidney recipients, where high levels of C-RP were maintained until the time of euthanasia in both heart and kidney recipients. In artery patch recipients, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were elevated with no IS, while IL-6 was not. With IS, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were reduced, but IL-6 was elevated. Elevated IL-6 levels were observed as early as 2 weeks in artery patch recipients. While IS was associated with reduced thrombin activation, fibrinogen and C-RP levels were increased when IS was given. There was a significant positive correlation between C-RP, IL-6, and fibrinogen levels. Additionally, absolute numbers of monocytes were significantly increased when IS was given, but not without IS. This was associated with increased CD40 and TF expression on CD14+ monocytes and lineageneg CD11c+ DC, with increased differentiation of the pro-inflammatory CD14+ CD11c+ monocyte population. At the time of euthanasia, C-RP deposition in kidney and heart xenografts, C-RP positive cells in artery patch xenograft and native lungs were detected. Finally, high levels of both pig and baboon C-RP mRNA were detected in heart and kidney xenografts.Conclusions
Inflammatory responses precede activation of coagulation after organ xenotransplantation. Early upregulation of C-RP and IL-6 levels may amplify activation of coagulation through upregulation of TF on innate immune cells. Prevention of systemic inflammation in xenograft recipients (SIXR) may be required to prevent dysregulation of coagulation and avoid excessive IS after xenotransplantation.
Background
The pig-to-non-human primate model is the standard choice for in vivo studies of organ and cell xenotransplantation. In 1998, Lambrigts and his colleagues surveyed the entire world literature and reported all experimental studies in this model. With the increasing number of genetically engineered pigs that have become available during the past few years, this model is being utilized ever more frequently.Methods
We have now reviewed the literature again and have compiled the data we have been able to find for the period January 1, 1998 to December 31, 2013, a period of 16 yr.ResultsThe data are presented for transplants of the heart (heterotopic and orthotopic), kidney, liver, lung, islets, neuronal cells, hepatocytes, corneas, artery patches, and skin. Heart, kidney, and, particularly, islet xenograft survival have increased significantly since 1998.DiscussionThe reasons for this are briefly discussed. A comment on the limitations of the model has been made, particularly with regard to those that will affect progression of xenotransplantation toward the clinic.
Baboons have natural antibodies against pig antigens. We have investigated whether there are differences in anti-non-Gal pig antibody levels between baboons maintained under specific pathogen-free (SPF) conditions and those housed under conventional conditions (non-SPF) that might be associated with improved outcome after pig-to-baboon organ transplantation. Baboons (n = 40) were housed indoors (SPF n = 8) or in indoor/outdoor pens (non-SPF n = 32) in colonies of similar size and structure. Non-SPF colonies harbor a number of pathogens common to non-human primate species, whereas many of these pathogens have been eliminated from the SPF colony. Complete blood cell counts (CBC), blood chemistry, and anti-non-Gal IgM and IgG levels were monitored. There were no significant differences in CBC or blood chemistry between SPF and non-SPF baboons. Anti-non-Gal IgM levels were significantly lower in the SPF baboons than in the non-SPF baboons (MFI 7.1 vs. 8.8, P < 0.05). One SPF and two non-SPF baboons had an MFI >20; if these three baboons are omitted, the mean MFIs were 4.8 (SPF) vs. 7.5 (non-SPF) (P < 0.05). Anti-non-Gal IgG was minimal in both groups (MFI 1.0 vs. 1.0). As their levels of anti-non-Gal IgM are lower, baboons maintained under SPF conditions may be beneficial for xenotransplantation studies as the initial binding of anti-pig IgM to an α1,3-galactosyltransferase gene-knockout pig organ may be less, thus resulting in less complement and/or endothelial cell activation. However, even under identical SPF conditions, an occasional baboon will express a high level of anti-non-Gal IgM, the reason for which remains uncertain.
Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications.
In this experiment, we replaced anti-CD154 antibody with a more clinically acceptable anti-CD40 antibody while keeping the rest of the immunosuppressive regimen and the donor pig genetics the same. This was carried out to evaluate the antibody's role in xenograft survival and prevention of coagulopathies. Two available clones of anti-CD40 antibody were tested. One mouse anti-human CD40 antibody, (clone 3A8), activated B lymphocytes in vitro and only modestly suppressed antibody production in vivo. Whereas a recombinant mouse non-human primate chimeric raised against macaque CD40, (clone 2C10R4), blocked B-cell activation in vitro and completely blocked antibody production in vivo.
The thrombotic complications seen with anti-CD154 antibody were effectively avoided but the graft survival, although extended, was not as prolonged as observed with anti-CD154 antibody treatment. The longest survival for the 3A8 antibody group was 27 days, and the longest graft survival in the 2C10R4 antibody group was 146 days. All of the grafts except two rejected and were explanted. Only two recipient baboons had to be euthanized due to unrelated complications, and the rest of the baboons remained healthy throughout the graft survival period or after graft explantation. In contrast to our anti-CD 154 antibody-treated baboons, the non-Gal antibody levels started to rise after B cells made their appearance around 8 weeks post-transplantation.
Anti-CD40 antibody at the current dose does not induce any coagulopathies but while effective, had reduced efficacy to induce similar long-term graft survival as with anti-CD154 antibody perhaps due to ineffective control of B-cell function and antibody production at the present dose. More experiments are required to determine antibody affinity and effective dose for inducing long-term cardiac xenograft survival.
Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM.
We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels.
A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene-either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay.
The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.
Background. Organ transplant recipients currently require lifetime immunosuppressive therapy, with its accompanying side effects. Biological agents that block T-cell costimulatory pathways are important components of strategies being developed to induce transplantation tolerance. The aim of this study was to test the effect of a novel chimeric anti-human CD40 monoclonal antibody (Chi 220), either alone or in combination with CTLA4-Ig, on the survival of renal allografts in a nonhuman primate model. Methods. Captive-bred adolescent male rhesus monkeys (Macaca mulatta) (4–10 kg) were used as recipients and donors. Four treatment protocols were tested: Chi220 monotherapy, CTLA4-Ig monotherapy, Chi220 combined with CTLA4-Ig, and H106 (anti-CD40L) combined with CTLA4-Ig. Control animals received human albumin. Recipients were followed for survival, renal allograft function as determined by measurement of serum blood urea nitrogen (BUN) and creatinine, chemistries (sodium, potassium, chloride, and bicarbonate), complete blood cell count (CBC) with differential, and the development of donor-specific alloantibody. Results. Treatment with Chi220 for 14 days prolonged renal allograft survival (MST 38.5 vs. 7 days in untreated controls). Notably, simultaneous blockade of the CD28/B7 pathway did not further augment graft survival but did suppress the development of donor-specific antibodies, an effect not achieved with Chi220 alone, despite peripheral B cell depletion. Finally, treatment with Chi220 suppressed the primary immune response to cytomegalovirus, resulting in severe systemic manifestations. Conclusions. Blockade of the CD40 pathway with anti-CD40 mAb is immunosuppressive in a large animal, preclinical renal transplant model. The potential effect of this therapy on viral immune responses will be important to consider for the design of safe clinical trials.
Background:
The increasing availability of genetically engineered pigs is steadily improving the results of pig organ and cell transplantation in non-human primates (NHPs). Current techniques offer knockout of pig genes and/or knockin of human genes. Knowledge of normal values of hematologic, biochemical, coagulation, and other parameters in healthy genetically engineered pigs and NHPs is important, particularly following pig organ transplantation in NHPs. Furthermore, information on parameters in various NHP species may prove important in selecting the optimal NHP model for specific studies.
Methods:
We have collected hematologic, biochemical, and coagulation data on 71 α1,3-galactosyltransferase gene-knockout (GTKO) pigs, 18 GTKO pigs additionally transgenic for human CD46 (GTKO.hCD46), four GTKO.hCD46 pigs additionally transgenic for human CD55 (GTKO.hCD46.hCD55), and two GTKO.hCD46 pigs additionally transgenic for human thrombomodulin (GTKO.hCD46.hTBM).
Results:
We report these data and compare them with similar data from wild-type pigs and the three major NHP species commonly used in biomedical research (baboons, cynomolgus, and rhesus monkeys) and humans, largely from previously published reports.
Conclusions:
Genetic modification of the pig (e.g., deletion of the Gal antigen and/or the addition of a human transgene) (i) does not result in abnormalities in hematologic, biochemical, or coagulation parameters that might impact animal welfare, (ii) seems not to alter metabolic function of vital organs, although this needs to be confirmed after their xenotransplantation, and (iii) possibly (though, by no means certainly) modifies the hematologic, biochemical, and coagulation parameters closer to human values. This study may provide a good reference for those working with genetically engineered pigs in xenotransplantation research and eventually in clinical xenotransplantation.
Islet cell transplantation (ICT) is a promising approach to cure patients with type 1 diabetes. We have implemented a new immunosuppression protocol with antithymoglobulin plus anti-inflammatory agents of anakinra and eternacept for induction and tacrolimus plus mycophenolate mofetil for maintenance [T-cell depletion with anti-inflammatory (TCD-AI) protocol], resulting in successful single-donor ICT.
Eight islet recipients with type 1 diabetes reported adverse events (AEs) monthly. AEs were compared between three groups: first infusion with the TCD-AI protocol (TCD-AI-1st) and first and second infusion with the Edmonton-type protocol (Edmonton-1st and Edmonton-2nd).
The incidence of symptomatic AEs within the initial three months in the TCD-AI-1st group was less than in the Edmonton-1st and Edmonton-2nd groups, with a marginally significant difference (mean ± SE: 5.5 ± 0.3, 7.5 ± 0.5, and 8.3 ± 1.3, respectively; p = 0.07). A significant reduction in liver enzyme elevation after ICT was found in the TCD-AI-1st group compared with the Edmonton-1st and Edmonton-2nd groups (p < 0.05). Because of AEs, all patients in the Edmonton protocol eventually converted to the TCD-AI protocol, whereas all patients tolerated the TCD-AI protocol.
TCD-AI protocol can be tolerated for successful ICT, although this study includes small cohort, and large population trial should be taken.
CD154 blockade-based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3-galactosyltransferase gene-knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade.
Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti-CD154mAb-based (Group2), or CTLA4-Ig-based (Group3) immunosuppressive therapy. Anti-pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration.
Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti-CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti-thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti-CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4-Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration.
(i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti-CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti-CD154mAb by CTLA4-Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti-pig antibodies.
Rejection of xenografts is associated with vascular-based inflammation, thrombocytopenia and the consumption of coagulation factors that may evolve into disseminated intravascular coagulation (DIC). Similarly, bone marrow-derived cellular xenotransplantation procedures are associated with endothelial cell activation and thrombotic microangiopathic injury. These complications generally develop despite the best available measures for depletion of xenoreactive natural antibody, inhibition of complement activation and suppression of T- and B-cell mediated immune responses. The mechanisms underlying the DIC and thrombotic microangiopathy associated with xenotransplantation are unclear. A proposed primary biological dysfunction of xenografts with respect to regulation of clotting could amplify vascular injury, promote immunological responses and independently contribute to graft failure. Disordered thromboregulation could have deleterious effects, comparable to unregulated complement activation, in the pathogenesis of xenograft rejection and may therefore represent a substantive barrier to xenotransplantation.
Cooper DKC, Ekser B, Burlak C, Ezzelarab M, Hara H, Paris L, Tector AJ, Phelps C, Azimzadeh AM, Ayares D, Robson SC, Pierson RN III. Clinical lung xenotransplantation – what donor genetic modifications may be necessary? Xenotransplantation 2012; 19: 144–158. © 2012 John Wiley & Sons A/S.
Abstract: Barriers to successful lung xenotransplantation appear to be even greater than for other organs. This difficulty may be related to several macro anatomic factors, such as the uniquely fragile lung parenchyma and associated blood supply that results in heightened vulnerability of graft function to segmental or lobar airway flooding caused by loss of vascular integrity (also applicable to allotransplants). There are also micro-anatomic considerations, such as the presence of large numbers of resident inflammatory cells, such as pulmonary intravascular macrophages and natural killer (NK) T cells, and the high levels of von Willebrand factor (vWF) associated with the microvasculature. We have considered what developments would be necessary to allow successful clinical lung xenotransplantation. We suggest this will only be achieved by multiple genetic modifications of the organ-source pig, in particular to render the vasculature resistant to thrombosis. The major problems that require to be overcome are multiple and include (i) the innate immune response (antibody, complement, donor pulmonary and recipient macrophages, monocytes, neutrophils, and NK cells), (ii) the adaptive immune response (T and B cells), (iii) coagulation dysregulation, and (iv) an inflammatory response (e.g., TNF-α, IL-6, HMGB1, C-reactive protein). We propose that the genetic manipulation required to provide normal thromboregulation alone may include the introduction of genes for human thrombomodulin/endothelial protein C-receptor, and/or tissue factor pathway inhibitor, and/or CD39/CD73; the problem of pig vWF may also need to be addressed. It would appear that exploration of every available therapeutic path will be required if lung xenotransplantation is to be successful. To initiate a clinical trial of lung xenotransplantation, even as a bridge to allotransplantation (with a realistic possibility of survival long enough for a human lung allograft to be obtained), significant advances and much experimental work will be required. Nevertheless, with the steadily increasing developments in techniques of genetic engineering of pigs, we are optimistic that the goal of successful clinical lung xenotransplantation can be achieved within the foreseeable future. The optimistic view would be that if experimental pig lung xenotransplantation could be successfully managed, it is likely that clinical application of this and all other forms of xenotransplantation would become more feasible.
Transgenic expression of human complement regulatory proteins reduces the frequency of hyperacute rejection (HAR) in Gal-positive cardiac xenotransplantation. In this study, we examined the impact of human CD55 (hCD55) expression on a Gal knockout (GTKO) background using pig-to-primate heterotopic cardiac xenotransplantation.
Cardiac xenotransplantation was performed with GTKO (group 1; n=6) and GTKO.hCD55 (group 2; n=5) donor pigs using similar immunosuppression. Cardiac biopsies were obtained 30 min after organ reperfusion. Rejection was characterized by histology and immunohistology. Intragraft gene expression, serum non-Gal antibody, and antibody recovered from rejected hearts were analyzed.
HAR of a GTKO heart was observed. Remaining grafts developed delayed xenograft rejection. Median survival was 21 and 28 days for groups 1 and 2, respectively. Vascular antibody deposition was uniformly detected 30 min after organ reperfusion and at explant. A higher frequency of vascular C5b deposition was seen in GTKO organs at explant. Serum non-Gal antibody, antibody recovered from the graft, and intragraft gene expression were similar between the groups.
HAR of GTKO hearts without hCD55 may occur. Expression of hCD55 seemed to restrict local complement activation but did not improve graft survival. Chronic vascular antibody deposition with evidence of protracted endothelial cell activation was seen. These observations suggest that non-Gal antibody-induced chronic endothelial cell activation coupled to possible hemostatic incompatibilities may be the primary stimulus for delayed xenograft rejection of GTKO hearts. To avoid possible HAR, future clinical studies should use donors expressing human complement regulatory proteins in the GTKO background.
Gene profiling methods have been widely useful for delineating changes in gene expression as an approach for gaining insight into the mechanism of rejection or disease pathology. Herein, we use gene profiling to compare changes in gene expression associated with different orthotopic cardiac xenotransplantation (OCXTx) outcomes and to identify potential effects of OCXTx on cardiac physiology.
We used the Affymetrix GeneChip Porcine Genomic Array to characterize three types of orthotopic cardiac xenograft outcomes: 1) rejected hearts that underwent delayed xenograft rejection (DXR); 2) survivor hearts in which the xenograft was not rejected and recipient death was due to model complications; and 3) hearts which failed to provide sufficient circulatory support within the first 48 h of transplant, termed "perioperative cardiac xenograft dysfunction" (PCXD). Gene expression in each group was compared to control, not transplanted pig hearts, and changes in gene expression > 3 standard deviations (±3SD) from the control samples were analyzed. A bioinformatics analysis was used to identify enrichments in genes involved in Kyoto Encyclopedia of Genes and Genomes pathways and gene ontogeny molecular functions. Changes in gene expression were confirmed by quantitative RT-PCR.
The ±3SD data set contained 260 probes, which minimally exhibited a 3.5-fold change in gene expression compared to control pig hearts. Hierarchical cluster analysis segregated rejected, survivor and PCXD samples, indicating a unique change in gene expression for each group. All transplant outcomes shared a set of 21 probes with similarly altered expression, which were indicative of ongoing myocardial inflammation and injury. Some outcome-specific changes in gene expression were identified. Bioinformatics analysis detected an enrichment of genes involved in protein, carbohydrate and branched amino acid metabolism, extracellular matrix-receptor interactions, focal adhesion, and cell communication.
This is the first genome wide assessment of changes in cardiac gene expression after OCXTx. Hierarchical cluster analysis indicates a unique gene profile for each transplant outcome but additional samples will be required to define the unique classifier probe sets. Quantitative RT-PCR confirmed that all transplants exhibited strong evidence of ongoing inflammation and myocardial injury consistent with the effects of cytokines and vascular antibody-mediated inflammation. This was also consistent with bioinformatic analysis suggesting ongoing tissue repair in survivor and PCXD samples. Bioinformatics analysis suggests for the first time that xenotransplantation may affect cardiac metabolism in survivor and rejected samples. This study highlights the potential utility of molecular analysis to monitor xenograft function, to identify new molecular markers and to understand processes, which may contribute to DXR.
In 2002, we introduced the anti-CD20 chimeric antibody, rituximab, for ABO-incompatible kidney transplantation (ABO-IKT). Here, we report the 5-year outcome obtained using rituximab as part of the preoperative regimen for ABO-IKT.
Between January 2002 and December 2008, 408 patients underwent living-related kidney transplantation at our department. The patients were divided into three groups: group A (n=280), ABO-compatible kidney transplantation (ABO-CKT); group B (n=63), ABO-IKT without rituximab induction; and group C (n=50), ABO-IKT with rituximab induction. Basic immunosuppression was the same in all three groups except for the use of rituximab, which was administered at 100 mg (n=6), 200 mg (n=26), and 500 to 1000 mg (n=18).
The graft survival rates in groups A, B, and C were 99.2%, 96.8%, and 100% at 1 year, 93.8%, 94.9%, and 100% at 3 years, and 88.4%, 90.3%, and 100% at 5 years after transplantation, respectively. Serum creatinine levels in the three groups were not different at 1, 3, and 5 years after transplantation. The numbers of episodes of acute antibody-mediated rejection in groups A, B, and C were 7 (2.5%), 10 (15.9%), and 2 (4.0%), respectively (P=0.651), and acute cellular rejection was observed in 40 (14.3%), 6 (9.5%), and 2 (4.0%) patients, respectively (P=0.0957). There was no increased risk of cytomegalovirus infection in group C.
In the long term, inclusion of rituximab in the preoperative regimen yielded an even better outcome than that of ABO-CKT and rituximab-untreated ABO-IKT, without any increase in the risk of infection.
The role of the innate immune system in the development of thrombotic microangiopathy (TM) after alpha1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain.
Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out.
Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft.
Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.