Non-human Primate Xenotransplantation Research Articles

Partial heart xenotransplantation: Aresearch protocol in non-human primates.

Partial heart transplantation is a new type of transplant that delivers growing heart valve replacements for babies. Partial heart transplantation differs from orthotopic heart transplantation because only the part of the heart containing the heart valve is transplanted. It also differs from homograft valve replacement because viability of the graft is preserved by tissue matching, minimizing donor ischemia times, and recipient immunosuppression. This preserves partial heart transplant viability and allows the grafts to fulfill biological functions such as growth and self-repair. These advantages over conventional heart valve prostheses are balanced by similar disadvantages as other organ transplants, most importantly limitations in donor graft availability. Prodigious progress in xenotransplantation promises to solve this problem by providing an unlimited source of donor grafts. In order to study partial heart xenotransplantation, a suitable large animal model is important. Here we describe our research protocol for partial heart xenotransplantation in nonhuman primates.

Preclinical rationale and current pathways to support the first human clinical trials in cardiac xenotransplantation

Recent initiation of the first FDA-approved cardiac xenotransplantation suggests xenotransplantation could soon become a therapeutic option for patients unable to undergo allotransplantation. Until xenotransplantation is widely applied in clinical practice, consideration of benefit versus risk and approaches to management of clinical xenografts will based at least in part on observations made in experimental xenotransplantation in non-human primates. Indeed, the decision to proceed with clinical trials reflects significant progress in last few years in experimental solid organ and cellular xenotransplantation. Our laboratory at the NIH and now at University of Maryland contributed to this progress, with heterotopic cardiac xenografts surviving more than two years and life-supporting cardiac xenografts survival up to 9 months. Here we describe our contributions to the understanding of the mechanism of cardiac xenograft rejection and development of methods to overcome past hurdles, and finally we share our opinion on the remaining barriers to clinical translation. We also discuss how the first in human xenotransplants might be performed, recipients managed, and graft function monitored.

Anti-pig IgE and IgA Antibodies in Naive Primates and Nonhuman Primates With Pig Xenografts

Natural preformed anti-pig IgM/IgG antibodies in primates play an important role in xenograft rejection. As it is not clear how IgE and IgA engage in the immune system in xenotransplantation, we investigated natural preformed and elicited anti-pig IgE/IgA in naive primates and after xenotransplantation in nonhuman primates. The binding of IgM/IgG/IgE/IgA antibodies to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/cytidine monophospho-N-acetylneuraminic acid hydroxylase gene-knockout/β-1,4 N-acetylgalactosaminyltransferase 2 gene-knockout (ie, triple-knockout pigs) pigs were measured by flow cytometry in naive human (n = 50) and baboon (n = 14) sera. Antibody binding to WT and GTKO pig RBCs (pRBCs) was also measured in the sera of baboons (nonsensitized n = 7, sensitized n = 2) and rhesus monkeys (nonsensitized n = 2, sensitized n = 11) following WT or GTKO pig organ/tissue xenotransplantation. Deposition of IgM/IgG/IgE/IgA in the grafts was detected by immunohistochemistry. The majority of humans had natural preformed IgM/IgG/IgE/IgA to WT and GTKO pRBCs. In contrast, IgM/IgG/IgE/IgA to triple-knockout pRBCs were present at lower levels and frequency (P < 0.01). Baboons also had IgM/IgG/IgE/IgA antibodies against WT pRBCs, but fewer to GTKO and triple-knockout (P < 0.01). After xenotransplantation into nonhuman primates, when IgM/IgG increased, IgE/IgA also increased, but to a lesser extent. In addition to IgM/IgG, IgE or IgA deposition was observed in rejected pig xenografts. Primates develop serum anti-pig IgE/IgA antibodies both naturally and during xenograft rejection. The pathophysiological role, if any, of anti-pig IgE/IgA antibodies remains unknown.

Future direction of immunosuppressive treatment in organ transplantation

Since the first success of kidney transplantation in 1954, significant advances have been achieved in the field of organ transplantation. It was possible with the introduction of immunosuppressive drugs belonging to the class of calcineurin inhibitors (CNIs) such as cyclosporine and tacrolimus, the advances in surgical techniques and perioperative management, the monitoring and management infections, and the highly sensitive and specific antibody detection techniques. Despite recent progress, we currently encounter the limitation of better long-term transplant outcomes mainly because of paradoxical CNI toxicity and failure to control antibody or antibody-mediated rejections. The future direction of immunosuppression can be continued by optimizing immunosuppressive regimens with currently available immunosuppressants for better control of antibodies while avoiding CNI toxicity and by using biological therapeutics such as costimulation blockade agents that provide effective control of antibodies along with a reduction in usage or avoidance of CNIs and may develop as new immunosuppressants in the near future. Moreover, a tolerance induction through transplantation of donor hematopoietic stem cells or an infusion of regulatory cells using various sources of immune cells can also be a promising strategy as it can fundamentally escape from the complications of immunosuppressants. Over and above, it is important to note that the results of clinically applicable immunosuppressants from research in the non-human primate xenotransplantation model at the forefront of the future development of immunosuppressants can be a good opportunity to selectively apply to allo-transplants. No immunosuppressants can be risk-free, and therefore, all new immunosuppressants should be evaluated under the considerations for the risk/benefit ratio in various clinical conditions.

Profiling natural serum antibodies of non-human primates with a carbohydrate antigen microarray.

Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection. This study analyzed serum from seven naïve cynomolgus monkeys (blood type O/B/AB=3/2/2) for the intensity of natural IgM and IgG signals using carbohydrate antigen microarray, which included historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications. The median (range) of IgM and IgG signals were 12.71 (7.23-16.38) and 9.05 (7.23-15.90), respectively. The highest IgM and IgG signals with narrowest distribution were from mono- and disaccharides, followed by modified structures. Natural anti-α-Gal antibody signals were medium to high in IgM (11.2-15.9) and medium in IgG (8.5-11.6) spectra, and was highest with Lac core structure (Galα1-3Galβ1-4Glc, iGb3) and lowest with LacNAc core structure (Galα1-3Galβ1-4GlcNAc). Similar signal intensities (up to 15.8 in IgM and up to 11.8 in IgG) were observed for historically detected natural non-α-Gal antigens, which included Tn antigen, T antigen, GM2 glycolipid, and Sda antigen. The hierarchical clustering analysis revealed the presence of clusters of anti-A antibodies and was capable of distinguishing between the blood group B and AB non-human primates. The results presented here provide the most comprehensive evaluation of natural antibodies present in cynomolgus monkeys.

Vascularized Thymosternal Composite Tissue Allo- and Xenotransplantation in Nonhuman Primates: Initial Experience

Background:Vascularized composite allotransplantation is constrained by complications associated with standard immunosuppressive strategies. Vascularized thymus and bone marrow have been shown to promote prolonged graft survival in composite organ and soft-tissue vascularized composite allotransplantation models. We report development of a nonhuman primate vascularized thymosternal composite tissue transplant model as a platform to address donor-specific immune tolerance induction strategies.Methods:Vascularized thymosternal allograft (skin, muscle, thymus, sternal bone) was transplanted between MHC-mismatched rhesus monkeys (feasibility studies) and baboons (long-term survival studies), with end-to-side anastomoses of the donor aorta and SVC to the recipient common femoral vessels. A male allograft was transplanted to a female’s lower abdominal wall, and clinically applicable immunosuppression was given. Skin biopsies and immunological assays were completed at regular intervals, and chimerism was quantified using polymerase chain reaction specific for baboon Y chromosome.Results:Four allo- and 2 xenotransplants were performed, demonstrating consistent technical feasibility. In 1 baboon thymosternal allograft recipient treated with anti-CD40–based immunosuppression, loss of peripheral blood microchimerism after day 5 was observed and anticipated graft rejection at 13 days. In the second allograft, when cutaneous erythema and ecchymosis with allograft swelling was treated with anti-thymocyte globulin starting on day 6, microchimerism persisted until immunosuppression was reduced after the first month, and the allograft survived to 87 days, 1 month after cessation of immunosuppression treatment.Conclusions:We established both allo- and xeno- composite vascularized thymosternal transplant preclinical models, which will be useful to investigate the role of primarily vascularized donor bone marrow and thymus.

Selection of Patients for Initial Clinical Trials of Solid Organ Xenotransplantation.

Several groups have reported extended survival of genetically engineered pig organs in nonhuman primates, varying from almost 10 months for life-supporting kidney grafts and more than 2 years for non-life-supporting heart grafts to less than 1 month for life-supporting liver and lung grafts. We have attempted to define groups of patients who may not have an option to wait for an allograft. These include kidney, heart, and lung candidates who are highly-allosensitized. In addition, some kidney candidates (who have previously lost at least 2 allografts from rapid recurrence of native kidney disease) have a high risk of further recurrence and will not be offered a repeat allotransplant. Patients with complex congenital heart disease, who may have undergone previous palliative surgical procedures, may be unsuitable for ventricular assist device implantation. Patients dying of fulminant hepatic failure, for whom no alternative therapy is available, may be candidates for a pig liver, even if only as a bridge until an allograft becomes available. When the results of pig organ xenotransplantation in nonhuman primates suggest a realistic potential for success of a pilot clinical trial, highly selected patients should be offered participation.

Systemic inflammation in xenograft recipients precedes activation of coagulation.

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. 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. In 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 lineage(neg) 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. 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.

Molecular basis of cross-species reactivities of human versus porcine CTLA-4

The binding motif of human CTLA-4 is well known to be MYPPPY and for porcine CTLA-4 the binding motif is LYPPPY. Is this single amino acid difference of methionine (M) versus leucine (L) critical for the CTLA-4 binding? Recently, we have reported that the recombinant soluble porcine CTLA-4 was incapable of binding to human CD80. In this study we mutated L to M in the binding motif of the soluble porcine CTLA-4 and mutated M to L in the binding motif of the soluble human CTLA-4. We then analyzed how these mutations affected the binding affinity of the mutants to both porcine and human CD80(+) cells. The soluble porcine CTLA-4-L97M mutant partially lost its binding affinity to porcine CD80 compared to the wild-type and conferred very weak binding ability to human CD80. These results indicate that the L in the binding motif of porcine CTLA-4 is important for determining its binding ability to porcine CD80. Wild-type soluble human CTLA-4 binds to both human and porcine CD80 with comparable affinity, however, the soluble human CTLA-4-M97L mutant almost lost its binding ability to human CD80 and increased its binding ability to porcine CD80. These results indicate that M in the human CTLA-4 binding motif is extremely critical for its binding to human CD80. Those data suggest that the human CTLA-4 based recombinant protein drugs such as human CTLA-4-Ig can be used and/or tested in a porcine model. Conversely, the use of porcine CTLA-4 based recombinant protein drugs such as porcine CTLA-4-Ig is restricted to swine models. The difference in binding specificity of CTLA-4 observed in this study may be useful for studies such as pig to nonhuman primate xeno-transplantation. Porcine CTLA-4- and human CTLA-4-M97L mutant-based recombinant protein drugs can be used to specifically block the direct presentation by donor antigen presenting cells in pig to nonhuman primate xeno-transplantation. Human CTLA-4-M97L mutant-based recombinant protein drugs will be more ideal as it is without immunogenicity to human being.

298 Novel Cardiopulmonary Bypass with Reduced Priming Volume for Pig-to-Baboon Heart Transplantation Studies

In the pig-to-baboon cardiac xenotransplantation model the possibilities for transfusion of blood products are limited. Besides low intraoperative blood loss and short cardiopulmonary bypass time, a low priming volume is essential for a good postoperative condition of the recipients. We designed a novel setup of the cardiopulmonary bypass (CPB) with a low priming volume to meet the special needs of non-human primate xenotransplantation.

Islet xenotransplantation: current status of preclinical studies in the pig-to-nonhuman primate model

Porcine islets are a major focus of current research in nonhuman primate xenotransplantation models. Major advances have been obtained recently and these are briefly described. Reports by three independent centres have described 6-month porcine islet xenograft survival in nonhuman primates. Two of these have obtained such results by interfering with the CD40/CD154 co-stimulatory pathway. While these results are groundbreaking, the immunosuppressive regimens used are not viewed as clinically applicable and will need to be modified before islet xenotransplantation can be considered for clinical trials. In contrast, preliminary results by Gianello and colleagues have demonstrated 6-month survival of diabetic nonhuman primates transplanted subcutaneously with encapsulated porcine islets, in the complete absence of immunosuppression. The confirmation and full assessment of these results are eagerly awaited. Importantly, to date, no evidence of xenozoonoses has been observed following porcine islet xenotransplantation to nonhuman primates. Of the possible organ candidates for xenotransplantation, porcine islets are the closest to a possible clinical application. Prior to the initiation of ethical and safe clinical trials, however, further efforts to generate additional efficacy and safety data in the nonhuman primate model will be indispensable.

Prolonged Survival of Porcine Hepatocytes in Cynomolgus Monkeys

Management of patients with liver failure can be a significant medical challenge, and transplantation of the liver is the only definitive therapy. Whole liver allotransplantation is limited by a shortage of human donors and the risks of the surgery in those most ill. Transplants consisting of xenogeneic hepatocytes might overcome these problems, and work in rodents indicates that such transplants can correct some metabolic deficiencies and can prevent the complications and mortality associated with hepatic failure. As a prelude to clinical application, we tested the feasibility of hepatocyte xenotransplantation in nonhuman primates. One to 2 billion hepatocytes from outbred swine were transplanted into the spleens of cynomolgus monkeys using conventional immunosuppression to control rejection. Duration of graft function was determined based on assay for porcine albumin. Following a single infusion, xenogeneic hepatocytes functioned for more than 80 days and, following re-transplantation, for more than 253 days. Engraftment in the spleen was confirmed 40 days after transplantation by asialoglycoprotein receptor-directed nuclear scanning. The humoral immune response to the transplanted porcine cells had no discernible impact on the survival of the grafts. Xenotransplantation of hepatocytes should be explored as a readily available, minimally invasive form of therapy for hepatic failure.