Xenogeneic thymus transplantation in a pig-to-baboon model

BACKGROUND: We have tested whether fetal porcine thymic tissue transplantation can lead to tolerance across a discordant (pig-to-baboon) xenogeneic barrier. METHODS: Six baboons underwent a conditioning regimen with thymectomy, splenectomy, and anti-monkey CD3 antibody conjugated to a diphtheria toxin binding site mutant (FN18-CRM9). Porcine fetal or neonatal thymic tissue was transplanted into three baboons. Three control baboons received either no transplanted pig tissue (n=1) or adult pig lymph node (n=2). Cellular responses and skin xenografts were used to test for tolerance. RESULTS: After T-cell depletion and thymic transplantation, recovery of thymus-dependent na?ve-type CD4 cells (CD4/CD45RA ) and in vitro xenogeneic hyporesponsiveness were observed. No sensitization of alpha-galactosyl antibody responses was observed. The thymic grafts survived up to 48 days. Porcine skin xenografts were performed in two of these animals with survival of 22 and 24 days. Only two of these animals were completely T-cell depleted, and both failed to recover thymus-dependent T cells (CD4/CD45RA ). In one animal, general in vitro hyporesponsiveness was observed, with subsequent death from infection. The second animal demonstrated delayed recovery of T cells and prolonged general hyporesponsiveness in vitro. Neither animal demonstrated prolongation of porcine skin grafts compared with allografts (both rejected by day 13). CONCLUSIONS: Porcine thymic tissue is able to induce xenogeneic hyporesponsiveness. More efficient thymic engraftment may allow this approach to induce xenograft tolerance.     

Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness

BACKGROUND: The potential of xenotransplantation for clinical application will require overcoming barriers of humoral and cellular rejection, through strategies using immune suppression or tolerance induction. This laboratory has previously reported the induction of tolerance in the discordant xenogeneic model of pig-to-rodent thymic transplantation. We also have described a miniature swine model of fully mismatched allogeneic composite vascularized thymokidney transplantation that induced transplantation tolerance. We tested a combination of these approaches in a clinically relevant pig-to-primate model of xenotransplantation. METHODS: Composite thymokidney grafts were prepared 40 to 80 days before transplantation by the autologous implantation of thymic tissue under the renal capsule of human decay-accelerating factor transgenic swine. Baboons received xenotransplants of both human decay-accelerating factor composite thymokidneys and omental implants of thymic tissue. Recipients were treated with an immunosuppressive-conditioning regimen including thymectomy or thymic irradiation, extracorporeal immunoadsorption of anti-alphaGal antibodies and T-cell depletion. Recipients were followed for indicators of xenograft rejection, T-cell depletion and reconstitution, anti-alphaGal antibody levels, and mixed lymphocyte responses. Immunologic responses were studied in those animals that survived for more than 3 weeks. RESULTS: Thymokidney xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall's corpuscles under the renal capsule and in the omental implants, and with evidence of few host lymphocytes. Three animals demonstrated donor-specific unresponsiveness, while maintaining normal alloresponses, in mixed-lymphocyte-response assays performed after immunosuppression had been stopped. Rejected grafts demonstrated humoral damage without evidence of cellular infiltrates. After graftectomy, one animal maintained donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels for more than 2 months. CONCLUSIONS: We concluded that composite thymokidney and thymic-tissue xenotransplantation from swine to baboons can induce donor-specific cellular unresponsiveness and stable anti-alphaGal antibody levels, suggesting avoidance of sensitization after xenotransplantation. The presence of viable donor-swine thymic epithelium could have a role in the development of donor-specific T-cell tolerance. Further strategies to address humoral rejection could prolong graft survival and result in long-term tolerance to xenografts.     

Xenogeneic islet transplantation

Simeonivic C J. Xenogeneic islet transplantation. Xenotransplantation 1999; 6: 00‐00. ©Munksgaard, Copenhagen.     

Xenogeneic transplantation of porcine hepatocytes into the CCl4 cirrhotic rat model

Liver support using extracorporeal devices and hepatocyte transplantation has received renewed interest for the management of acute and chronic liver failure. The aim of this study was to determine whether xenogeneic porcine hepatocytes could integrate into the liver parenchyma of cirrhotic Lewis rats when administered by an intrasplenic route. Cirrhosis was induced by carbon tetrachloride (CCl4) inhalation and confirmed histologically. Freshly isolated porcine hepatocytes were infused directly into the splenic pulp at laparotomy over a 5-15-min interval. Using (111)In-labeled hepatocytes, the degree of localization of porcine hepatocytes to the spleen and liver was found to be greater than 60% in both control and cirrhotic rats. Integration of porcine hepatocytes into the rat liver parenchyma was determined by immunohistochemical staining for porcine albumin in rat liver sections. Further confirmation was provided by in situ hybridization using a porcine-specific probe that binds to a distinct repetitive element (PRE) in porcine DNA. Evidence of integrated porcine hepatocytes was seen for over 50 days in animals under cyclosporine immunosuppression. These data demonstrate the integration of xenogeneic porcine hepatocytes into the liver of the cirrhotic rat and their ability to produce porcine albumin for up to 50 days.     

Xenogeneic embryonic stem cell-derived cardiomyocyte transplantation

PURPOSE: The purpose of this study was to investigate the survival of xenogeneic embryonic stem cell (ES cell)-derived cardiomyocytes transplanted into the normal myocardium. MATERIAL AND METHODS: Undifferentiated mouse ES cells carrying the enhanced green fluorescent protein (EGFP) were cultured in hanging drops and then plated onto dishes. These cells were identified as cardiomyocytes by the expression of cardiac-specific genes, recording of action potential, and immunostaining with anti-sarcomeric myosin antibody. Donor cells were injected into the normal myocardium, with cyclosporine administered daily. One week after the transplantation, we investigated donor cell survival by examining EGFP expression, hematoxylin and eosin staining, and immunostaining with anti-sarcomeric myosin antibody. RESULTS: In vitro donor cells derived from ES cells expressed myosin light chain-2v and alpha-myosin heavy chain genes, had action potentials of a ventricular myocyte type, and were stained by anti-sarcomeric myosin antibody. In vivo 1 week after transplantation, EGFP-expressed cells were detected in the cell transplanted area. No lymphocytic infiltration was observed around these cells. CONCLUSIONS: ES cell-derived cardiomyocytes survived in the normal myocardium after the transplantation, even in a discordant xenogeneic transplantation model. These results indicate that cell transplantation using cardiomyocytes derived from ES cells, even if xenogeneic represents an attractive strategy for treating heart disease.     

Xenogeneic bone marrow transplantation: II. Porcine-specific growth factors enhance porcine bone marrow engraftment in an in vitro primate microenvironment

Abstract: Establishment of mixed bone marrow chimerism in pig-to-primate transplantation, as a means of inducing specific immune tolerance, will require that both immune and nonimmune barriers be overcome. As a preliminary step in evaluating nonimmune barriers in this system, we have developed an in vitro model of engraftment in which long-term culture of porcine bone marrow-derived hematopoietic cells is supported on preformed primate bone marrow stromal layers. In the absence of cytokine supplementation, primate stromal cells were unable to support long-term porcine hematopoiesis in these cultures. Supplementation with porcine Steel Factor was required for long-term maintenance of hematopoietic progenitor cell content and total hematopoietic activity. Addition of porcine IL-3, in combination with porcine Steel Factor, increased long-term progenitor cell content and hematopoietic activity on primate stroma to levels comparable to that obtained in cultures on porcine stroma. The combination of porcine GM-CSF and Steel Factor increased progenitor cell content and hematopoietic activity early in the cultures, but had little effect in long-term cultures. The Steel Factor and IL-3 combination was species-specific in its action in these cultures, as the corresponding human cytokines were unable to effectively support long-term porcine hematopoiesis. Likewise, the combination of porcine cytokines had only minimal effects on long-term bone marrow culture of primate CD34+ cells I on primate stroma.     

A heterotopic primate model for facial composite tissue transplantation

The purpose of this study was to develop a nonhuman primate model for heterotopic composite tissue facial transplantation in which to study the natural history of facial transplantation and evaluate immunosuppressive regimens.A composite oromandibular facial segment transplant based on the common carotid artery was evaluated. Flaps from 7 cynomolgus monkeys were transplanted to the groins of 7 recipients at the superficial femoral artery and vein. The immunosuppressive regimen consisted of thymoglobulin, rapamycin, and tacrolimus. Allograft survival ranged from 6 to 129 days. Histology performed in the long-term survivor at the time of necropsy revealed extensive inflammation and necrosis of the allograft skin; however, muscle and bone elements were viable, with minimal inflammation.This heterotopic facial transplantation model avoids the potential morbidity of mandibular resection and orthotopic facial transplantation. Our work also concurs with the work of other groups who found that the skin component is the most antigenic.     

Xenogeneic transplantation of porcine islets: an overview

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.     

Tolerance in a concordant nonhuman primate model

BACKGROUND: We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS: After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS: In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS: Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.     

Vascularized thymic lobe transplantation in a pig-to-baboon model: a novel strategy for xenogeneic tolerance induction and T-cell reconstitution

BACKGROUND: This laboratory has previously demonstrated the induction of allogeneic tolerance by vascularized thymic lobe (VTL) transplantation in miniature swine. We report here our initial attempt to induce tolerance by VTL transplantation in the clinically relevant, discordant, pig-to-baboon model of xenotransplantation. METHODS: Six baboons received xenografts of hDAF VTLs. Four of these baboons also received omental thymic tissue implants. All recipients were treated with an immunosuppressive conditioning regimen that included thymectomy, splenectomy, extracorporeal immunoadsorption of anti-alpha Gal antibodies, and T-cell depletion. Two control baboons received sham operations, of which one also received 5x10 hDAF porcine thymocytes/kg intravenously. RESULTS: Transplanted VTL grafts supported early thymopoiesis of recipient-type immature thymocytes, and facilitated engraftment of nonvascularized thymic omental implants. Recipients of the VTL grafts demonstrated donor-specific unresponsiveness in MLR assays, development of peripheral CD45RAhigh/CD4 double positive (DP) cells, and positive cytokeratin staining of thymic stroma in the grafts for 2 months following xenotransplantation. The control baboons did not show these markers of thymic reconstitution. The eventual return of Gal natural antibodies led to the destruction of graft epithelial cells and the rejection of all VTL grafts by 3 months posttransplantation. CONCLUSIONS: VTL transplantation from hDAF swine to baboons induced early thymopoiesis in the recipients and donor-specific cellular unresponsiveness in vitro. When coupled with additional strategies aimed at silencing humoral rejection, VTL transplantation may significantly prolong xenograft survival and result in long-term tolerance.     

A study of neurotrophism in a primate model

This study investigated the existence of neurotrophism in a primate model. In eight adult cynomolgus monkeys the sensory component of the femoral nerve was sectioned and introduced into the proximal channel of a silicone Y chamber. The proximal stump was given distal choices of various tissues inserted into the remaining arms of the silicone Y chamber. The targets presented were combinations of tendon, muscle, intact distal nerve, distal nerve graft, or an empty silicone channel. After 6 weeks, ultrastructural analysis confirmed axonal growth toward distal nerve tissue, while minimal or no nerve regeneration was directed toward tendon, muscle, or the empty silicone channel. The results showed that either a distal nerve stump or a nerve graft will act as a specific target to the regenerating primate proximal nerve stump.