The ability to transplant pig organs into humans would resolve the current crisis in the supply of cadaveric human organs for the treatment of end stage disease. Several immunologic barriers need to be overcome if pig-to-primate transplantation is to be successful. The presence of preformed antibodies in humans, apes and Old World monkeys directed against galactose epitopes on pig vascular endothelium provides the major barrier, as binding of antibody to antigen leads to graft destruction by complement activation and other mechanisms. Hyperacute rejection can result from the action of complement. If this is prevented, delayed antibody-mediated rejection develops, which can be associated with a state of consumptive coagulopathy (disseminated intravascular coagulation, DIC). Efforts being made to overcome antibody-mediated rejection include depletion of antibody by extracorporeal immunoadsorption, prevention of an induced antibody response by co-stimulatory blockade, B-cell and/or plasma cell depletion, depletion or inhibition of complement, or the use of organs from pigs transgenic for a human complement regulatory protein, such as hDAF. The ultimate solution would be the induction of both B- and T-cell tolerance to the transplanted pig organ, which is being explored by attempting to induce haematopoietic cell chimerism. One complication of this is a thrombotic microangiopathy, similar to thrombotic thrombocytopenic purpura. The many and diverse roles in which pharmacotherapy is involved in attempts to overcome the barriers of xenotransplantation are reviewed and current progress, particularly in our own laboratory, is discussed.
512 In allograft models, the induction of mixed hematopoietc cell chimerism by bone marrow (BM) or peripheral blood stem cells (PBSC) transplantation (Tx) leads to donor-specific tolerance of subsequently transplanted organs. We are investigating the induction of mixed chimerism in the discordant pig-to-baboon model. METHODS. Leukopheresis of cytokine-stimulated (pIL3, pSCF, hGCSF) mobilised PBSC in pigs results in the collection of 30-90×10⁁10 cells. Pig leukocytes (15-35×10⁁10) were transplanted into 3 groups of splenectomized baboons. Group 1 (n=2) received no preparative therapy. Group 2 (n=2) received whole body (300cGy) and thymic (700cGy) irradiation, ATG, cyclosporine, mycophenolate mofetil, cobra venom factor, pig-specific cytokines, and extracorporeal immunoadsorption to remove anti-Gal antibodies before PBSC Tx. Group 3 (n=6) received the above regimen combined with prostacyclin, low-dose heparin and methylprednisolone (PHM) +/− antiCD40L mAb (20mg/kg ×2 or ×8 doses). RESULTS. Baboons in Groups 1 and 2 developed severe thrombocytopenia (<10,000cu.mm) requiring multiple platelet transfusions, marked schistocytosis (>12hpf), increase in plasma LDH (<25,000U/L), loss of high molecular von Willebrand factor (vWF), and mild anemia, transient neurologic changes, renal insufficiency and clinical purpura. Two baboons died of these complications; autopsy confirmed extensive platelet thrombi in the microcirculation. (The Group 2 conditioning regimen alone - without PBSC Tx - does not induce these changes.) Group 3 baboons developed moderate thrombocytopenia (not requiring transfusion), mild schistocytosis (<3hpf), mild increase in LDH (<1000U/L), with no other sequelae, despite loss of vWF. CONCLUSIONS. These data are in keeping with a thrombotic thrombocytopenic purpura-like state induced in baboons by porcine PBSC Tx, which can be fatal. Prophylactic therapy with PHM, particularly when combined with anti-CD40L mAb, reduces the sequelae of endothelial activation, markedly reduces microangiopathic hemolysis, and facilitates the induction of mixed chimerism.
Introduction. Attempts to achieve immunological tolerance to porcine tissues in nonhuman primates through establishment of mixed hematopoietic chimerism are hindered by the rapid clearance of mobilized porcine leukocytes, containing progenitor cells (pPBPCs), from the circulation. Eighteen hours after infusing 1–2×1010 pPBPC/kg into baboons that had been depleted of circulating anti-αGal and complement, these cells are almost undetectable by flow cytometry. The aim of the present study was to identify mechanisms that contribute to rapid clearance of pPBPCs in the baboon. This was achieved by depleting, or blocking the Fc-receptors of, cells of the phagocytic reticuloendothelial system (RES) using medronate liposomes (MLs) or intravenous immunoglobulin (IVIg), respectively. Methods. Baboons (preliminary studies, n=4) were used in a dose-finding and toxicity study to assess the effect of MLs on macrophage depletion in vivo. In another study, baboons (n=9) received a nonmyeloablative conditioning regimen (NMCR) aimed at inducing immunological tolerance, including splenectomy, whole body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700 cGy), T-cell depletion, complement depletion with cobra venom factor, mycophenolate mofetil, anti-CD154 monoclonal antibody, and multiple extracorporeal immunoadsorptions of anti-αGal antibodies. The baboons were divided into three groups: Group 1 (n=5) NMCR+pPBPC transplantation; Group 2 (n=2) NMCR+ML+pPBPC transplantation; and Group 3 (n=2) NMCR+IVIg+pPBPC transplantation. Detection of pig cells in the blood was assessed by fluorescence-activated cell sorter and polymerase chain reaction (PCR). Results. Preliminary studies: ML effectively depleted macrophages from the circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were detected 2 hr postinfusion of 1×1010 pPBPC/kg. After 18 hr, there were generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of pig cell chimerism (55–78%) were detected 2 hr postinfusion, even when a smaller number (0.5–1×1010/kg) of pPBPCs had been infused, and these levels were better sustained 18 hr later (10–52%). Group 3: In one baboon, 4.4% pig cells were detected 2 hr after infusion of 1×1010 pPBPC/kg. After 18 hr, however, 7.4% pig cells were detected. A second baboon died 2 hr after infusion of 4×1010 pPBPC/kg, with a total white blood cell count of 90,000, of which 70% were pig cells. No differences in microchimerism could be detected between the groups as determined by PCR. Conclusions. This is the first study to report an efficient decrease of phagocytic function by depletion of macrophages with MLs in a large-animal model. Depletion of macrophages with MLs led to initial higher chimerism and prolonged the survival of circulating pig cells in baboons. Blockade of macrophage function with IVIg had a more modest effect. Cells of the RES, therefore, play a major role in clearing pPBPCs from the circulation in baboons. Depletion or blockade of the RES may contribute to achieving mixed hematopoietic chimerism and induction of tolerance to a discordant xenograft.
The pig is being investigated as an organ donor for humans. Induction of immunologic tolerance to pig tissues in primates would overcome the major immunologic barriers to xenotransplantation. A proven method of inducing tolerance to allografts is by the induction of mixed hematopoietic chimerism by bone marrow transplantation. We are therefore investigating induction of mixed hematopoietic chimerism in the pig-to-baboon model.To obtain large numbers of pig hematopoietic cells, leukapheresis was used to collect blood cell products in miniature swine (n = 5) after progenitor cell mobilization by use of a course of hematopoietic growth factors (cytokines), consisting of porcine interleukin 3, porcine stem cell factor, and human granulocyte colony-stimulating factor.Cytokine therapy and leukapheresis were well tolerated. Cytokine therapy increased the total white blood cell count and allowed large numbers of leukocytes (60 x 10(10)) to be obtained by apheresis, of which approximately 0.1% were granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units (CFU-GEMMs), which are considered to be representative of hematopoietic progenitors with multi-lineage potential.The combination of cytokine therapy and leukapheresis enables hematopoietic progenitor cells to be obtained safely from miniature swine.
Alwayn, I. P.J.; Basker, M.; Buhler, L.; Rieben, R.; Harper, D.; Appel, J. Z. III; Awwad, M.; Down, J.; White-Scharf, M.; Sachs, D. H.; Thall, A.; Cooper, D. K.C. Author Information
Background. In an attempt to induce mixed hematopoietic chimerism and transplantation tolerance in the pig-to-primate model, we have infused high-dose porcine peripheral blood progenitor cells (PBPC) into baboons pretreated with a nonmyeloablative regimen and anti-CD154 monoclonal antibody (mAb). Methods. Group 1 baboons (n=2) received a nonmyeloablative regimen including whole body irradiation, pharmacological immunosuppression, porcine hematopoietic growth factors, and immunoadsorption of anti-Gal[alpha]1,3Gal (Gal) antibody before infusion of high doses of PBPC (2.7-4.6×1010cells/kg). In group 2 (n=5), cyclosporine was replaced by anti-CD154 mAb. Group 3 (n=3) received the group 1 regimen plus anti-CD154 mAb. Results. In group 1, pig chimerism was detected in the blood by flow cytometry (FACS) for 5 days (with a maximum of 14%), and continuously up to 13 days by polymerase chain reaction (PCR). In group 2, pig chimerism was detectable for 5 days by FACS (maximum 33%) and continuously up to 28 days by PCR. In group 3, initial pig chimerism was detectable for 5 days by FACS (maximum 73%). Two of three baboons showed reappearance of pig cells on days 11 and 16, respectively. In one, in which no anti-Gal IgG could be detected for 30 days, pig cells were documented in the blood by FACS on days 16-22 (maximum 6% on day 19) and pig colony-forming cells were present in the blood on days 19-33, which we interpreted as evidence of engraftment. Microchimerism was continuous by PCR up to 33 days. Conclusions. These results suggest that there is no absolute barrier to pig hematopoietic cell engraftment in primates, and that this may be facilitated if the return of anti-Gal IgG can be prevented.
To achieve successful xenotransplantation, it is necessary to overcome the immunological barriers that evolution has built up between different species. In contrast to allotransplantation, where the cellular response is the main hurdle, in xenotransplantation both humoral and cellular responses have to be overcome. In attempts to achieve successful pig-to-human xenotransplantation, several approaches are currently being evaluated. Genetic engineering techniques are being applied to the problems of xenotransplantation. To achieve successful xenotransplantation, it will probably be necessary to combine several therapeutic techniques and/or agents, as is the case with allotransplantation today. Xenotransplantation offers the first opportunity for modifying the donor as opposed to the recipient, which opens up new possibilities in this era of rapidly developing techniques such as genetic engineering, gene transfer, and cloning. The breeding of a pig with a vascular endothelial structure against which humans have no preformed antibodies would be a major advance. In the recipient, however, it will still be necessary to inhibit the production of induced antibodies, as well as the strong cellular response, either by some form of immunosuppressive therapy or by the induction of tolerance.
