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Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond.
Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific immunosuppressive agents.
Group A grafts (n = 8) survived for an average of 70 days, with the longest survival of 236 days. Some animals in this group (n = 3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark.
Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) achieved long-term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.
Patients with end-stage organ failure waiting for donor organs have limited treatment options. For those with cardiac failure, mechanical assist devices provide one solution, but various complications associated with these devices have reduced their effectiveness.
Until we learn to grow organs via tissue engineering, which is unlikely in the near future, xenotransplantation seems to be a valid approach to supplement human organ availability. Despite many setbacks over the years, 2 major recent developments have helped revitalized progress in the xenotransplantation field. First is the ability to produce genetically engineered (GE) pigs
in which certain genes that are immunogenic to humans are knocked out and human transgenes, such as complement regulatory proteins (human complement regulatory protein) and human thromboregulatory protein, are expressed on pig cells.
Because of the cost of these experiments and scarce research funding, it is not feasible to address each genetic and immunosuppressive manipulation individually. Therefore, laboratories performing experiments in the field of xenotransplantation have selectively picked specific genetic modifications in pigs and immunosuppressive drug combinations to perform xenotransplantation experiments. In this report, we have summarized our results from multiple experiments to show the impact of these GE pigs and target-specific immune suppression focusing on recipient B-cell depletion and costimulation blockade.
Animal Models and Genetic Modifications
Specific pathogen-free (SPF) baboons weighing 7 to 15 kg from the University of Oklahoma (Norman, Okla) were housed in a clean pathogen-free facility. These SPF baboons are known to have lower levels of both anti–non-Gal immunoglobulin (Ig)G and IgM.
Genetically modified pigs (aged 4-8 weeks) that were alpha galactosidase transferase knockout and hCD46 transgenic (GTKO.hCD46), with or without thrombomodulin (TBM) expression (Revivicor Inc, Blacksburg, Va), were used as heart donors. The weights of donor pigs were matched to the baboon recipient to ensure adequate accommodation of the heterotopic heart. All animals were used in compliance with guidelines provided by the National Heart, Lung, and Blood Institute Animal Care and Use Committee. All transplant procedures were performed at a National Heart, Lung, and Blood Institute core surgical facility.
Donor pig hearts were transplanted into recipient SPF baboons in a heterotopic position, as described previously.
Briefly, the recipient baboon's infrarenal aorta and inferior vena cava were exposed through a midline abdominal incision. Side-biting clamps were applied; an aortotomy and venotomy were made, and the end-to-side anastomosis was performed between the donor and recipient aorta and the donor pulmonary artery with the recipient inferior vena cava.
A detailed description of the immunosuppressive regimen is shown in Figure 1. In brief, it includes induction with antithymocyte globulin, anti-CD20 antibody (Rituxan; Genentech, Inc, South San Francisco, Calif), and costimulation blockade with anti-CD154 (5C8) or anti-CD40 (3A8 or 2C10R4) monoclonal antibody (mAb). Cobra venom factor was used to inhibit the complement activation. Mycophenolate mofetil and costimulation blockade antibody were also used daily and weekly as described in Figure 1 to prevent immune rejection. All recipient baboons received continuous heparin infusion to maintain the activated clotting time (ACT) level at twice the baseline. Ganciclovir was administered intravenously (IV) daily to prevent any potential viral infections. Erythropoietin (200 U/kg) was administered IV daily from day −7 to 7, and cefazolin (250 mg) IV was administered twice per day for 7 days.
The experimental groups are described in Table 1, and the duration of immunosuppression is illustrated in Figure 1. The main difference among the 4 groups was the type, strength, and duration of antibody used for costimulation blockade. In group A, anti-CD154 (5C8) (20 mg/kg) IV was used for the entire period of graft survival. In group B, anti-CD40 (3A8) (20 mg/kg) IV was used for a maximum of 60 days. In group C, anti-CD40 (2C10) (20 mg/kg) was tapered off in 60 days. In group D, anti-CD40 (2C10) (50 mg/kg) was continued for 1 year (n = 2) or reduced (25 mg/kg) after 100 days (n = 2).
Table 1Description of the 4 experimental groups: The difference in the 4 experimental groups is described, including the antibody for costimulation blockade and B-cell depletion in each group
If rejection was suspected by diminution of xenograft function, rescue therapy was initiated with intravenous methyl prednisolone (10-15 mg/kg) for 6 days. Heparin was also used to prevent thrombus formation, and activated clotted time (ACT) was maintained at twice the baseline.
Measurement of Graft Survival
Telemetry, manual palpation, and noninvasive ultrasonography were used to monitor the xenograft function. A telemetry device was implanted at the time of transplantation to monitor the baboon recipient's temperature, graft left ventricular pressure (LVP), and electrocardiogram (EKG). The telemetry device data were transmitted wirelessly to a receiver attached to the animal's cage (RMISS, Wilmington, Del). The parameters were recorded and included the peak systolic pressure, end-diastolic pressure, LVP, heart rate based on LVP, EKG, heart rate based on EKG, and recipient's body temperature. Heart function was continuously evaluated by telemetry, and a decrease in LVP to less than 60 mm Hg was correlated with the initiation of the rejection process, which affected the graft contractility. An LVP less than 10 mm Hg was an indicator of complete cessation of graft contractility.
Recipients were sedated weekly for the first 2 months post-transplant and biweekly thereafter for blood collection. Palpation and ultrasound were also done on this schedule. On the basis of xenograft palpation, contractility of the heart was scored as ++++ (fully functional) to 0 (nonfunctional). Blood flow and wall motion were analyzed by echocardiography.
White blood cell (WBC) count, hematocrit, red blood cell count, hemoglobin, platelets, neutrophils, and monocytes were analyzed. Blood chemistry was performed weekly for the first 2 months and then biweekly until the graft was explanted or rejected. ACT, prothrombin time, and troponin levels were measured at the same intervals.
Paraffin sections from biopsies and explanted xenografts were stained with hematoxylin–eosin for light microscopy. Sections were analyzed for cardiomyocyte viability and the presence of hemorrhage, microvasculature thrombosis, and cellular infiltrates.
Graft survival curves for all groups are shown in Figure 2, A. Median graft survivals of the 4 groups are shown in Figure 2, B, and the longest survival in each group is shown in Figure 2, C. Among all groups, group D, receiving the high dose of anti-CD40mAb, had the longest median and individual survival. In group A, most of the grafts were still contracting at the time of recipient death or euthanasia because of various complications. All grafts in groups B and C were rejected. Two of 5 grafts in group D stopped contracting on postoperative days 146 and 159, but the other 3 grafts are still contracting at more than 200 to 500 days at the time of this submission.
Measurement of Graft Contractility
Echocardiography was performed only in groups C and D (echocardiography machine not available for earlier experiments) receiving 2C10R4mAb and GTKO.hCD46 (group C) or GTKO.hCD46.hTBM (group D) pig heart xenografts. Excellent graft contractility was observed in all animals. The original wall thickness was maintained to the end, but in animals that stopped functioning in both groups, left ventricular and left atrial walls were considerably thickened. Of note, there was no thrombus observed in group D receiving grafts with human TBM expression and high-dose anti-CD40 mAb (2C10R4) treatment.
Telemetry was used to measure LVP and EKG. The heart rate was calculated by the software on the basis of the number of LVP peaks and QRS complexes per minute. Because of limited battery life, telemetry was useful for 200 to 400 days post-transplantation. Three grafts in group D were still functioning to the day of the submission of the article, and telemetry was useful in determining graft function for 230, 250, and 400 days in each. All of the grafts in group C rejected within 146 days, and telemetry documented a loss of LVP and heart contractility. Manual graft palpation scores were consistent with the findings of ultrasound and telemetry.
Depletion of T and B Cells and the Effect on Non-Gal Antibody Production
Initial treatment with ATG significantly reduced the number of circulating T cells, but they were not totally eliminated. However, treatment with 4 weekly doses of anti-CD20 effectively depleted all of the circulating B cells, and they stayed depleted for at least 60 days, after which they were slowly restored to pretreatment levels.
Although anti-CD20 has no direct affect on plasma cells, the non-Gal antibody production was maintained at a low level by this treatment.
Complete Blood Count, Chemistry, and Coagulation Profile
Figure 3 shows the comparison of WBC count, platelet count, hematocrit, and hemoglobin levels. The treatment regimen was effective in keeping WBC counts low in all 4 groups. The median platelet numbers were maintained better with 2C10R4 mAb treatment, but only group C platelet counts were significantly better than in the other 3 groups. The hemoglobin level and hematocrit percentage were kept close to normal in all groups, and there was no significant difference among the groups. Anemia and thrombocytopenia were treated with a transfusion of packed red blood cells as needed.
Graft histology in group A showed small areas of normal myocardium along with significant areas of hemorrhage, coagulative necrosis, and fibrosis. Some thrombotic microangiopathy (TM) was observed in this group. No TM was observed in groups B, C, or D. Rejected grafts in groups B, C, and D showed some myofibril loss with fibrosis, but there was only minimal hemorrhage present. Figure 4 shows a representative hematoxylin–eosin staining of a left ventricle from the group A graft, showing widespread areas of fibrosis, along with some normal myocytes, and a biopsy specimen from a group D heart taken at 182 days postoperatively, showing primarily normal myocytes and a small area of fibrosis.
Most animals in group A had to be euthanized or died of various complications (eg, abdominal bleeding, aspiration pneumonia, and consumptive coagulopathy). Infections were rare in all the groups and if noticed were managed by antibiotic treatment after culture and sensitivity testing was performed. Few complications were seen in groups B, C, and D. In these groups, most of the heart xenografts were explanted after rejection, and recipient baboons survived.
The availability of GE pigs has transformed the field of xenotransplantation and rejuvenated the hopes for clinical xenotransplantation. However, there are parts of this puzzle that still need to be solved.
Our laboratory, in collaboration with other groups, has made several inroads in this field. Conventional immunosuppression has been tried in this model, and these agents reasonably prolonged the graft survival.
We have tried to improve on the conventional immunosuppression with the use of target-specific immunosuppressive agents, which, along with the use of newly available GE pigs, have significantly improved the graft survival of pig cardiac xenografts in baboons.
The first step toward improvement in graft survival was observed with B-cell depletion via anti-CD20 mAb. The possible mechanisms of depletion include direct lysis via natural killer cells/macrophages, antibody-mediated cell cytotoxicity, and complement-dependent cytotoxicity.
Only 4 doses of anti-CD20 mAb on days −7, 0, 7, and 14 were able deplete the B-cell population for 2 months. The research group at the Mayo Clinic (Rochester, Minn) has shown that 1 dose can deplete circulating B cells effectively, but 4 doses are required to deplete B cells from the lymphoid organs (Gerry Byrne, personal communication, October 2011). Although this anti–B-cell treatment was not effective for plasma cell depletion, because plasma cells do not express CD20, the levels of both anti–non-Gal IgM and IgG remained at very low levels because of this treatment, and signs of antibody-mediated rejection were not seen in any animals receiving anti-CD20 mAb treatment. Of note, we have reported that the cardiac xenografts do show some minor deposition of anti–non-Gal antibodies.
There was a moderate increase in non-Gal antibody levels after 60 days of survival, but no obvious signs of rejection were observed. Perhaps the xenograft is “accommodated” by this time or a state of “immune tolerance” is induced. Currently, studies are being performed to evaluate these mechanisms. There may be a benefit of continuous suppression of B cells by anti-CD20 antibody beyond 60 days. The anti-CD40 antibody used in these experiments also suppresses the B-cell response and may be sufficient to keep the B-cell response under control after 60 days.
The costimulation blockade pathway was critical to graft survival, because suppression of this pathway modulates the response of both B and T cells. In the initial experiments, we targeted the CD40-CD40Ligand pathway by using anti-CD154 mAb, which yielded the previous longest cardiac xenograft survival of 236 days. The use of this antibody in some cases, because of expression of CD154 on platelets and binding of anti-CD154 to the platelets resulting in their activation and aggregation, was accompanied by fatal coagulopathies leading to euthanasia or death of recipient baboons before graft rejection.
However, the experiments with this antibody demonstrated the beneficial effect of costimulation blockade in suppressing the xenogeneic immune response and extending xenograft survival. The use of anti-CD40 mAb allowed blockade of the same CD40-CD40L costimulation pathway without encountering the complications of thrombosis. Two available anti-CD40 mAbs were used for this purpose, one generated in mouse (clone 3A8) and one in recombinant mouse-rhesus chimeric antibody (clone 2C10R4). Neither clones had the thrombogenic characteristics observed with the anti-CD154 mAb (5C8). The antibody from the 3A8 clone was not able to extend graft survival beyond 28 days.
However, the graft survival was significantly prolonged with the 2C10R4 clone, irrespective of the pig genetics used. A higher dose of this antibody was required in these experiments to produce consistent long-term graft survival, perhaps because of lower affinity of this antibody for baboon cells. We also observed a quicker recovery time from surgery in groups using anti-CD40 mAb. No complications were observed in groups C or D. A humanized form of anti-CD40 antibody is under production and will be available for clinical use soon (Keith Reimann, personal communication, April 2014).
Additional donor pig genetic modifications used, in addition to alpha Gal knockout, were transgenic expression of human CD46 and TBM. Our results indicate that TBM expression and anti-CD40 treatment were crucial in long-term graft survival. TBM expression successfully avoided TM and other coagulation issues. It is difficult to dissect the preferential role of either of the 2 modifications used, and further experiments are required for this clarification.
Infections were rarely seen in our animals despite the long-term infusion of maintenance drugs via a jugular catheter. More complications were related to technical problems with the tether system than the recipient baboon themselves. No perioperative graft dysfunction was observed. Some pig viruses, such as cytomegalovirus (CMV), have been shown to affect graft survival in pig allograft models, but we have not seen any active CMV disease in any of our baboons except 1 in which CMV inclusion bodies were found in the testes on necropsy. The source of these inclusion bodies was not determined, and no evidence of active CMV disease was found in this baboon. Other complications experienced over the length of the project included abdominal bleeding, gastrointestinal bleeding, aspiration pneumonia, seizures, thrombocytopenia, and anemia.
For example, infections were detected early by monitoring recipient temperature via telemetry. Video monitoring allowed continuous surveillance of the baboons and triggered prompt response to emergencies such as infusion pump malfunction, tether/jacket problems, and seizures. These methods retrospectively enabled us to evaluate the onset of any complication.
Significant improvement in graft survival from 179 days
and to more than 1 year as we reported, by using newly available GE pigs and an immunosuppression regimen based on an nonhuman primate-mouse chimeric anti-CD40 mAb (2C10R4), along with the histology showing preservation of heart architecture in long-term grafts, is encouraging and indicates that these xenograft hearts can be tested in the orthotopic position for their ability to sustain life.
The authors thank the following people and programs for their support: Billeta Lewis, BS, for animal care; Tannia Clak, DVM, for help in performing echocardiography; Michael Eckhaus, DVM, for pathology; Caleb Seavey, for developing analyzing software; Suyapa Ball and Carol Phelps, PhD (Revivicor, Inc), for the production of GE pigs; Eckhard Wolf and Nikolai Klymiuk (Ludwig-Maximillians University, Munich), for the collaboration on GE pig cells; Keith Reimann, PhD, for providing the anti-CD40 mAb; the Animal Surgery Resource and National Heart, Lung, and Blood Institute, for help with the surgical procedure; the Flow Cytometry Core, National Heart, Lung, and Blood Institute, Division of Veterinary Resource, and National Institutes of Health, for animal care; and Patricia Jackson, for administrative help.
Postoperative care and complications after ventricular assist device implantation.
Best Pract Res Clin Anaesthesiol.2012; 26: 231-246