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Ann Thorac Surg 2000;70:131-138
© 2000 The Society of Thoracic Surgeons

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Original articles: Cardiovascular

Mixed hematopoietic chimerism induces long-term tolerance to cardiac allografts in miniature swine

^a Division of Cardiac Surgery and Transplantation Biology Research Center, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

Address reprint requests to Dr Madsen, Division of Cardiac Surgery, Massachusetts General Hospital, EDR 105, 55 Fruit St, Boston, MA 02114
e-mail: madsen{at}helix.mgh.harvard.edu

Presented at the Thirty-sixth Annual Meeting of the Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. Tolerance to cardiac allografts has not been achieved in large animals using methods that are readily applicable to human recipients. We investigated the effects of mixed hematopoietic chimerism on cardiac allograft survival and chronic rejection in miniature swine

Methods. Recipients were T-cell depleted using a porcine CD3 immunotoxin, and each received either of two nonmyeloablative preparative regimens previously demonstrated to permit the establishment of stable mixed hematopoietic chimerism across MHC-matched, minor-antigen–mismatched histocompatibility barriers. Five to 12 months after the chimerism was induced, hearts from the original cell donors were heterotopically transplanted into the stable mixed chimeras.

Results. Cardiac allografts transplanted into untreated recipients across similar minor antigen barriers were rejected within 44 days (within 21, 28, 35, 39, 44 days among individual study subjects). In contrast, hearts transplanted into the mixed chimeras were all accepted long term ( > 153, > 225, > 286, > 362 days) without immunosuppressive drugs and developed minimal vasculopathy.

Conclusions. Mixed hematopoietic chimerism, established in miniature swine using clinically relevant, nonmyeloablative conditioning regimens, permits long-term cardiac allograft survival without chronic immunosuppressive therapy, significant vasculopathy, or graft-versus-host disease.

	Introduction

Top Abstract Introduction Material and methods Results Comment References

 
Dramatic success has been achieved in cardiac transplantation over the past 15 years through the use of powerful immunosuppressive agents, including but not limited to prednisone, cyclosporine (CyA), and azathioprine. However, these nonspecific immunosuppressive agents are associated with serious complications, such as malignancy, end organ toxicity, and infectious diseases. Furthermore, neither these agents nor even newer T-cell–directed therapies have been able to prevent cardiac allograft vasculopathy (CAV), a manifestation of chronic rejection and the leading cause of death and graft loss after the first posttransplant year. A superior alternative to nonspecific immunosuppressive therapy would be the induction of donor-specific transplantation tolerance, which could result in permanent graft survival without the need for long-term immunosuppressive therapy. Indeed, we have recently shown that the induction of rapid and stable tolerance can prevent the development of CAV [1].

A state of mixed chimerism is achieved when bone marrow transplantation results in the stable coexistence of multilineage hematopoietic cells from the donor and host (reviewed recently by Wekerle and Sykes [2]). This state confers permanent tolerance for solid organs of donor (bone marrow) type while maintaining normal immune responses to third-party grafts and pathogens [3]. The major obstacle preventing the clinical application of mixed chimerism to tolerance induction has been the severe toxicity associated with host myeloablative conditioning regimens, which have generally utilized lethal whole-body irradiation (WBI). Lethal WBI has been required to deplete host T cells and create "space" for the engraftment of allogeneic stem cells following donor bone marrow transplantation in most large-animal models. However, this limitation has been overcome in miniature swine by the development of nonmyeloablative conditioning regimens made possible by two recent advances. The first was to substitute a novel swine CD3 immunotoxin, pCD3–CRM9, for lethal WBI to achieve T-cell depletion in the host. The immunotoxin pCD3–CRM9 is extremely effective in depleting mature T cells from the peripheral blood, lymph nodes, and thymus of miniature swine [4]. The second was the use of high doses of peripheral blood stem cells (PBSCs) instead of bone marrow to reconstitute the T-cell–depleted hosts. Our laboratory has shown that cytokine mobilization and apheresis of miniature swine blood allows the collection of PBSC capable of full hematopoietic reconstitution in lieu of bone marrow [5]. Using nonmyeloablative conditioning regimens that include pCD3–CRM9 and high-dose PBSC, either with nonlethal WBI [6] or without WBI [7], our laboratory has recently demonstrated the safe and reliable induction of stable multilineage mixed chimerism and skin-graft tolerance without the toxicity of lethal whole-body irradiation.

In order to apply the mixed chimerism approach to patients, large-animal models are required, both to understand the mechanism and to optimize the treatment protocol. Partially inbred miniature swine have been developed in this laboratory as a large-animal preclinical model for studies of transplantation immunobiology; the swine are very similar to humans in this regard [8]. The ability of mixed chimerism protocols to confer tolerance to cardiac allografts has not previously been demonstrated in large animals. In this report, we use animals available from other studies dealing with the induction of mixed chimerism in miniature swine [6, 7] to test whether or not mixed hematopoietic chimerism, established with clinically relevant, nonmyeloablative conditioning regimens, permits long-term cardiac allograft survival in the absence of immunosuppressive therapy and abrogates CAV.

	Material and methods

Top Abstract Introduction Material and methods Results Comment References

 
Animals
Transplant donors and recipients aged 2 to 3 months that were matched for MHC (SLA) but mismatched for minor antigens were selected from our herd of Massachusetts General Hospital MHC inbred miniature swine. The immunogenetic characteristics of this herd and intra-MHC recombinants have been described previously [9] (Fig 1). All animal care and procedures were performed in compliance with both the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the institute of Laboratory Animal Resources and published by the National Institutes of Health, revised 1996.

View larger version (38K): [in this window] [in a new window]   Fig 1. Origin of available SLA haplotypes in partially inbred miniature swine. (SLA = swine leukocyte antigen.)

Irradiation
Animals received intravenous 0.15 mg/kg of Telazol (AHP, Madison, NJ) for sedation and were then placed in the supine position on a plastic cradle and secured into position. Recipients #13131 and #13235 received sublethal whole body irradiation (150 cGy) on days -4 and -3 and thymic irradiation (700 cGy) on day -2 as previously described [6]. Recipients #12757 and 12963 received 700 cGy of thymic irradiation alone on day -2 and no whole-body irradiation.

Recipient T-cell depletion
All recipients in the mixed chimera group underwent T-cell depletion using the newly described diphtheria-toxin–based swine CD3 immunotoxin, pCD3–CRM9 [4]. This swine CD3 immunotoxin was made by conjugating the diphtheria toxin binding site mutant, CRM9, to the antiporcine CD3 mAb, 898H2–6–F15. On day -2, 0.05 mg/kg of pCD3–CRM9 was administered intravenously to recipient animals.

CyA treatment
Cyclosporine (Sandimmune oral solution, Novartis, Summit, NJ) was administered through a gastric tube at 30 mg · kg^-1 · d^-1 in divided doses from day -1 to day 30.

Antibodies and flow cytometry
Flow cytometry (Becton Dickinson FACScan, San Jose, CA) was used to monitor the presence of donor cell populations in each pig following pCD3–CRM9 administration and PBSC infusion, as previously described [6]. Briefly, peripheral blood or thymic cell suspensions were incubated with an anti-CD3 monocolonal antibody (898H2–6–15) together with the donor-specific biotin-conjugated 1038H–10-9 (B10.PD1, IgMK) monoclonal antibody specific for swine pig allelic antigen (PAA) for 30 minutes followed by streptavidin phycoerythrin (Pharmingen, San Diego, CA). Red blood cells were lysed and the cells fixed using a fluorescent activated cell sorter (FACS) lysing solution (Becton Dickinson, San Jose, CA) before acquisition. Data were analyzed using Winlist list mode analysis software (Verity Software House, Topsham, ME).

Skin grafts
Skin grafts were performed by a previously published technique [1]. Briefly, split-thickness skin was harvested from the donor and placed on a deep split-thickness bed on the recipient’s dorsal thorax. Grafts were examined daily until rejection occurred. Rejection was determined macroscopically and defined as diffuse cyanosis and induration of the graft.

Cardiac transplantation
The technique of heterotopic heart transplantation has been described previously [10]. In brief, after the induction of general anesthesia, both the donor and recipient animal were heparinized with 300 U/kg of heparin. The donor heart was harvested after the administration of cold crystalloid cardioplegia solution (Plegisol, Abbott Laboratories, Chicago, IL). The heart was prepared for implantation by creating an atrial septal defect and by defunctionalizing the mitral valve to minimize left ventricular atrophy and intracavitary thrombus formation. The donor pulmonary artery was anastomosed end-to-side to the recipient’s inferior vena cava. The ascending aorta of the donor heart was then anastomosed to the recipient’s abdominal aorta. Iridium-tipped ventricular electrodes (Model 6500 pacing lead, Medtronic, Minneapolis, MN) were implanted into each ventricle and brought out through the skin for long-term electrocardiographic monitoring. Allograft rejection was defined as lack of a ventricular impulse on palpation, an R-wave amplitude of less than 3 mm on epicardial electrocardiograph, a lack of ventricular contraction on echocardiography or palpation, or any combination of those signs. Serial open biopsies were performed using Tru-cut needles (Baxter, Deerfield, IL).

Histopathological examination
Heart tissue either from biopsies performed on approximately 30, 70, and 150 days after the procedure or from autopsy specimens was fixed in 10% formalin. The tissue specimens were imbedded in paraffin, and cut sections were stained with hematoxylin-and-eosin and elastin stains. The severity of interstitial rejection and intimal proliferation was evaluated by a blinded cardiac pathologist. Scoring of the acute rejection in the cardiac allograft was based on International Society for Heart and Lung Transplantation criteria [11], and the degree of intimal thickening was based on computerized morphometry.

Computerized morphometry
Epicardial and myocardial arteries and veins were examined morphometrically using an image analysis system. Images of histologic sections were captured to a Power Macintosh 7300/200 computer (Apple, Cupertino, CA) by a Hitachi 3-CCD Color Camera (model HV–C20) (Hitachi Denshi, Rodgau, Germany) attached to a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan). With digital image analysis (IPLab Spectrum, Signal Analytics Corporation, Vienna, VA), the images were then analyzed by manual color segmentation, tracing the endothelial surface (intima), internal elastic lamina, and external elastic lamina of each vessel. Computed measurements from segmented image provided calculation of intima-to-media ratio and percent occlusion of each vessel lumen. Analysis of vessel size and mean intimal thickness allowed for further characterization of the extent of vasculopathy.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Creation of mixed allogeneic chimeras using CD3 immunotoxin and high-dose PBSC
Our laboratory has recently developed relatively nontoxic conditioning regimens that permit the reliable induction of stable and long lasting mixed allogeneic chimerism. A detailed phenotypic characterization of the multilineage peripheral blood and thymic chimerism achieved in animals undergoing these nonmyeloablative conditioning protocols is described elsewhere [6, 7]. Host T lymphocytes were depleted using the swine CD3 mutant diphtheria toxin conjugate pCD3–CRM9. Previous dose-response analyses demonstrated that a single intravenous dose of 0.05 mg/kg of pCD3–CRM9 resulted in the elimination of more than 99.8% of peripheral T cells with no major side effects [4]. Therefore, recipients were given one dose of 0.05 mg/kg pCD3–CRM9 48 hours before the infusion of cytokine-mobilized PBSC. This allowed maximal host T-cell depletion before donor-cell infusion. Two different nonlethal irradiation protocols were used to complete the conditioning regimen. The first included two animals (#13131 and #13235) that received nonmyeloablative whole-body irradiation along with thymic irradiation (TI). These recipients were transfused with 20 x 10⁸ donor PBSC/kg on day 0. The second protocol included two animals (#12757 and #12963) that received TI alone. Eliminating WBI completely made this protocol truly nonmyelosuppressive. These recipients were transfused with 100 x 10⁸ donor PBSC/kg on day 0. Figure 2 is a schematic diagram of the two conditioning regimens used for the induction of mixed hematopoietic chimeras in these experiments.

View larger version (18K): [in this window] [in a new window]   Fig 2. Experimental protocols. (A) Timeline for the nonmyeloablative conditioning regimen which included WBI and TI. (B) Timeline for the nonmyelosuppressive conditioning regimen in which WBI is eliminated. *Note that swine #13235 did not receive CyA. (CyA = cyclosporine; TI = thymic irradiation; WBI = whole-body irradiation.)

View larger version (40K): [in this window] [in a new window]   Fig 3. Donor lymphoid chimerism in mixed hematopoietic chimeras. The two-color scatter plots demonstrate mononuclear cell staining with FITC-conjugated anti-CD3 antibody versus biotin-conjugated PAA antibody. Double-stained cells (upper right quadrant of each scatter plot) represent PAA-positive T lymphocytes of donor origin. Percentage lymphocyte chimerism denotes the number of double positive stained cells over the total number of mononuclear cells acquired by FACS. (A) Representative scatter plot from recipient #13131 (WBI/TI and pCD3–CRM9 conditioning regimen) 13 days before cardiac transplantation and 126 days following cardiac ransplantation. (B) Representative scatter plot from recipient #12963 (TI and pCD3–CRM9 conditioning regimen) on the day of cardiac transplantation (postoperative day 0) and 116 days following cardiac transplantation. (pCD3–CRM9 = porcine CD3 immunotoxin; FACS = fluorescent activated cell sorter; FITC = fluorescein isothiocyanate; PAA = pig allelic antigen; TI = thymic irradiation; WBI = whole-body irradiation.)

Given the significant prolongation achieved in the survival of donor-specific skin grafts, we sought to evaluate the effects of mixed chimerism on the acute and chronic rejection of vascularized, whole-organ cardiac allografts. Long-term, stable mixed chimeras that had previously received donor-specific and third party skin grafts were transplanted with the hearts from their respective PBSC donors. The survival of these donor-specific grafts were compared with the survival of MHC-matched, minor antigen–mismatched cardiac allografts in untreated control animals. The chimeric animals received their hearts 146, 160, 175, and 355 days following PBSC transfusion (Fig 2). At the time of cardiac transplantation, these recipients had all maintained stable chimerism that ranged between 10% and 52% in the peripheral blood and 10% and 63% in the thymus (Table 1). Also, at the time of transplantation, cell-mediated lympholysis assays demonstrated that recipient T cells were unresponsive to cells from the PBSC donor (donor-specific cells) but that they generated appropriate cytotoxic responses to cells from animals that were matched to the donor MHC but mismatched for minor antigens (third-party controls) [6, 7].

We have previously reported that MHC-matched, minor antigen–mismatched hearts transplanted into untreated recipients were all acutely rejected within 44 days (Table 2) [10]. Postmortem specimens revealed extensive mononuclear cell infiltrates, myocyte necrosis, and interstitial hemorrhage consistent with a florid acute rejection response (Fig 4A). These hearts exhibited grade 3b and 4 rejection according to the International Society for Heart and Lung Transplantation scoring system [10]. Of note, none of the donor hearts exhibited intimal proliferation within the coronary artery walls. In contrast, hearts transplanted into the mixed chimeras were all accepted long term without immunosuppressive therapy (Table 2). Serial biopsies revealed no evidence of interstitial rejection and no evidence of coronary vasculopathy at any time point during the life of the recipient. Three late deaths occurred in long-term mixed chimeras with beating donor hearts (#12963, #12757, #13131). Each death was due to the spontaneous rupture of the donor left atrium. This complication may have resulted from our practice of opening the atrial septum and defunctionalizing the mitral valve before implantation to avoid the formation of left ventricular thrombosis. This resulted in progressive dilatation of the donor left atrium in long-term survivors. Postmortem specimens from these three donor hearts showed ischemic changes that were probably related to late cardiac rupture. However, the donor hearts had minimal interstitial infiltrates (Fig 4B), in stark contrast to the heart allografts, which were acutely rejected in the untreated controls (Fig 4A). Hearts explanted from the chimeric recipients at autopsy, exhibited mild intimal proliferation within the walls of a small number of coronary arteries, a situation consistent with chronic rejection (Fig 4C). This unexpected result led us to perform a detailed computer-based morphologic analysis of the coronary artery walls in each explanted donor heart. Table 3 shows that of the thousands of epicardial and intramyocardial arteries examined, only 1.0% to 6.7% exhibited any intimal proliferation. These vascular lesions seemed to be equally distributed among the epicardium and myocardium; when present, they resulted in an average 44.7% to 68.7% occlusion of the vessel’s cross-sectional lumen.

View larger version (118K): [in this window] [in a new window]   Fig 4. Three donor heart autopsy specimens stained with hematoxylin and eosin. (A) Section from recipient #10607 scored as ISHLT grade 4 rejection shows no evidence of intimal proliferation (magnification, 200x). (B) Section from recipient #12757 scored as ISHLT grade 1A rejection shows no evidence of intimal proliferation (magnification, 400x). (C) Section from recipient #13131 scored as ISHLT grade 0 rejection but shows evidence of mild intimal proliferation within intramyocardial arteries (magnification, 400x). (ISHLT = International Society for Heart and Lung Transplantation.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

The establishment of mixed chimerism induces central deletional tolerance by actively "tricking" the recipient’s immune system into treating donor antigens as self-antigens [2]. To achieve this goal, the host has, until recently, received some form of WBI in order to make "space" for a subsequent donor bone marrow transfusion. Once hematopoietic stem cells contained in the donor bone marrow engraft, they coexist with recipient stem cells and give rise to cells of all hematopoietic lineages. In addition, hematopoietic progenitor cells seed the thymus, giving rise to both T cells and dendritic cells [12]. Since hematopoietic cells from both the recipient and the donor colocate to the thymus, both self-reactive and donor-reactive T cells are eliminated by negative selection (the process that defines the phrase "central deletional tolerance") [13]. Consequently, the newly developing T-cell repertoire in mixed chimeras is tolerant towards the donor and remains so as long as chimerism persists.

Ildstad and Sachs demonstrated that the induction of mixed chimerism could result in long-lasting tolerance in rodents [3]. Since then, the ability of mixed chimerism protocols to induce tolerance specifically to cardiac allografts has been demonstrated in rodent models [14]. However, clear immunobiological differences exist between rodents and larger animals, including humans. For instance, we have demonstrated that rodents do not constitutively express MHC class II antigens on the endothelium of their coronary arteries, whereas large animals (including pigs and humans) do express these molecules [15]. Given the crucial role that MHC class II antigens play in allograft rejection, these interspecies differences may be important in both early and late alloresponses. Furthermore, many methods by which solid-organ transplantation tolerance can be induced in rodents have failed when applied to large animals or to patients. There have been a limited number of studies examining mixed chimerism in dogs and cynomologous monkeys that demonstrated successful achievement of mixed chimerism and, in the monkeys, tolerance to kidney transplants [16–18]. We have found no previous studies examining the effects of mixed chimerism protocols on cardiac allografts in large animals. The subject has clinical relevance, because in experimental models, cardiac allografts are more immunogenic than kidney allografts and thus more difficult to transplant successfully [1]. In this study we confirm that establishment of mixed chimerism can also induce donor-specific tolerance to cardiac allografts in large animals.

We [1] and others [19] have shown that the induction of tolerance can mitigate the development of CAV. Using a MHC class I disparate strain combination, we demonstrated that when hearts transplanted into miniature swine were treated with a short course of cyclosporine, florid CAV developed and the hearts were rejected within 55 days. However, when a donor-specific kidney was cotransplanted with the heart allograft, recipients became tolerant to donor antigen and accepted both allografts long term. Furthermore, the tolerogenic state induced by heart and kidney cotransplantation prevented the development of CAV [1]. Thus, we were surprised to observe a small, albeit real, number of vascular lesions in the postmortem specimens of donor heart from long-term mixed chimeras. The pathogenesis of these lesions in these mixed chimerism protocols is unclear but may be related to aberrant healing following a transient, low-level acute rejection response that may be a prerequisite for the induction of stable tolerance in the mixed chimerism protocol and not the heart-kidney cotransplantation protocol. Alternatively, the low-grade vasculopathy observed in the mixed chimeras may have been due to an immune response directed toward tissue-specific antigens shared by the skin grafts and endothelial antigens present in the heart [20]. The fact that CAV was never observed in naïve or CyA-treated recipients of MHC-matched, minor antigen–mismatched hearts without skin grafts supports this latter hypothesis [10]. In either case, the prevalence and severity of these late vascular lesions had no impact on graft survival.

The extrapolation of tolerance strategies, such as bone marrow chimerism, to clinical transplantation depends on developing safe and reliable nonmyeloablative conditioning regimens. The development of a successful nonmyeloablative regimen in large animals has proved difficult. Recently our laboratory has utilized the newly described CD3 immunotoxin pCD3–CRM9 [4] together with high-dose, cytokine-mobilized PBSC (as a source of donor hematopoietic cells) for reliable induction of long-term mixed chimerism in miniature swine without the toxic effects of lethal WBI or GvHD [6, 7]. Furthermore, whole-body irradiation has been successfully eliminated from the conditioning regimen by increasing the dose of allogeneic hematopoietic cells administered [7]. The elimination of WBI from the preparative regimen has further reduced toxicity and allows this approach to tolerance induction to be more clinically acceptable. The clinical potential of hematopoietic chimerism is illustrated by reports of patients who have undergone allogeneic bone marrow transplantation for hematological indications, and who subsequently received a kidney transplant from the same donor. These patients accepted the renal graft without immunosuppressive therapy, even across major MHC barriers [21–23]. Even more exciting is the fact that Spitzer and colleagues at the Massachusetts General Hospital have recently published the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen used to treat a hematological malignancy and to provide allotolerance for a solid-organ transplant [24]. The patient remains clinically well and is off all immunosuppressive therapy 15 months following kidney transplantation (T. R. Spitzer, personal communication).

In conclusion, we have shown that mixed chimerism can induce long-term survival of cardiac allografts without chronic immunosuppressive therapy in a preclinical large-animal model. Recently, studies in our research center have demonstrated the successful induction of mixed chimerism across a full MHC mismatch barrier in miniature swine [7]. The possibility that T-cell costimulatory blockade may replace T-cell depletion in achieving high levels of chimerism and central T-cell tolerance in the pig model is also being investigated. These nonmyeloablative protocols may allow the safe and reliable induction of long term tolerance in human recipients of whole-organ allografts and xenografts.

	Acknowledgments

 
Doctor Schwarze is a Claude E. Welch surgical research fellow at Massachusetts General Hospital and is a recipient of the Research Fellowship Award from the International Society of Heart and Lung Transplantation. Doctor Menard is an Edward D. Churchill Surgical Research Fellow at Massachusetts General Hospital and a recipient of the Roche Surgical Scientist Award from the American Society of Transplantation. The authors are indebted to Mr J. Scott Arn for herd management and quality control typing. The authors also acknowledge the generosity of the Novartis Pharmaceutical Corporation, which kindly provided cyclosporine, and of Schering-Plough Animal Health for providing flunixamine.

This work was supported in part by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (2RO1–HL54211–04) and the Thoracic Surgery Foundation For Research & Education.

	Footnotes

 
This article has been selected for the discussion forum on the STS Web site: http://www.sts.org/section/atsdiscussion/

	References

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