Stuart Houser, Kwabena Mawulawde, Kenneth S. Allison, David H. Sachs and Joren C. Margaret L. Schwarze, Matthew T. Menard, Yasushi Fuchimoto, Christene A. Huang,
in miniature swineMixed hematopoietic chimerism induces long-term tolerance to cardiac allografts
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Mixed Hematopoietic Chimerism InducesLong-Term Tolerance to Cardiac Allografts inMiniature SwineMargaret L. Schwarze, MD, Matthew T. Menard, MD, Yasushi Fuchimoto, MD,Christene A. Huang, PhD, Stuart Houser, MD, Kwabena Mawulawde, MD,Kenneth S. Allison, BA, David H. Sachs, MD, and Joren C. Madsen, MDDivision of Cardiac Surgery and Transplantation Biology Research Center, Department of Surgery, Massachusetts GeneralHospital, Harvard Medical School, Boston, Massachusetts
Background. Tolerance to cardiac allografts has notbeen achieved in large animals using methods that arereadily applicable to human recipients. We investigatedthe effects of mixed hematopoietic chimerism on cardiacallograft survival and chronic rejection in miniatureswine
Methods. Recipients were T-cell depleted using a por-cine CD3 immunotoxin, and each received either of twononmyeloablative preparative regimens previously dem-onstrated to permit the establishment of stable mixedhematopoietic chimerism across MHC-matched, minor-antigen–mismatched histocompatibility barriers. Five to12 months after the chimerism was induced, hearts fromthe original cell donors were heterotopically trans-planted into the stable mixed chimeras.
Results. Cardiac allografts transplanted into untreated
recipients across similar minor antigen barriers wererejected within 44 days (within 21, 28, 35, 39, 44 daysamong individual study subjects). In contrast, heartstransplanted into the mixed chimeras were all acceptedlong term ( > 153, > 225, > 286, > 362 days) withoutimmunosuppressive drugs and developed minimalvasculopathy.
Conclusions. Mixed hematopoietic chimerism, estab-lished in miniature swine using clinically relevant, non-myeloablative conditioning regimens, permits long-termcardiac allograft survival without chronic immunosup-pressive therapy, significant vasculopathy, or graft-versus-host disease.
Dramatic success has been achieved in cardiac trans-plantation over the past 15 years through the use of
powerful immunosuppressive agents, including but notlimited to prednisone, cyclosporine (CyA), and azathio-prine. However, these nonspecific immunosuppressiveagents are associated with serious complications, such asmalignancy, end organ toxicity, and infectious diseases.Furthermore, neither these agents nor even newer T-cell–directed therapies have been able to prevent cardiacallograft vasculopathy (CAV), a manifestation of chronicrejection and the leading cause of death and graft lossafter the first posttransplant year. A superior alternativeto nonspecific immunosuppressive therapy would be theinduction of donor-specific transplantation tolerance,which could result in permanent graft survival withoutthe need for long-term immunosuppressive therapy. In-deed, we have recently shown that the induction of rapidand stable tolerance can prevent the development ofCAV [1].
A state of mixed chimerism is achieved when bone
marrow transplantation results in the stable coexistenceIof multilineage hematopoietic cells from the donor andhost (reviewed recently by Wekerle and Sykes [2]). Thisstate confers permanent tolerance for solid organs ofdonor (bone marrow) type while maintaining normalimmune responses to third-party grafts and pathogens[3]. The major obstacle preventing the clinical applicationof mixed chimerism to tolerance induction has been thesevere toxicity associated with host myeloablative condi-tioning regimens, which have generally utilized lethalwhole-body irradiation (WBI). Lethal WBI has been re-quired to deplete host T cells and create “space” for theengraftment of allogeneic stem cells following donorbone marrow transplantation in most large-animal mod-els. However, this limitation has been overcome in min-iature swine by the development of nonmyeloablativeconditioning regimens made possible by two recent ad-vances. The first was to substitute a novel swine CD3immunotoxin, pCD3–CRM9, for lethal WBI to achieveT-cell depletion in the host. The immunotoxin pCD3–
Presented at the Thirty-sixth Annual Meeting of the Society of ThoracicSurgeons, Fort Lauderdale, FL, Jan 31–Feb 2, 2000.
Address reprint requests to Dr Madsen, Division of Cardiac Surgery,Massachusetts General Hospital, EDR 105, 55 Fruit St, Boston,MA 02114; e-mail: [email protected].
This article has been selected for the discussion forumon the STS Web site:
CRM9 is extremely effective in depleting mature T cellsfrom the peripheral blood, lymph nodes, and thymus ofminiature swine [4]. The second was the use of highdoses of peripheral blood stem cells (PBSCs) instead ofbone marrow to reconstitute the T-cell–depleted hosts.Our laboratory has shown that cytokine mobilization andapheresis of miniature swine blood allows the collectionof PBSC capable of full hematopoietic reconstitution inlieu of bone marrow [5]. Using nonmyeloablative condi-tioning regimens that include pCD3–CRM9 and high-dose PBSC, either with nonlethal WBI [6] or without WBI[7], our laboratory has recently demonstrated the safeand reliable induction of stable multilineage mixed chi-merism and skin-graft tolerance without the toxicity oflethal whole-body irradiation.
In order to apply the mixed chimerism approach topatients, large-animal models are required, both to un-derstand the mechanism and to optimize the treatmentprotocol. Partially inbred miniature swine have beendeveloped in this laboratory as a large-animal preclinicalmodel for studies of transplantation immunobiology; theswine are very similar to humans in this regard [8]. Theability of mixed chimerism protocols to confer toleranceto cardiac allografts has not previously been demon-strated in large animals. In this report, we use animalsavailable from other studies dealing with the induction ofmixed chimerism in miniature swine [6, 7] to test whetheror not mixed hematopoietic chimerism, established withclinically relevant, nonmyeloablative conditioning regi-mens, permits long-term cardiac allograft survival in theabsence of immunosuppressive therapy and abrogatesCAV.
Material and Methods
AnimalsTransplant donors and recipients aged 2 to 3 months thatwere matched for MHC (SLA) but mismatched for minorantigens were selected from our herd of MassachusettsGeneral 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 performedin compliance with both the “Principles of LaboratoryAnimal Care” formulated by the National Society forMedical Research and the “Guide for the Care and Use ofLaboratory Animals” prepared by the institute of Labo-ratory Animal Resources and published by the NationalInstitutes of Health, revised 1996.
Peripheral Blood Stem Cell CollectionA stem-cell mobilizing regimen consisting of daily treat-ments with recombinant porcine stem cell factor (pSCF,100 mg/kg) in combination with recombinant porcineinterleukin 3 (pIL-3, 100 mg/kg), both from BioTransplant(Boston, MA) and with or without recombinant humangranulocyte colony-stimulating factor (rhu G-CSF, 10mg/kg), was administered subcutaneously. Collection ofperipheral blood stem cell (PBSC) was achieved by leu-kapheresis (COBE Spectra Apheresis System, GambroBTC, Lakewood, CO) beginning on day 5 of cytokinetherapy and continuing daily until sufficient numbers ofcells were collected. The PBSC collection, either fresh orfrozen and quickly thawed, were adjusted to a concen-tration of 2.0 3 108 mL and infused intravenously onday 0.
IrradiationAnimals received intravenous 0.15 mg/kg of Telazol(AHP, Madison, NJ) for sedation and were then placed inthe supine position on a plastic cradle and secured intoposition. Recipients #13131 and #13235 received sublethalwhole body irradiation (150 cGy) on days 24 and 23 andthymic irradiation (700 cGy) on day 22 as previouslydescribed [6]. Recipients #12757 and 12963 received 700cGy of thymic irradiation alone on day 22 and nowhole-body irradiation.
Recipient T-Cell DepletionAll recipients in the mixed chimera group underwentT-cell depletion using the newly described diphtheria-toxin–based swine CD3 immunotoxin, pCD3–CRM9 [4].This swine CD3 immunotoxin was made by conjugatingthe diphtheria toxin binding site mutant, CRM9, to theantiporcine CD3 mAb, 898H2– 6 –F15. On day 22,0.05 mg/kg of pCD3–CRM9 was administered intrave-nously to recipient animals.
CyA TreatmentCyclosporine (Sandimmune oral solution, Novartis,Summit, NJ) was administered through a gastric tube at30 mg z kg21 z d21 in divided doses from day 21 to day 30.
Antibodies and Flow CytometryFlow cytometry (Becton Dickinson FACScan, San Jose,CA) was used to monitor the presence of donor cellpopulations in each pig following pCD3–CRM9 adminis-tration and PBSC infusion, as previously described [6].Briefly, peripheral blood or thymic cell suspensions wereincubated with an anti-CD3 monocolonal antibody
Fig 1. Origin of available SLA haplotypes in partially inbredminiature swine. (SLA 5 swine leukocyte antigen.)
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(898H2–6–15) together with the donor-specific biotin-conjugated 1038H–10-9 (B10.PD1, IgMK) monoclonal an-tibody specific for swine pig allelic antigen (PAA) for 30minutes followed by streptavidin phycoerythrin (Pharm-ingen, San Diego, CA). Red blood cells were lysed andthe cells fixed using a fluorescent activated cell sorter(FACS) lysing solution (Becton Dickinson, San Jose, CA)before acquisition. Data were analyzed using Winlist listmode analysis software (Verity Software House, Top-sham, ME).
Skin GraftsSkin grafts were performed by a previously publishedtechnique [1]. Briefly, split-thickness skin was harvestedfrom the donor and placed on a deep split-thickness bedon the recipient’s dorsal thorax. Grafts were examineddaily until rejection occurred. Rejection was determinedmacroscopically and defined as diffuse cyanosis andinduration of the graft.
Cardiac TransplantationThe technique of heterotopic heart transplantation hasbeen described previously [10]. In brief, after the induc-tion of general anesthesia, both the donor and recipientanimal were heparinized with 300 U/kg of heparin. Thedonor heart was harvested after the administration ofcold crystalloid cardioplegia solution (Plegisol, AbbottLaboratories, Chicago, IL). The heart was prepared forimplantation by creating an atrial septal defect and bydefunctionalizing the mitral valve to minimize left ven-tricular atrophy and intracavitary thrombus formation.The donor pulmonary artery was anastomosed end-to-side to the recipient’s inferior vena cava. The ascendingaorta of the donor heart was then anastomosed to therecipient’s abdominal aorta. Iridium-tipped ventricularelectrodes (Model 6500 pacing lead, Medtronic, Minne-apolis, MN) were implanted into each ventricle andbrought out through the skin for long-term electrocar-diographic monitoring. Allograft rejection was defined aslack of a ventricular impulse on palpation, an R-waveamplitude of less than 3 mm on epicardial electrocardio-graph, a lack of ventricular contraction on echocardiog-raphy or palpation, or any combination of those signs.Serial open biopsies were performed using Tru-cut nee-dles (Baxter, Deerfield, IL).
Histopathological ExaminationHeart tissue either from biopsies performed on approx-imately 30, 70, and 150 days after the procedure or fromautopsy specimens was fixed in 10% formalin. The tissuespecimens were imbedded in paraffin, and cut sectionswere stained with hematoxylin-and-eosin and elastinstains. The severity of interstitial rejection and intimalproliferation was evaluated by a blinded cardiac pathol-ogist. Scoring of the acute rejection in the cardiac allo-graft was based on International Society for Heart andLung Transplantation criteria [11], and the degree ofintimal thickening was based on computerizedmorphometry.
Computerized MorphometryEpicardial and myocardial arteries and veins were exam-ined morphometrically using an image analysis system.Images of histologic sections were captured to a PowerMacintosh 7300/200 computer (Apple, Cupertino, CA) bya Hitachi 3-CCD Color Camera (model HV–C20) (HitachiDenshi, Rodgau, Germany) attached to a Nikon EclipseE600 microscope (Nikon, Tokyo, Japan). With digitalimage analysis (IPLab Spectrum, Signal Analytics Corpo-ration, Vienna, VA), the images were then analyzed bymanual color segmentation, tracing the endothelial sur-face (intima), internal elastic lamina, and external elasticlamina of each vessel. Computed measurements fromsegmented image provided calculation of intima-to-media ratio and percent occlusion of each vessel lumen.Analysis of vessel size and mean intimal thickness al-lowed for further characterization of the extent ofvasculopathy.
Results
Creation of Mixed Allogeneic Chimeras Using CD3Immunotoxin and High-dose PBSCOur laboratory has recently developed relatively non-toxic conditioning regimens that permit the reliable in-duction of stable and long lasting mixed allogeneic chi-merism. A detailed phenotypic characterization of themultilineage peripheral blood and thymic chimerismachieved in animals undergoing these nonmyeloablativeconditioning protocols is described elsewhere [6, 7]. HostT lymphocytes were depleted using the swine CD3 mu-tant diphtheria toxin conjugate pCD3–CRM9. Previousdose-response analyses demonstrated that a single intra-venous dose of 0.05 mg/kg of pCD3–CRM9 resulted inthe elimination of more than 99.8% of peripheral T cellswith no major side effects [4]. Therefore, recipients weregiven one dose of 0.05 mg/kg pCD3–CRM9 48 hoursbefore the infusion of cytokine-mobilized PBSC. Thisallowed maximal host T-cell depletion before donor-cellinfusion. Two different nonlethal irradiation protocolswere used to complete the conditioning regimen. Thefirst included two animals (#13131 and #13235) that re-ceived nonmyeloablative whole-body irradiation alongwith thymic irradiation (TI). These recipients were trans-fused with 20 3 108 donor PBSC/kg on day 0. The secondprotocol included two animals (#12757 and #12963) thatreceived TI alone. Eliminating WBI completely made thisprotocol truly nonmyelosuppressive. These recipientswere transfused with 100 3 108 donor PBSC/kg on day 0.Figure 2 is a schematic diagram of the two conditioningregimens used for the induction of mixed hematopoieticchimeras in these experiments.
As indicated in Table 1, each animal that completedone of the two nonmyeloablative conditioning regimensand received cytokine-mobilized PBSC did successfullyengraft and develop stable and long-lasting chimerism[6, 7]. Furthermore, all animals recovered without anyevidence of graft-versus-host disease (GvHD). Chimer-ism was detected by flow cytometry using our PAA mAb,
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which recognized a pig allelic antigen present on swineleukocytes. Donor and recipient animals were selected asPAA positive and PAA negative, respectively, to facilitatechimerism detection. Figure 3A is a representative FACSflow cytometric scan showing the percentage of donorlymphocytes detected in the peripheral blood of recipient#13131, which was conditioned with the WBI-TI andpCD3–CRM9 protocol. Following PBSC transplantation,the two recipients in the WBI/TI and pCD3–CRM9 pro-tocol developed stable, long-term peripheral lymphocytechimerism that ranged from 40% to 60% for recipient#13131 and from 20% to 30% for recipient 13235. Thymicchimerism in these animals ranged between 47% to 60%for recipient #13131 and between 26% to 43% for recipient#13235 [6]. Figure 3B is a representative FACS scanshowing the percentage of donor cells detected in theperipheral blood of recipient #12963, which was condi-tioned with TI and pCD3–CRM9 protocol. FollowingPBSC transplantation, the two recipients in the TI andpCD3–CRM9 protocol also developed stable, long termperipheral lymphocyte chimerism that ranged from 10%
to 20% for recipient #12963 and from 20% to 30% forrecipient #12757. Thymic chimerism ranged from 3.5% to4.5% for recipient #12963 and from 9% to 10% for recip-ient #12757 [7]. Of note, clinically relevant leukopenia orthrombocytopenia did not develop in animals condi-tioned with either nonmyeloablative regimen. Further-more, the general condition of these recipients remainedexcellent, and occult infections were not observed.
Effect of Mixed Chimerism on TransplantationToleranceTo determine the efficacy and the specificity of theimmunologic unresponsiveness induced by mixed chi-merism, recipients were grafted with skin from the ani-mals that had donated PBSC (donor-specific skin) andfrom animals that were matched to the donor MHC butmismatched for minor antigens (third-party controls). Aspreviously reported [6, 7], all chimeras showed eitherindefinite acceptance or significant prolongation of do-nor-specific skin grafts, compared with donor-MHCmatched, minor antigen–mismatched skin grafts (Table
Fig 2. Experimental protocols. (A) Timelinefor the nonmyeloablative conditioning regi-men which included WBI and TI. (B) Time-line for the nonmyelosuppressive conditioningregimen in which WBI is eliminated. *Notethat swine #13235 did not receive CyA.(CyA 5 cyclosporine; TI 5 thymic irradia-tion; WBI 5 whole-body irradiation.)
Table 1. Skin Graft Survival and Peripheral Donor Chimerism in Chimeric Miniature Swine
a Assessed within 30 days of heart transplantation. b Assessed 100–130 days following heart transplantation. c Result has been reported previously[7]. d Result has been reported previously [6].
1). Recipients #12757 and #13131 accepted donor-specificskin grafts indefinitely but rejected minor antigen–mismatched control skin grafts on days 9 and 22, respec-tively. Recipients #12963 and #13235 accepted donor skingrafts for 45 days but rejected control skin grafts on 9days and 10 days, respectively. There was no correlationbetween the percentage of donor chimerism and skin-graft survival in these minor-mismatched, mixed chi-meric animals [6, 7].
Given the significant prolongation achieved in thesurvival of donor-specific skin grafts, we sought to eval-uate the effects of mixed chimerism on the acute andchronic rejection of vascularized, whole-organ cardiacallografts. Long-term, stable mixed chimeras that hadpreviously received donor-specific and third party skingrafts were transplanted with the hearts from their re-spective PBSC donors. The survival of these donor-specific grafts were compared with the survival of MHC-matched, minor antigen–mismatched cardiac allograftsin untreated control animals. The chimeric animals re-ceived their hearts 146, 160, 175, and 355 days followingPBSC transfusion (Fig 2). At the time of cardiac trans-plantation, these recipients had all maintained stablechimerism that ranged between 10% and 52% in theperipheral blood and 10% and 63% in the thymus (Table1). Also, at the time of transplantation, cell-mediatedlympholysis assays demonstrated that recipient T cellswere unresponsive to cells from the PBSC donor (donor-specific cells) but that they generated appropriate cyto-toxic responses to cells from animals that were matchedto the donor MHC but mismatched for minor antigens(third-party controls) [6, 7].
We have previously reported that MHC-matched, mi-nor antigen–mismatched hearts transplanted into un-treated recipients were all acutely rejected within 44 days
(Table 2) [10]. Postmortem specimens revealed extensivemononuclear cell infiltrates, myocyte necrosis, and inter-stitial hemorrhage consistent with a florid acute rejectionresponse (Fig 4A). These hearts exhibited grade 3b and 4rejection according to the International Society for Heartand Lung Transplantation scoring system [10]. Of note,none of the donor hearts exhibited intimal proliferationwithin the coronary artery walls. In contrast, heartstransplanted into the mixed chimeras were all acceptedlong term without immunosuppressive therapy (Table 2).Serial biopsies revealed no evidence of interstitial rejec-tion and no evidence of coronary vasculopathy at anytime point during the life of the recipient. Three latedeaths occurred in long-term mixed chimeras with beat-ing donor hearts (#12963, #12757, #13131). Each death wasdue to the spontaneous rupture of the donor left atrium.This complication may have resulted from our practice ofopening the atrial septum and defunctionalizing themitral valve before implantation to avoid the formationof left ventricular thrombosis. This resulted in progres-sive dilatation of the donor left atrium in long-termsurvivors. Postmortem specimens from these three donorhearts showed ischemic changes that were probablyrelated to late cardiac rupture. However, the donor heartshad minimal interstitial infiltrates (Fig 4B), in stark con-trast to the heart allografts, which were acutely rejectedin the untreated controls (Fig 4A). Hearts explanted fromthe chimeric recipients at autopsy, exhibited mild intimalproliferation within the walls of a small number ofcoronary arteries, a situation consistent with chronicrejection (Fig 4C). This unexpected result led us toperform a detailed computer-based morphologic analy-sis of the coronary artery walls in each explanted donorheart. Table 3 shows that of the thousands of epicardialand intramyocardial arteries examined, only 1.0% to 6.7%
Fig 3. Donor lymphoid chimerism in mixedhematopoietic chimeras. The two-color scatterplots demonstrate mononuclear cell staining withFITC-conjugated anti-CD3 antibody versus bi-otin-conjugated PAA antibody. Double-stainedcells (upper right quadrant of each scatter plot)represent PAA-positive T lymphocytes of donororigin. Percentage lymphocyte chimerism denotesthe number of double positive stained cells overthe total number of mononuclear cells acquiredby FACS. (A) Representative scatter plot fromrecipient #13131 (WBI/TI and pCD3–CRM9 con-ditioning regimen) 13 days before cardiac trans-plantation and 126 days following cardiac rans-plantation. (B) Representative scatter plot fromrecipient #12963 (TI and pCD3–CRM9 condi-tioning regimen) on the day of cardiac trans-plantation (postoperative day 0) and 116 daysfollowing cardiac transplantation. (pCD3–CRM9 5 porcine CD3 immunotoxin; FACS 5fluorescent activated cell sorter; FITC 5 fluores-cein isothiocyanate; PAA 5 pig allelic antigen;TI 5 thymic irradiation; WBI 5 whole-bodyirradiation.)
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exhibited any intimal proliferation. These vascular le-sions seemed to be equally distributed among the epi-cardium and myocardium; when present, they resultedin an average 44.7% to 68.7% occlusion of the vessel’scross-sectional lumen.
Comment
In this report, we demonstrate for the first time that thesuccessful establishment of mixed chimerism inducesdonor-specific tolerance to cardiac allografts in largeanimals. Long-term mixed chimerism was achieved us-ing recently described nonmyeloablative conditioningregimens that are relatively nontoxic and thus havesignificant clinical relevance.
The establishment of mixed chimerism induces centraldeletional tolerance by actively “tricking” the recipient’simmune system into treating donor antigens as self-antigens [2]. To achieve this goal, the host has, untilrecently, received some form of WBI in order to make“space” for a subsequent donor bone marrow transfu-sion. Once hematopoietic stem cells contained in thedonor bone marrow engraft, they coexist with recipientstem cells and give rise to cells of all hematopoieticlineages. In addition, hematopoietic progenitor cells seedthe thymus, giving rise to both T cells and dendritic cells[12]. Since hematopoietic cells from both the recipientand the donor colocate to the thymus, both self-reactiveand donor-reactive T cells are eliminated by negativeselection (the process that defines the phrase “centraldeletional tolerance”) [13]. Consequently, the newly de-
Fig 4. Three donor heart autopsy specimens stained with hema-toxylin and eosin. (A) Section from recipient #10607 scored asISHLT grade 4 rejection shows no evidence of intimal proliferation(magnification, 2003). (B) Section from recipient #12757 scored asISHLT grade 1A rejection shows no evidence of intimal proliferation(magnification, 4003). (C) Section from recipient #13131 scored asISHLT grade 0 rejection but shows evidence of mild intimal prolifer-ation within intramyocardial arteries (magnification, 4003).(ISHLT 5 International Society for Heart and LungTransplantation.)
Table 2. Survival of MHC–Matched, MinorAntigen–Mismatched Cardiac Allografts
RecipientNo.
StrainCombination Conditioning Regimen
Survival(days)
2499a cc 3 cc None 352502a cc 3 cc None 39818a cc 3 cc None 28828a cc 3 cc None 4410607a dd 3 dd None 2113131b cc 3 cc WBI/TI and pCD3–CRM9 . 28613235 ac 3 ac WBI/TI and pCD3–CRM9 . 22512757b ac 3 ac TI and pCD3–CRM9 . 36212963b cc 3 cc TI and pCD3–CRM9 . 151
a Previously published [10]. b Died due to unrelated reasons with abeating heart.
MHC 5 major histocompatibility complex; pCD3–CRM9 5 swineCD3 immunotoxin; TI 5 thymic irradiation; WBI 5 nonlethalwhole-body irradiation.
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veloping T-cell repertoire in mixed chimeras is toleranttowards the donor and remains so as long as chimerismpersists.
Ildstad and Sachs demonstrated that the induction ofmixed chimerism could result in long-lasting tolerance inrodents [3]. Since then, the ability of mixed chimerismprotocols to induce tolerance specifically to cardiac allo-grafts has been demonstrated in rodent models [14].However, clear immunobiological differences exist be-tween rodents and larger animals, including humans. Forinstance, we have demonstrated that rodents do notconstitutively express MHC class II antigens on theendothelium of their coronary arteries, whereas largeanimals (including pigs and humans) do express thesemolecules [15]. Given the crucial role that MHC class IIantigens play in allograft rejection, these interspeciesdifferences may be important in both early and latealloresponses. Furthermore, many methods by whichsolid-organ transplantation tolerance can be induced inrodents have failed when applied to large animals or topatients. There have been a limited number of studiesexamining mixed chimerism in dogs and cynomologousmonkeys that demonstrated successful achievement ofmixed chimerism and, in the monkeys, tolerance tokidney transplants [16–18]. We have found no previousstudies examining the effects of mixed chimerism proto-cols on cardiac allografts in large animals. The subjecthas clinical relevance, because in experimental models,cardiac allografts are more immunogenic than kidneyallografts and thus more difficult to transplant success-fully [1]. In this study we confirm that establishment ofmixed chimerism can also induce donor-specific toler-ance to cardiac allografts in large animals.
We [1] and others [19] have shown that the induction oftolerance can mitigate the development of CAV. Using aMHC class I disparate strain combination, we demon-strated that when hearts transplanted into miniatureswine were treated with a short course of cyclosporine,florid CAV developed and the hearts were rejectedwithin 55 days. However, when a donor-specific kidneywas cotransplanted with the heart allograft, recipientsbecame tolerant to donor antigen and accepted bothallografts long term. Furthermore, the tolerogenic stateinduced by heart and kidney cotransplantation pre-vented the development of CAV [1]. Thus, we were
surprised to observe a small, albeit real, number ofvascular lesions in the postmortem specimens of donorheart from long-term mixed chimeras. The pathogenesisof these lesions in these mixed chimerism protocols isunclear but may be related to aberrant healing followinga transient, low-level acute rejection response that maybe a prerequisite for the induction of stable tolerance inthe mixed chimerism protocol and not the heart-kidneycotransplantation protocol. Alternatively, the low-gradevasculopathy observed in the mixed chimeras may havebeen due to an immune response directed toward tissue-specific antigens shared by the skin grafts and endothe-lial antigens present in the heart [20]. The fact that CAVwas never observed in naı̈ve or CyA-treated recipients ofMHC-matched, minor antigen–mismatched hearts with-out skin grafts supports this latter hypothesis [10]. Ineither case, the prevalence and severity of these latevascular lesions had no impact on graft survival.
The extrapolation of tolerance strategies, such as bonemarrow chimerism, to clinical transplantation dependson developing safe and reliable nonmyeloablative condi-tioning regimens. The development of a successful non-myeloablative regimen in large animals has proved dif-ficult. Recently our laboratory has utilized the newlydescribed CD3 immunotoxin pCD3–CRM9 [4] togetherwith high-dose, cytokine-mobilized PBSC (as a source ofdonor hematopoietic cells) for reliable induction of long-term mixed chimerism in miniature swine without thetoxic effects of lethal WBI or GvHD [6, 7]. Furthermore,whole-body irradiation has been successfully eliminatedfrom the conditioning regimen by increasing the dose ofallogeneic hematopoietic cells administered [7]. Theelimination of WBI from the preparative regimen hasfurther reduced toxicity and allows this approach totolerance induction to be more clinically acceptable. Theclinical potential of hematopoietic chimerism is illus-trated by reports of patients who have undergone allo-geneic bone marrow transplantation for hematologicalindications, and who subsequently received a kidneytransplant from the same donor. These patients acceptedthe renal graft without immunosuppressive therapy,even across major MHC barriers [21–23]. Even moreexciting is the fact that Spitzer and colleagues at theMassachusetts General Hospital have recently publishedthe first report of the deliberate induction of mixedlymphohematopoietic chimerism after a nonmyelo-ablative preparative regimen used to treat a hematolog-ical malignancy and to provide allotolerance for a solid-organ transplant [24]. The patient remains clinically welland is off all immunosuppressive therapy 15 monthsfollowing kidney transplantation (T. R. Spitzer, personalcommunication).
In conclusion, we have shown that mixed chimerismcan induce long-term survival of cardiac allografts with-out chronic immunosuppressive therapy in a preclinicallarge-animal model. Recently, studies in our researchcenter have demonstrated the successful induction ofmixed chimerism across a full MHC mismatch barrier inminiature swine [7]. The possibility that T-cell costimu-latory blockade may replace T-cell depletion in achieving
Table 3. Morphometric Analysis of Intimal Proliferation inCoronary Arteries of Donor Hearts From Mixed Chimeras
Variables
Recipient No.
12757 12963 13131
Arteries with CAV 109 17 13Arteries without CAV 1506 1638 359Total no. of arteries
high levels of chimerism and central T-cell tolerance inthe pig model is also being investigated. These nonmy-eloablative protocols may allow the safe and reliableinduction of long term tolerance in human recipients ofwhole-organ allografts and xenografts.
Doctor Schwarze is a Claude E. Welch surgical research fellow atMassachusetts General Hospital and is a recipient of the Re-search Fellowship Award from the International Society of Heartand Lung Transplantation. Doctor Menard is an Edward D.Churchill Surgical Research Fellow at Massachusetts GeneralHospital and a recipient of the Roche Surgical Scientist Awardfrom the American Society of Transplantation. The authors areindebted to Mr J. Scott Arn for herd management and qualitycontrol typing. The authors also acknowledge the generosity ofthe Novartis Pharmaceutical Corporation, which kindly pro-vided cyclosporine, and of Schering-Plough Animal Health forproviding flunixamine.
This work was supported in part by grants from the NationalHeart, Lung, and Blood Institute of the National Institutes ofHealth (2RO1–HL54211–04) and the Thoracic Surgery Founda-tion For Research & Education.
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DISCUSSION
DR CONSTANTINE MAVROUDIS (Chicago, IL): This is anexcellent study. I was curious as to the eventual fate of the nativeand engrafted leukocytes. What was the percentage of engraftedleukocytes in your study animals and did this percentage changeover time? And my other question was, did you assess ventric-
ular function by echocardiography? We performed similar ex-periments in our laboratory. We used rats and hamsters acrossa major barrier and had similar results, although the resultsremain unpublished. Your results in a larger animal model likethis is really good news. Congratulations.
138 SCHWARZE ET AL Ann Thorac SurgMIXED CHIMERISM AND HEART ALLOGRAFT TOLERANCE 2000;70:131–9
by on May 31, 2013 ats.ctsnetjournals.orgDownloaded from
Stuart Houser, Kwabena Mawulawde, Kenneth S. Allison, David H. Sachs and Joren C. Margaret L. Schwarze, Matthew T. Menard, Yasushi Fuchimoto, Christene A. Huang,
in miniature swineMixed hematopoietic chimerism induces long-term tolerance to cardiac allografts
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http://ats.ctsnetjournals.org/cgi/content/full/70/1/131including high-resolution figures, can be found at:
Supplementary Material http://ats.ctsnetjournals.org/cgi/content/full/70/1/131/DC1
