American Journal of Transplantation 2004; 4: 79–86 Blackwell Munksgaard
C Blackwell Munksgaard 2003 Copyright
doi: 10.1046/j.1600-6143.2003.00295.x
Composite ‘Thymoheart’ Transplantation Improves Cardiac Allograft Survival Matthew T. Menarda , Margaret L. Schwarzea , James S. Allana,c , Douglas R. Johnstona , Kwabena Mawulawdea , Akira Shimizua , Kazuhiko Yamadaa , Stuart L. Housera,d , Kenneth S. Allisona , David H. Sachsa and Joren C. Madsena,b,∗ a
Transplantation Biology Research Center, Division of Cardiac Surgery, and c Division of Thoracic Surgery, Department of Surgery, and d Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA ∗ Corresponding author: Joren C. Madsen,
[email protected] b
We have generated a novel composite organ, the thymoheart, which facilitates the contemporaneous transfer of fully vascularized and functional donor thymic tissue to the host at the time of cardiac transplantation. Composite thymoheart allografts were prepared in MHC-inbred miniature swine by implanting autologous thymic tissue into donor hearts 60–90 days before organ procurement. Thymoheart allografts and unmanipulated control hearts were then transplanted into three groups, each treated with the same 12 days of cyclosporine. MHC-matched thymohearts transplanted into euthymic recipients had a minimum survival ranging between 72 and 194 days vs. 42–64 days for unmanipulated control hearts (p = 0.02). MHC class I-disparate thymohearts transplanted into euthymic recipients had a minimum survival ranging between 64 and 191 days vs. 30–55 days for unmanipulated control hearts (p = 0.01). MHC class I-disparate thymohearts transplanted into thymectomized recipients survived between 41 and 70 days vs. 8–27 days for unmanipulated control hearts (p = 0.01). Cellular and humoral functional assays, and skin grafting, confirmed the presence of donor-specific hyporesponsiveness in long-term thymoheart allografts recipients. The transfer of vascularized, functional donor thymic tissue to the host at the time of cardiac transplantation may provide a novel approach to the induction of tolerance in human heart transplant recipients. Key words: plantation
Heart, swine, thymus, tolerance, trans-
Received 21 April 2003, revised 29 August and accepted for publication 3 September 2003
The importance of the thymus in the induction of central tolerance to self-antigens is well established (1). In this process, T lymphocytes that are potentially autoreactive are clonally eliminated or anergized, following exposure to self-antigens in the context of either bone marrow-derived dendritic cells or thymic epithelial cells (2). A growing body of literature suggests that comparable thymic-dependent mechanisms are relevant in the induction of donor-specific tolerance to alloantigens following experimental organ transplantation (3,4). In studying the mechanisms of tolerance induction in a miniature swine, we have demonstrated that the presence of an intact thymus is crucial for the induction of rapid and stable tolerance (5,6). Tolerance has been achieved by grafting nonvascularized allogeneic thymic tissue into a variety of host sites (7–9). However, in those experiments, fetal or neonatal thymic tissue grafts were used and the recipients were either nude, total lymphoid irradiated (TLI) or T- and NK-celldepleted mice (7–9). While tolerance induction using nonvascularized thymic grafts has been well established in immuno deficient mice, efforts to induce transplantation tolerance by grafting fragments of donor-derived thymic tissue into immunocompetent recipients or large animal recipients of solid organ allografts have not been successful. This may be as a result of early necrosis of the nonvascularized thymic graft. A recent report from our colleagues support this theory, as renal allografts containing vascularized donor thymic tissue successfully induce tolerance across several histocompatibility barriers in juvenile miniature swine, whereas simultaneous transplantation of nonvascularized thymic tissue could not induce a tolerant state when cotransplanted with a kidney allograft using the same immunosuppressive regimen (10). Based on the these findings, we hypothesized that a state of tolerance might likewise be achieved in porcine heart transplant recipients if fully vascularized and functional thymic tissue from the donor were transferred to the recipient at the time of donor heart implantation. To test this hypothesis, we developed the ‘thymoheart’ allograft, a novel composite organ produced by implanting thymic autografts into the donor heart several months before organ transplantation (11). Transplantation of the thymoheart allograft allowed functional, vascularized donor thymic tissue to be cotransplanted along with the cardiac allograft. Our
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initial studies demonstrated reliable intracardiac engraftment of the autologous thymic tissue (11). In this study, we show that transplantation of composite thymoheart allografts could prolong allograft survival and diminish the development of chronic vascular lesions. These data suggest that, with modifications made based on this study, the strategy of transplanting vascularized donor thymus at the time of cardiac allotransplantation may have important clinical utility as part of evolving tolerance protocols, especially in children.
Recipient thymectomy. Under the above-described general anesthesia, recipients underwent a complete thymectomy via median sternotomy 21 days before receiving an allograft (heart or thymoheart). Care was taken to ensure that the thymectomy was complete, including resection of the cervical and pericardial extensions of the thymus gland. Skin grafting. Under the above-described general anesthesia, selected recipients underwent skin grafting as a test of immunologic tolerance. Skin was harvested as a split-thickness graft using a dermatome at a thickness of 0.020 inches, and sewn to a similarly prepared dermal bed.
Immunosuppression
Materials and Methods Animals A selective breeding program has been used for the past 30 years to develop and maintain a herd of miniature swine with defined MHC loci (12). At present, three homozygous MHC haplotypes [designated swine leukocyte antigen (SLA)a , SLAc , and SLAd ] and four intra-MHC recombinant haplotypes (designated SLAg , SLAh , SLAj , and SLAk ), derived from spontaneous recombination events during the breeding of heterozygotes (13), are available for study. Swine (ages 2–11 months) were used in this study. Recipients were divided into three experimental groups: (1) euthymic recipients receiving MHC-matched, minor antigen-mismatched thymohearts (n = 3) or unmanipulated control hearts (n = 3); (2) euthymic recipients receiving MHC class I-mismatched thymohearts (n = 3) or unmanipulated control hearts (n = 7); and (3) thymectomized recipients receiving MHC class I-mismatched thymohearts (n = 4) or unmanipulated control hearts (n = 3). All recipients demonstrated significant in vitro antidonor cytotoxic activity (>20% percent specific lysis) by cell-mediated lympholysis assay (see below) before organ transplantation. All animal care and procedures were in compliance with the Principles of Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (NRC 1996).
Cyclosporine A (CyA) was generously provided by Novartis Pharmaceutical Corporation (Hanover, NJ). Cyclosporine A was given to all recipients as a daily infusion over 1 h at a dose of 10–13 mg/kg for 12 consecutive days starting on the day of transplantation. The CyA dose was adjusted to achieve blood trough levels of 400–800 ng/mL as determined by a fluorescence polarization immunoassay (Abbott Laboratories, Dallas, TX), which measures the parent compound but not metabolites. No further immunosuppression was used after postoperative day (POD) #11.
Histological and immunohistochemical analysis Cardiac biopsies and necropsy tissue were fixed in 10% formalin and evaluated using hematoxylin and eosin (H&E) stain and van Gieson’s elastin stain. The specimens were then scored under light microscopy by a blinded observer to determine the severity of acute rejection and degree of intimal proliferation. Scoring of interstitial rejection in the heart allograft was based on the International Society for Heart and Lung Transplantation classification system (16). The degree of vasculopathy was scored using a modification of the system described by Lurie et al. (14,17). The average grade of intimal thickening of all small, medium and large arterial vessels examined from each heart at necropsy was designated the mean average involvement score and recorded. Thymic biopsy specimens were stained with hematoxylin and eosin as well as immunohistochemical stains using monoclonal antibodies (mAbs) to cytokeratin, and recipient class I MHC antigens.
Surgical procedures
Cell-mediated lympholysis assay
Preparation of the thymoheart allograft. Thymohearts were prepared as previously described (11). Briefly, after the induction of general anesthesia, the donor thymus and heart were exposed using a median sternotomy. Thymic tissue was harvested and finely minced in culture medium (RPMI 1640; Gibco BRL, Carlsbad, CA). Approximately 5–10 cc of minced autologous thymus was then injected through a 14-gauge catheter into the atrial appendages, with the subepicardial fat pads overlying the right and left atrioventricular junctions, and the aortopulmonary window of the same animal. The sternum was then closed. Sixty to 90 days later, the composite thymoheart graft was excised and transplanted heterotopically into the allogeneic recipient in a manner identical to that employed for na¨ıve control hearts.
The presence of cytotoxic T lymphocytes (CTLs) in the recipient was monitored by cell-mediated lympholysis (CML) assay at frequent intervals using previously described techniques (14,15). Briefly, lymphocyte cultures containing 4 × 106 /mL responder and 4 × 106 /mL stimulator peripheral blood lymphocytes (PBLs) (irradiated with 2500 cGy) were incubated for 6 days at 37 ◦ C in 7.5% CO2 and 100% humidity in tissue culture media. Bulk cultures were harvested and effectors were tested for cytotoxic activity on lymphoblast targets labeled with 51 Cr (Amersham Corp., Arlington Heights, IL). Effector cells were incubated for 5.5 h with target cells at E/T ratios of 100 : 1, 50 : 1, 25 : 1 and 12.5 : 1. Three target cells were tested in each assay: PBLs SLA-matched to the responder (negative control), PBLs SLA-matched to the donor, and third-party PBLs. Supernatants were then harvested using the Skatron collection system (Skatron, Sterling, VA), and 51 Cr release was determined on a gamma-counter (Micromedics, Huntsville, AL). The results were expressed as percent specific lysis (PSL), calculated as:
Heterotopic heart and thymoheart transplantation. The surgical procedures used for heart and thymoheart transplantation have been described in detail previously (14,15). In brief, using the above-described anesthetic regimen, the donor graft was implanted by anastomozing the donor pulmonary artery end-to-side to the recipient inferior vena cava using continuous 6–0 polypropylene sutures (Prolene; Ethicon, Inc., Somerville, NJ) and then similarly anastomozing the donor ascending aorta end-to-side to the recipient abdominal aorta (14). Graft function was monitored daily by direct transabdominal palpation, electrocardiography (EK/5 A; Burdick Corp., Milton, WI) and/or surface echocardiography (Hewlett-Packard Sonos 1500; Andover, MA). Allograft rejection was defined by the loss of ventricular impulse on palpation, the lack of ventricular contraction on surface echocardiography, or the diminution of the R wave amplitude on ECG to less than 3 mm (0.3 mV).
80
experimental release c.p.m. − spontaneous release c.p.m./ maximum release c.p.m. − spontaneous release c.p.m. × 100%
Alloantibody detection The presence of antidonor class I antibody (IgM and IgG) in the serum of recipient swine was detected by indirect flow cytometry using PBL from SLA recombinant swine to determine the SLA-binding specificity of the antibody as described previously (14).
American Journal of Transplantation 2004; 4: 79–86
Composite Thymoheart Transplantation Statistical analysis Minimum graft survival times were analyzed using a log-rank test. A p-value 1373 >1942,3
1 Results
of these animals have been reported previously (14). grafts placed on POD #140. 3 Sacrificed electively with beating allografts. PM = postmortem. 2 Skin
American Journal of Transplantation 2004; 4: 79–86
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Menard et al. Table 2: Euthymic MHC class I-mismatched recipients Histology at week Animal number
Strain comb.
Organ
110651
gg→dd
Heart
111681
gg→dd
Heart
117491
gg→dd
Heart
117761
gg→dd
Heart
12049
jj→cc
Heart
12354
jj→cc
Heart
12539
jj→cc
Heart
12570
gg→dd
Thymoheart
12632
gg→dd
Thymoheart
13087
gg→dd
Thymoheart
2 Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial
0 1b 0 1b 0 0 0 1a
4
2 1b 2 3a 3 4 2 3b 3 4 2 3b 2 3b 0 1a
8
3 1a
12
0 1a
16
3 1a
20
0 1b
PM 3 4 3 3a 3 3b 3 4 3 4 2 4 3 4 2 4 3 3a 0 4
Survival (days) 35 45 55 47 46 46 30 64 >1912,3 63
1 Results
of these animals have been reported previously (19). grafts placed on POD #140. 3 Sacrificed electively with beating allografts. PM = postmortem. 2 Skin
Figure 1: Hemotoxylin and eosin stain of class I-disparate thymoheart #12632 on postoperative day (POD) #191 demonstrating CAV.
Figure 2: Immunohistochemical analysis of medullary thymic tissue from the thymoheart of swine #12632 postoperative day (POD) #191]. Thymic tissue is double stained for cytokeratin (light stain) and recipient class I antigens (dark stain).
recovered late from animal #12632 demonstrated epithelial cells staining for cytokeratin in close association with cells of host origin (recipient class I positive) in a medullary zone (Figure 2). This finding suggests that host-type cells were still circulating through the viable donor thymic graft late in its course. The alternative explanation that these host cells represented inflammatory cells rejecting the donor thymic
tissue is less likely as there was no evidence of necrosis or damage to the thymic architecture and no host type cells in thymic cortex (Figure 2).
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CML assays were performed using lymphocytes from the peripheral blood of the thymoheart recipient obtained preoperatively and at the time of each biopsy. Throughout American Journal of Transplantation 2004; 4: 79–86
Composite Thymoheart Transplantation
#12570 80
60
60
40
40
POD #0
20 100:1 50:1 25:1 12.5:1
Specific Percent Lysis
0 80
60
60
40
40
POD #31
20
POD #30
20 100:1 50:1 25:1 12.5:1
100:1 50:1 25:1 12.5:1
0 80
0 80
60
60
40
40
POD #64
20 0
POD #0
20 100:1 50:1 25:1 12.5:1
0 80
70
thymoheart rejection, similar to that seen in control animals (14,15). In contrast, no alloantibody was ever detected from the long-term thymoheart survivor (#12632) (Figure 4).
#12632
80
100:1 50:1 25:1 12.5:1
POD #64
20 0
100:1 50:1 25:1 12.5:1
80 60 40
POD #159
20
100:1 50:1 25:1 12.5:1
0
Skin Grafts
80
Donor Third Party
60 40
POD #177
20
100:1 50:1 25:1 12.5:1
0
Effector:Target Ratio Figure 3: CML assay of antidonor reactivity in euthymic class I-disparate thymoheart recipients. Swine #12570 demonstrates antidonor CML reactivity on par with third-party controls throughout duration of experiment. Swine #12632, whose graft exhibited long-term survival, demonstrated donor-specific CML unresponsiveness until the time of skin-grafting on postoperative day (POD) #159.
the postoperative period, CTLs from swines #13087 and #12570 (Figure 3) demonstrated specific lysis for donortype cells on par with the cytolysis of third-party controls. In contrast, the CML assays performed for animal #12632 demonstrated donor-specific hyporesponsiveness throughout the entire postoperative period until skin grafts were placed on POD #159. Peripheral blood lymphocytes obtained from this animal 2 days after the rejection of the donor-matched skin demonstrated donor-specific lysis identical to that of third-party controls (Figure 3) IgM and IgG alloantibodies were both present in the sera of swines #12570 (Figure 4) and #13087 at the time of #12570 IgM
#12632 IgG
IgM
At autopsy, three of the thymohearts (#13092, #13154, and #12291) showed some scarring of the thymic implants; however, there were remaining areas of immature thymocytes on H&E staining. Swine #12991 also had moderate preservation of thymic epithelial cells when stained immunohistochemically with cytokeratin. The thymic graft of the remaining thymectomized recipient (#13027) demonstrated excellent viability with normal thymic architecture, including intact Hassal’s corpuscles. Immunohistochemical staining of this tissue revealed good preservation of cytokeratin-positive thymic stromal cells (data not shown).
IgG POD #0
POD #0 POD #30 POD #21 POD #78 POD #31 POD #159 POD #64 POD #191
Figure 4: Flow cytometric analysis for IgM and IgG antidonor alloantibody in the sera of class I-disparate thymoheart recipients. IgM and IgG are detectable by postoperative day (POD) #64 in swine #12570. No IgM or IgG alloantibody is detected in long-term survivor #12632.
American Journal of Transplantation 2004; 4: 79–86
Thymoheart transplantation in thymectomized MHC class I mismatched recipients The inability of thymic grafts to induce tolerance to hearts in euthymic recipients could have resulted from alloreactive T cells generated in host thymus causing partial or full rejection of the thymic graft (10). To test this hypothesis, total thymectomies were performed 3 weeks before thymoheart transplantation. We have previously shown that transplantation of an unmanipulated, class I disparate heart into a thymectomized recipient treated with CyA results in accelerated cardiac allograft rejection as compared with identical transplants in euthymic recipients (Table 3; 5). When thymohearts were transplanted into a thymectomized miniature swine treated with a 12-day course of CyA, significant prolongation of thymoheart survival was observed relative to isolated hearts transplanted into CyAtreated thymectomized recipients (41–70 days vs. 8–27 days, p = 0.01) (Table 3). However, long-term survival was not achieved. Most of the thymohearts transplanted into thymectomized recipients developed severe rejection (grades 3–4) by POD #30, a finding that persisted until the time of autopsy. The presence of CAV at autopsy was variable and did not appear to correlate with either graft survival or the severity of interstitial rejection.
CML assays were performed using lymphocytes obtained from the thymectomized thymoheart recipient preoperatively and at the time of each biopsy. All four recipients in this group demonstrated responsiveness on par with MHC-matched controls preoperatively, but then developed donor-specific unresponsiveness by POD #36. Recipients #12991 and #13092 regained donor-specific responsiveness at autopsy (after rejection) while #13154 remained hyporesponsive to donor type just before allograft rejection (Figure 5). IgM and IgG alloantibody were found in swine #13092, while only IgG was found in swine #12991. No alloantibody was found in either swine #13027 or swine #13154 (data not shown). 83
Menard et al. Table 3: Thymectomized MHC class I-mismatched recipients Histology at week Animal number
Strain comb.
Organ
12208
gg→dd
Heart
12398
gg→dd
Heart
12627
gg→dd
Heart
12991
gg→dd
Thymoheart
13092
gg→dd
Thymoheart
13027
gg→dd
Thymoheart
13154
gg→dd
2 Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial Vascular Interstitial
Thymoheart
4
8
PM
0 4
3 3a 0 4 2 3a 0 4 3 4 3 3a 0 4
3 3a 0 4 2 3a 1 3b 3 4 1
1b 0 3a
Survival (days) 27 8 22 48 41 >542 70
1 Insufficient
vascular tissue to characterize. was removed before it rejected owing to poor animal health. Although scattered foci of grade 3a rejection were present, the predominate histologic finding was grade 1b interstitial rejection. PM = postmortem.
2 Graft
Specific
#13092
#13154
80
80
60
60
40
40
20 100:1 50:1 25:1 12.5:1
0 80
POD #0
Percent Lysis
60
20 100:1 50:1 25:1 12.5:1
0 80
POD #0
60
40
40
20 100:1 50:1 25:1 12.5:1
POD #29
20 100:1 50:1 25:1 12.5:1
0 80
0 80
60
60
40
POD #29
40
20 0
Donor Third Party
100:1 50:1 25:1 12.5:1
POD #41
20 0
POD #64
100:1 50:1 25:1 12.5:1
Effector:Target Ratio
Figure 5: CML assays of antidonor reactivity in thymectomized class I-disparate thymoheart recipients (#13092 and #13154) demonstrating donor-specific hyporesponsiveness until time of graft rejection.
Discussion We have developed a novel strategy, which allows the transfer of donor thymic tissue to the host at the time of cardiac transplantation. Using the composite thymoheart technique, we have demonstrated that fully vascularized and functional donor thymus cotransplanted with a donor heart can prolong cardiac allograft survival and diminish the development of CAV in large animals. However, thymoheart allografts transplanted under a short course of CyA did not induce a state of donor-specific tolerance, as evidenced by the lack of sustained donor-specific unresponsiveness in in vitro assays and the failure to fully prevent CAV. Further information on the immune status of porcine recipients of heart transplants can be gleaned by comparing the results of donor skin grafting. We have found previously 84
that donor heart plus kidney cotransplantation induces a robust tolerance and that the delayed rejection of a donor skin grafts in long-term heart/kidney recipients has no effect on the organ allografts (15). However, donor skin grafting of long-term double heart recipients leads to the acute rejection of the donor skin and the rejection of both cardiac allografts (19). This suggests that heart/kidney transplantation induces a robust tolerance whereas double heart transplantation prolongs graft survival without achieving a tolerant state. The ISHLT grade 3a rejection that developed in the long term MHC-matched thymoheart recipient (#13184) after it had rejected a donor skin graft (Table 1) supports our conclusion that this particular strategy of thymoheart transplantation could not induce a state of tolerance. In contrast, other investigators in this laboratory have recently prepared a composite organ, known as the ‘thymokidney’ (20), which was capable of inducing tolerance to a renal allografts (10). Why thymokidneys but not thymohearts were able to induce tolerance in a miniature swine is unclear. One explanation for this disparity may relate to the known difference in the ease with which tolerance is induced to a kidney vs. a cardiac allograft. It is clear that a cardiac allograft poses a greater barrier to tolerance induction in a miniature swine than a kidney allograft transplanted across the same histoincompatibility (15). Alternatively, this difference could be explained by the lesser load of thymic tissue conferred to the host by the thymoheart vs. the thymokidney allograft. It would not be surprising if the induction of tolerance (and abrogation of CAV) in this model requires the successful transplantation of a critical mass of vascularized donor thymus. This could explain why recipient 13027 (Table 3) developed foci of grade 3a rejection despite the presence of a viable donor thymus. To address this issue, LaMattina and colleagues (21) American Journal of Transplantation 2004; 4: 79–86
Composite Thymoheart Transplantation
have developed a microsurgical technique for vascularized thymic lobe transplantation in a miniature swine. The contransplantation of a vascularized thymic lobe and unmanipulated heart allograft will greatly augment the amount of donor thymus conferred to the host. Finally, it is possible that mature alloreactive T cells circulating in the recipient at the time of transplantation may contribute to graft loss. We are currently investigating the effects of T-cell depletion on the induction of tolerance following thymoheart transplantation (22). The mechanism by which thymic grafting prolongs the survival of cardiac allografts is unclear. We have shown previously that a DST given on POD 0 along with the same 12-day course of CyA does not prolong cardiac allograft survival (23). However, cotransplanting a donor-specific kidney (15) or a second donor-matched heart (19) does prolong cardiac allograft survival in this model. These data suggest that specialized cells unique to the kidney allograft (24) or the augmentation of donor-antigen load (19) can contribute to the prolongation of cardiac allograft survival in a miniature swine. It is possible that the mechanisms of graft prolongation following thymoheart transplantation are similar to the mechanisms underlying graft prolongation induced by a cotransplanted donor kidney or heart. However, based on ongoing studies in a model of thymokidney transplantation, we believe that the mechanisms are instead related directly to the function of the donor thymus (10,21). For instance, other studies have demonstrated that thymic epithelial cells can take part in both positive and negative selection of host progenitor thymocytes (10,25), or can induce anergy (26). Indeed, the presence of recipienttype thymocytes within the medulla but not cortex of a viable donor thymus in long-term thymohearts allografts (Figure 2) suggests that functional thymopoiesis was occurring in the vascularized donor thymic grafts as opposed to simply infiltration of the graft by alloaggressive cells (i.e. rejection). Alternatively, it is possible that thymic emigrants from the thymic grafts developed regulatory properties, which were able to suppress the rejection response (10). It is recognized that CD4+ CD25+ regulatory T cells are generated primarily in the thymus, with CD25 appearing during the transition of CD4+ CD8+ double-positive cells to singlepositive CD4+ cells (27). Clearly, thymoheart transplantation is not applicable to cadaveric human cardiac allotransplantation given the time needed to create the composite organ. However, our findings represent an important proof-of-principal for incorporating vascularized donor thymus transplantation in new and evolving tolerance strategies related to heart transplantation. To that end, we have recently developed a new technique to transplant a vascularized thymic lobe en bloc with a cardiac allograft (28). This technique makes the transfer of large amounts of vascularized donor thymus feasible in human recipients of cadaveric donor hearts with minimal modification of current operative and immunosuppressive regimens. Experiments to determine the effects American Journal of Transplantation 2004; 4: 79–86
of en bloc heart and thymus cotransplantation, with host thymectomy and T-cell depletion will be critical in advancing this tolerance strategy. In summary, our results indicate that transplantation of vascularized and functional donor thymic tissue at the time of heart transplantation can prolong allograft survival in a clinically relevant large-animal model of heart transplantation. Attempts to optimize this strategy and induce full tolerance are in progress. Techniques that allow the transfer of a whole vascularized thymic lobes en-bloc with cadaveric donor hearts are now available for application to human transplantation. Due to the process of thymic involution (29), we believe that donor thymus cotransplantation will be most effective in pediatric and adolescent heart transplant recipients, the group of patients that would benefit most from a protocol that induces stable tolerance (30). Future application could include pig-to-human xenotransplantation.
Acknowledgments This work was supported in part by grants from the National Heart, Lung, and Blood Institute (PO1 HL18646, RO1 HL54211, F32 HL69645) and the National Institute of Allergy and Infectious Disease (PO1 AI50157) of the National Institutes of Health. Dr Menard is an Edward D. Churchill Surgical Research Fellow, Massachusetts General Hospital, and is a recipient of the Roche Surgical Scientist Award from the American Society of Transplantation. Dr Schwarze is a Claude E. Welch Surgical Research Fellow, Massachusetts General Hospital, and is a recipient of the Research Fellowship Award from the International Society of Heart and Lung Transplantation. Dr Johnston is an Edward D. Churchill Surgical Research Fellow, Massachusetts General Hospital, and is a recipient of the American College of Surgeons Resident Research Scholarship. The authors are indebted to Mr J. Scott Arn for herd management and Drs Christene Huang and Robert Cina for critical review of the manuscript. The authors also acknowledge the generosity of the Novartis Pharmaceutical Corporation, which kindly provided cyclosporine.
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