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Optimizing chimerism level through bone marrow transplantation and irradiation to induce long-term tolerance to composite tissue allotransplantation
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Optimizing chimerism level through bone marrow transplantation and irradiation to induce long-term tolerance to composite tissue allotransplantation Jeng-Yee Lin, MD,a,b Feng-Chou Tsai, MD, PhD,b Christopher Glenn Wallace, MBChB, MS,c Wei-Chao Huang, MD, PhD,a,d Fu-Chan Wei, MD,a,c and Shuen-Kuei Liao, PhDa,e,* a
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan Division of Plastic Surgery, Department of Surgery, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan c Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan d Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chiyi, Taiwan e Cancer Immunotherapy Program, Cancer Center, Taipei Medical University Hospital and Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan b
article info
abstract
Article history:
Background: Mixed chimerism with long-term composite tissue allotransplant (CTA)
Received 29 November 2011
acceptance can be achieved through allogeneic bone marrow transplantation (BMT). The
Received in revised form
present study investigated the optimal chimerism level by giving different irradiation
28 February 2012
dosages to recipients to induce tolerance to CTA.
Accepted 29 February 2012
Methods: Chimera were prepared using Brown-Norway and Lewis rats with strong major
Available online 17 March 2012
histocompatibility complex incompatibility. The Lewis rats received 5 mg antilymphocyte
Keywords:
groups 1, 2, 3, 4, and 5 according to the day 1 irradiation dosage: 0, 200, 400, 600, and 950 cGy,
Graft-versus-host disease
respectively. The Lewis rats were then reconstituted with 100 106 T-celledepleted Brown-
Bone marrow transplantation
Norway bone marrow cells (day 0) and received vascularized Brown-Norway-CTA on day 28.
T-cell depletion
Chimerism was assessed monthly by flow cytometry starting on day 28 after BMT. Graft-
Composite tissue allotransplant
versus-host disease (GVHD) was assessed clinically and histologically.
globulin (day 1 and 10) and 16 mg/kg cyclosporine (day 0e10) and were separated into
Results: Chimerism, 4 weeks after BMT, averaged 0.2%, 9.2%, 30.7%, 58%, and 99.3% in groups 1 to 5, respectively. GVHD occurred as follows: groups 1 and 2, none; group 3, 1 case of GVHD; group 4, 7 cases of GVHD (of which 3 died); and group 5, 10 cases of GVHD (of which 6 died). The percentage of long-term CTA acceptance was 0%, 0%, 90%, 70%, and 40% in groups 1 to 5, respectively. The percentage of regulatory T cells was significantly lower in high-chimerism (20%, n ¼ 15) than in low-chimerism (<20%, n ¼ 5) rats that accepted CTA long-term . Conclusions: The chimerism level correlated positively with GVHD occurrence and longterm CTA acceptance but correlated negatively with regulatory T-cell levels. Optimal chimerism for CTA acceptance through pre-CTA BMT and irradiation occurs at 20e50% at day 28 after BMT in the rat model. ª 2012 Elsevier Inc. All rights reserved.
* Corresponding author. Cancer Center, Taipei Medical University Hospital, 252 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: þ886 2 2739 8278. E-mail address: [email protected] (S.-K. Liao). 0022-4804/$ e see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.064
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1.
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Introduction
Recent clinical application of composite tissue allotransplantation (CTA) has ushered in a new era for the restoration of massive tissue loss, including nerves [1], abdominal walls [2], tendons [3], bone and joints [4], larynx [5], hands [6e8], scalp and ear [9], and face [10]. However, the balance between the risks and benefits of reconstructive CTA remains controversial, especially regarding the problems of lifelong immunosuppressive therapy. First, current immunosuppression protocols do not completely protect transplants from acute rejection episodes. Second, long-term transplant function is still compromised by chronic rejection that is not responsive to current immunosuppressant agents [11,12]. Third, nonspecific lifelong immunosuppressive therapy is associated with the development of opportunistic infection, drug toxicity, and malignancy [13,14]. One approach that might overcome many of the disadvantages of immunosuppression is transplantation tolerance induction. Current research has focused on 3 main strategies to induce donor-specific tolerance in the recipient, including genetic matching, co-stimulatory blockade [15,16], and mixed chimerism [17,18] (usually by creating mixed allogeneic chimerism through bone marrow transplantation [BMT]). Chimerism refers to a state in which 2 genetically different hematopoietic systems coexist harmoniously in 1 organism. Mixed allogeneic chimerism has been shown to prevent chronic rejection in transplanted grafts while maintaining immunocompetence in the recipients [19,20]. Clinical application of mixed chimerism to induce tolerance was further encouraged by a pilot series of successful immunosuppression-free renal transplantations that were co-transplanted with donor bone marrow [21]. Although the level of chimerism that is needed to induce tolerance remains a topic of discussion, our previous work has demonstrated that the level of chimerism in the initial stage correlated with CTA acceptance in the cyclosporine-based vascularized allogeneic BMT (allo-BMT) rat model [22,23]. Moreover, a greater percentage of donor T lymphocytes circulating in recipients was associated with a greater percentages of graft-versus-host disease (GVHD) [22]. Regulatory T (Treg) cells play an important role in regulating the immune responses to self- and allogeneic antigens. The recent finding that Treg cells infiltrate the donor skin of long-term CTA in the mixed chimeric recipient implies a role in maintaining allograft survival [24]. Introducing Treg cells might, therefore, assist in tailoring immunosuppressive management as a part of the preconditioning regimen when creating mixed chimerism [25]. From these findings and inferences, our study aimed to investigate whether mixed allogeneic chimerism is related to GVHD occurrence, long-term allograft acceptance rate, and Treg cell level in the conventional allo-BMT rat model using differing radiation doses.
2.
Materials and methods
2.1.
Rats
Five- to eight-week-old (weight 200e350 g) male BrownNorway (RT1Ac) and Lewis (RT1Al) rats were used as donors and recipients for CTA. The rats were purchased from the
National Laboratory Animal Center (Taipei, Taiwan) and housed in a pathogen-free facility. The rats were cared for according to the Institutional Animal Care and Use Committee guidelines at the Laboratory Animal Care-approved Research and Resource Center of Chang Gung Memorial Hospital and University.
2.2.
Treatment groups
The Lewis recipients groups 1 to 5 (n ¼ 10, each group) received 0, 200, 400, 600, and 950 cGy of radiation, respectively (liner accelerator, 21EX, 6-MV X-ray; Varian Medical Systems, Palo Alto, CA). The myeloablative dose of total body irradiation in the rat is 950 cGy (group 5). The immunosuppression regimen included 5 mg of goat anti-rat lymphocyte serum (Accurate Chemical, Westbury, NY) by intraperitoneal injection 1 d before and 10 d after BMT. Cyclosporine (Sandimmune; Novartis Taiwan, Taipei, Taiwan) was administered at 16 mg/kg/d intramuscularly from days 0 to 10 after BMT.
2.3.
Bone marrow preparation
The bone marrow was harvested from the femurs, tibias, and humeri of Brown-Norway rats by flushing with Media 199 (Life Technologies, Rockville, MD) containing 10 mg/mL gentamycin using a 22-gauge needle. The cells were resuspended using an 18-gauge needle and filtered through sterile nylon mesh to produce a single cell suspension. The bone marrow cells were washed, centrifuged (500 g for 10 min at 4 C), resuspended, and counted before T-cell depletion (TCD).
2.4. Immunomagnetic bead depletion of T lymphocytes and BMT TCD before BMT was performed to reduce the incidence of GVHD. The bone marrow cells were incubated with purified anti-ab and anti-gd T-cell receptor (TCR) monoclonal antibodies (R73; mouse IgG1; Pharmingen, San Diego, CA) for 30 min at 4 C in the dark. Secondary antibody with coated beads (Dynabeads M-450; goat anti-mouse IgG; Dynal, Lake Success, NY) were then added to bind the primary monoclonal antibodies and incubated for 60 min at 4 C in the dark at a bead/T-cell ratio of 6:1. To negatively select T cells, tubes with bone marrow cell suspensions were placed in a magnetic cell separator (BioMag Separator; Advanced Magnetics, Cambridge, MA) for 5 min. The unbound cells were aspirated, and cell separation was repeated. T-lymphocyteedepleted bone marrow cells were washed, counted, and re-suspended in Dulbecco’s modified Eagle medium solution to achieve a concentration of 100 106 bone marrow cells/mL. The adequacy of TCD was confirmed by flow cytometry. The recipients were reconstituted within 24 h after total body irradiation with 100 106 T-cell-depleted bone marrow cells from Brown-Norway rats diluted in 1 mL of Dulbecco’s modified Eagle medium by way of the penile vein.
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2.5.
Composite tissue allotransplantation
At 28 d after BMT, hindlimb osteomyocutaneous flap CTAs from Brown-Norway donors were performed, as previously described [26]. Inhalational isoflurane (Halocarbon, Products Corporation, River Edge, NJ) provided continuous anesthesia for both donors and recipients. In brief, a longitudinal incision was made from thigh to ankle on the medial leg. A circumferential skin incision was made at the mid-thigh and the ankle joint to isolate a 3 5-cm skin paddle on the lateral leg. The femoral vessels were mobilized up to the inguinal ligament. A complete osteotomy was performed on the femur and tibia to harvest an osteomyocutaneous flap based solely on the femoral vessels. Bone marrow cells were aggressively flushed out of the femur and tibia cut ends before transplantation. The harvested flap was wrapped with wet gauze and placed on iced saline. The procedure on the recipient chimeric rat (Brown-Norway/Lewis) began with a transverse incision in the inguinal region to expose and mobilize the femoral artery and vein before they were cut and clamped for microanastomoses (10-0 nylon, using a standard microsurgical technique) with the donor femoral vessels. All rats were housed individually in filter-top cages and fed autoclaved rat chow to minimize the risk of opportunistic infection.
2.6. Characterization of chimerism and donor cell lineage by flow cytometry Engraftment of the allogeneic bone marrow was assessed 2 weeks after BMT with flow cytometry. Flow cytometry was also used monthly after BMT to determine the chimerism level, defined as the percentage of peripheral blood lymphocytes bearing donor (Brown-Norway) major histocompatibility complex class I antigen. In brief, whole blood aliquots of 100 mL were stained with antieBrown-Norway (RT1Ac-FITC [OX27, mouse IgG2a; Serotec, Raleigh, NC]), B-cell (CD45RA-PE [OX33]), mouse IgG1 (BD Pharmingen, San Diego, CA), T-cell (TCR-PerCP [R73], mouse IgG1; BD Pharmingen), natural killer cell (antiNKR-P1A-APC [10/78], mouse IgG1; Caltag Laboratories, Bangkok, Thailand), helper T-cell (CD4-APC [W3/25], mouse IgG1; BD Pharmigen), cytotoxic T cell (CD8a-PerCP:OX-8; mouse IgG1; Pharmingen), and macrophage-granulocyte (MAC1FITC:OX42; mouse IgG2a; Serotec, Baveria, Germany) lineagespecific fluorescein isothiocyanate-labeled monoclonal antibodies for 30 min. Flow cytometry was performed using a FASCort flow cytometer (Becton Dickinson, Franklin, NJ).
2.7.
Assessment of limb rejection
The transplanted flaps were examined daily for any signs of rejection, including edema, desquamation, epidermolysis, hair loss, exudation, and flap necrosis or shrinkage. Routine skin biopsies were performed monthly after flap transplantation. Standard histopathologic features were used to assess the degree of acute allograft rejection in skin biopsies using the following histopathologic grading classification [27]: grade 0, normal epidermal appearance without evidence of rejection; grade 1, focal basal cell layer vacuolization; grade 2, dyskeratosis of squamous cells in the epidermis or hair follicle epithelium; grade 3, subepidermal clefting or microvesiculation; and
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grade 4, complete separation at the epidermaledermal junction. Composite tissue allograft survival acceptance was defined as complete survival and growth of hair in the allotransplant without any rejection signs at the endpoint (150 d).
2.8.
Secondary skin grafting
To confirm whether donor-specific transplantation tolerance existed in CTA-accepting recipients, donor, and third-party skin grafts were performed in all CTA-accepting rats. Full-thickness skin grafts were harvested from the abdominal wall of the donor (Brown-Norway) and third party (ACI) rats. The grafts were sutured to the dorsal neck in anesthetized recipients with a 3-mm skin bridge between them and secured with a tie-over bolster dressing. The bolster was removed on the seventh day, and the grafts were inspected daily for signs of rejection.
2.9.
Assessment of GVHD
The rats were weighed at BMT and at varying intervals thereafter. The diagnosis of GVHD was determined from clinical signs, including weight loss, unkempt appearance, diarrhea, diffuse erythema, hyperkeratosis of foot pads, and/ or dermatitis [28,29]. GVHD was graded clinically as none, mild, moderate, or severe [30]. At necropsy, an ear wedge biopsy was performed to examine for lymphoid infiltration, subepidermal cleft formation, and/or epidermal necrosis.
2.10.
Treg cells identification
The cell population marker of Treg cells (CD4þCD25þFoxP3þ) in the peripheral lymphoid gate of recipients was confirmed with flow cytometry and intracytoplasmic staining.
2.11.
Statistical Analysis
Statistical analyses were performed using Fisher’s exact test.
3.
Results
3.1.
Efficacy of TCD
Predepletion donor bone marrow contained a mean of 1.63% ab-TCRepositive and 0.87% gd-TCRepositive T cells. After bead depletion, the ab-TCRepositive cell count was reduced to an average of 0.05% and the gd-TCRepositive cell count to 0.03% of the lymphoid gate (Fig. 1A). This demonstrated adequate depletion of donor T lymphocytes before BMT.
3.2. Mixed chimerism and donor multilineage hematopoietic cells The chimerism level refers to the percentage of donor cells in the peripheral blood leukocytes of the recipients. Multilineage chimerism of both lymphoid and myeloid lineages, including donor CD4þ cells, CD8þ cells, B cells, natural killer cells, and dendritic cells, was present in all groups except group 1, suggesting that engraftment of pluripotent hematopoietic
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Fig. 1 e (A) Most T cells express ab-TCR and 5% of T cells express gd-TCR on their cell surfaces. Immunomagnetic beads for binding both ab- and gd-T cell receptors were used to remove the T cell repertoire effectively. Histograms showed pre- and post-T cell-depletion results of donor bone marrow. Reductions of ab TCR T cells from 1.63% ± 0.03% to 0.05% ± 0.03% and gd TCR T-cells from 0.87% ± 0.1% to 0.03% ± 0.01% were routinely achieved. (B) Mutlilineage analysis of the recipient peripheral whole blood 2 weeks after BMT (group 3) showed evidence of donor bone marrow engraftment, as determined by dual expression of donor-specific (RT1Ac) and lineage-specific markers in flow cytometry. Engraftment of at least 6 different lineages of donor cells shown (CD4D [ CD4D T cells; CD8D [ CD8D T cells; B [ B cells; DC [ dendritic cells; Mac/Gran [ macrophages/granulocytes; NK [ natural killer cells).
stem cells from the donor was successful in all irradiated recipients from groups 2 to 5 (Fig. 1B). The average chimerism level in each group was as follows: group 1 (no irradiation), 0.2%; group 2 (radiation dose 200 cGy), 9.2%; group 3 (radiation dose 400 cGy), 30.7%; group 4 (radiation dose 600 cGy), 58%; and group 5 (radiation dose 950 cGy), 99.3%. The radiation dosage in group 5 caused complete myeloablation. Thus, full chimerism rather than mixed chimerism was observed in group 5. Flow cytometry 28 d after BMT demonstrated that
groups with a greater radiation dosage had a greater donor chimerism level in the engrafting recipient.
3.3. Occurrence of GVHD and survival of hindlimb transplantation The percentage of GVHD reflected the radiation dosage. The rats in groups 1 and 2 developed no signs of GVHD. One rat in group 3 had minor signs of acute GVHD from 7 d after BMT that
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continued until day 45. Also, 3 rats from group 4 died of acute GVHD with continued weight loss, ear and claw erythema, and severe hair loss, and 3 rats in group 4 developed moderate signs of GVHD with ear and paw erythema, edema, failure to increase body weight, and unkempt hair that continued through to the endpoint of the study (day 150). Another rat in group 4 developed minor signs of GVHD that persisted through the endpoint of the study. Group 5 had 6 rats that died of GVHD. Another 4 rats had moderate signs of GVHD that continued to the endpoint of the study. In the groups with greater total body irradiation, a greater percentage of acute GVHD was observed (Fig. 2). In groups 1 (nonirradiated recipients) and 2 (200 cGy radiation), none of the CTAs survived beyond 29 and 45 d, respectively, after CTA. In group 3 (400 cGy radiation), 90% (9 of 10) of the rats accepted the CTA until the endpoint of study (day 150). The CTA acceptance rate for group 4 and 5 was 70% (7 of 10) and 40% (4 of 10), respectively.
3.4.
Secondary skin grafting
Mixed allogeneic chimeras (Brown-Norway/Lewis) permanently accepted donor (Brown-Norway) but rejected (mean survival 14 d) third-party (ACI) skin grafts.
3.5. Correlation of mixed chimerism level to GVHD, CTA acceptance, and Treg level To determine the relationship between chimerism level with GVHD and CTA acceptance, all chimera rats were classified according to their level of chimerism into groups of 10%
Fig. 2 e Chimerism level, GVHD rate, and CTA acceptance rate in each radiation group. Diagram shows that chimerism level, GVHD, and CTA rate correlated positively with total body radiation dosage. Note, however, that CTA acceptance rates deceased with greater radiation, groups 4 (600 cGy) and 5 (950 cGy), owing to GVHD-related deaths of recipient chimera. This showed that group 3 (400 cGy) had the greatest CTA acceptance rate with an acceptable GVHD rate. (Inset A) Long-term CTA acceptance in recipient chimera rats. (Inset B) Histologic findings of biopsy taken from accepted CTA shows allograft skin free of lymphocyte infiltration. (Color version of figure is available online.)
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increments. The results showed that the optimal chimerism level was about 20e50%, with this range producing a 100% CTA acceptance rate and no GVHD (Fig. 3). The percentage of Treg cells (CD4þ CD25þ) in the CD4þ cell population was significantly lower in the CTA acceptance recipients with greater chimerism (20%; n ¼ 15) at the endpoint of this study (day 150) compared with the CTA acceptance recipients with lower chimerism (<20%; n ¼ 5; P < 0.01; Fig. 4).
4.
Discussion
CTA has provided a solution to massive tissue loss such as major limb amputations and extensive face or tissue injuries. The long-term functional results of CTA might not be completely maintained because of chronic rejection occurring despite control of acute rejection episodes. Donor-specific tolerance that results from mixed chimerism has shown some promise in the search to solve this problem in CTA. The concept of mixed allogeneic chimerism has gained increasing popularity since the 1950s when Billingham et al. [31] first successfully performed skin grafting between dizygotic cattle. Durable mixed allogeneic chimerism through allo-BMT has been shown to induce tolerance to allogeneic transplantation of several tissues and organs in animal and human recipients. However, 2 facts could explain why it is considered more difficult to induce tolerance for CTA compared with solid organ transplantation. First, the skin tissue is thought to be more immunogenic than other tissues. Second, the engrafted lymphoid tissue (bone or lymph nodes) engrafted provides a constant source of donor antigens. Efforts using a variety of aforementioned methods independently have obtained good results to date. Our study, which combined the available methods and adjusted the radiation dosage to increase CTA success through the conventional allo-BMT model, revealed
Fig. 3 e Relationship of chimerism level to GVHD and CTA acceptance rates. Chimerism level correlated positively with GVHD and CTA acceptance rates. Note, CTA acceptance rate decreased in higher chimerism groups owing to GVHD-related deaths of chimera after BMT, suggesting that optimal chimerism level was about 20e50% with 100% CTA acceptance and 0% GVHD. (Color version of figure is available online.)
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Fig. 4 e Comparison of percentage of Treg (CD4DCD25 DFoxP3D) cells/CD4D cells in high-chimerism (‡20%, n [ 15) and low-chimerism (<20%, n [ 5) chimera recipients with long-term CTA acceptance. This histogram showed that the percentage of Treg cells/CD4D cells was significantly greater in rats with low chimerism that accepted CTAs (63.9% versus 31.3%, *P < 0.001).
(20%) group (n ¼ 15) that accepted allotransplants was observed to have a lower Treg cell percentage in the CD4þ cell population than the low-chimerism (<20%) group (n ¼ 5) that accepted allotransplants (31.3% versus 63.9%, P < 0.001). One explanation was that the acceptance model with a high chimerism level ensured the continuous contribution of donor and host cells to antigen-presenting cells that induced intrathymic deletion. In contrast, although in acceptance models in which only low levels of donor chimerism persist, Treg cells might have a significant role because the deletion of donor-reactive T cells appears to be less complete. The emergence of donor-reactive T cells from the thymus might then permit the expansion of regulatory mechanisms. However, this finding also implied augmentation of Treg cells might help tolerance induction in low levels of mixed chimerism. However, more studies are needed to investigate the mechanism that Treg cells play in low-level mixed chimerism. Although the preconditioning regimens are not yet clinically practical owing to possible host toxicity, graft failure, GVHD, and a waiting period for chimerism to develop, ongoing active research is bringing tolerance induction closer to clinical reality.
5. the relationship among GVHD, acceptance rate, Treg cell level, and the optimal radiation dose. It has been reported that TCD using anti-ab plus anti-gd TCR monodispersed antibodies and immunomagnetic beads effectively removed T cells from the donor bone marrow and effectively prevented GVHD in major histocompatibility complex-mismatched mice [32,33] but only partially prevented GVHD in rats [34,35]. That study confirmed that although adequate TCD before BMT was performed, an unacceptably high GVHD rate was observed in the chimeric recipient rats with a chimerism level of more than 50% at 28 d after BMT. Obviously, more sophisticated immune systems play a role in the rat model in which TCD could not prevent GVHD completely after allo-BMT. In our study, the rats with a mixed chimerism level of more than 20% at 28 d after BMT maintained a long-term CTA, suggesting that a threshold donor contribution to the hematopoietic stem cell pool might be required to ensure the constant presence of donor-derived thymic dendritic cells to contribute to negative selection. Therefore, it seems that a harmonious balance between GVHD and allotransplant acceptance can be achieved through titration of chimerism to its optimal level. Thus, to keep the chimerism level as low as possible to avoid GVHD but still greater than the threshold level to ensure allotransplant acceptance. Our chimeric rat model suggested that an optimal chimerism level of about 20e50% at 28 d after BMT resulted in a low GVHD rate and high CTA acceptance. A considerable body of data implicating CD25þCD4þ Treg cells in the maintenance of peripheral tolerance to organ-specific self-antigens has been described [36e39]. Although all published studies on mixed chimerism suggested only a limited role for Treg cells in tolerance induction [40,41], they did not preclude a role for suppression of donor reactive cells in protocols involving BMT in which only low levels of chimerism were established. In our study, the high-chimerism
Conclusions
Our experiments have offered a suitable rat model with durable mixed chimerism through BMT for studying CTA. The data showed that the chimerism level positively correlated with GVHD occurrence and long-term allograft acceptance. Titration of mixed chimerism to its optimal level (20e50%) at day 28 through pre-CTA BMT, and irradiation, therefore, provides an approach to reducing GVHD and increasing rejection-free CTA survival in the rat model.
Acknowledgments The present report was supported by grant 100-TMU-TMUH11 from the: Taipei Medical University Hospital.
references
[1] Bain JR. Peripheral nerve and neuromuscular allotransplantation: Current status. Microsurgery 2000;20: 384e8. [2] Levi DM, Tzakis AG, Kato T, et al. Transplantation of the abdominal wall. Lancet 2003;361:2173e6. [3] Guimberteau JC, Baudet J, Panconi B, et al. Human allotransplant of a digital flexion system vascularized on the ulnar pedicle: A preliminary report and 1-year follow-up of two cases. Plast Reconstr Surg 1992;89:1135e47. [4] Hofmann GO, Kirschner MH, Brauns L, et al. Vascularized knee joint transplantation in man: A report on the first cases. Transpl Int 1998;11(Suppl):S487e90. [5] Strome M, Stein J, Escalmado R, et al. Laryngeal transplantation and 40-month follow-up. N Engl J Med 2001; 344:1676e9. [6] Dubernard JM, Owen E, Herzberg G, et al. Human hand allograft: Report on first 6 months. Lancet 1999;353:1315e20.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) 4 8 7 e4 9 3
[7] Jones JW, Gruber SA, Barker JH, et al. Successful hand transplantation: One-year follow-up. Louisveille Hand Transplant Team. N Engl J Med 2000;343:468e73. [8] Cooney WP, Hentz VR. Successful hand transplantation: One year follow-up. N Engl J Med 2001;344:65. [9] Jiang HQ, Wang Y, Hu XB, et al. Composite tissue allograft transplantation of cephalocervical skin flap and two ears. Plast Reconstr Surg 2005;115:31ee5e. [10] Dubernard JM, Devauchelle B. Face transplantation. Lancet 2008;372:603e4. [11] Colson YL, Zadach K, Nalesnik M, et al. Mixed allogeneic chimerism in the rat: Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts. Transplantation 1995;60: 971e80. [12] Orloff MS, DeMara EM, Coppage ML, et al. Prevention of chronic rejection and graft arteriosclerosis by tolerance induction. Transplantation 1995;59:282e8. [13] Penn I. Depressed immunity and the development of cancer. Clin Exp Immunol 1981;46:459e74. [14] Bach FH, Sachs DH. Current concepts: Immunology, transplantation immunology. N Engl J Med 1987;317:489e92. [15] Zhu ZX, Fan LY, Wang Q. Simultaneous blockade of costimulatory signals CD28-CD80 and CD40-CD154 combined with monoclonal antibody against CD25 induced a stable chimerism and tolerance without graft-versus-host disease in rat. Eur Surg Res 2011;46:109e17. [16] Foster RD, Pham S, Li S, et al. Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade. Transplantation 2003;76:988e94. [17] Foster RD, Fan L, Neipp M, et al. Donor-specific tolerance induction in composite tissue allografts. Am J Surg 1998;176: 418e21. erratum in Am J Surg 1999;177:529. [18] Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med 1989;169:493e502. [19] Blom D, Morrissey NJ, Mesonero C, et al. Induction of specific tolerance through mixed hematopoietic chimerism prevents chronic renal allograft rejection in a rat model. Surgery 1996; 120:213e9. [20] Colson YL, Zadach K, Nalesnik M, Ildstad ST. Mixed allogeneic chimerism in the rat: Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allograft. Transplantation 1995;60:971e80. [21] Kawai T, Cosimi B, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 2008;358:353e61. [22] Huang WC, Lin JY, Wallace CG, et al. Vascularized bone grafts within composite tissue allotransplants can autocreate tolerance through mixed chimerism with partial myeloablative conditioning: An experimental study in rats. Plast Reconstr Surg 2010;125:1095e103. [23] Huang WC, Liao SK, Wallace CG, et al. Greater efficacy of tolerance induction with cyclosporine versus tacrolimus in composite tissue allotransplants with less myeloablative conditioning. Plast Reconstr Surg 2011;127:1141e8. [24] Bozulic LD, Wen Y, Xu H, et al. Evidence that FoxP3þ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants. Transplantation 2011;91:908e15.
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[25] Pilat N, Baranyi U, Klaus C, et al. Treg therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning. Am J Tansplant 2010;10:751e62. [26] Adamson LA, Huang WC, Breidenbach WC, et al. A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts. Microsurgery 2007; 27:630e6. [27] Saurat JH, Gluckman E, Bussel A, et al. The lichen planus-like eruption after bone marrow transplantation. Br J Dermatol 1975;93:675e9. [28] Foster RD, Ascher NL, McCalmont TH, et al. Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers. Transplantation 2001;72:791e9. [29] Beschorner WE, Tutschka PJ, Santos GW. Sequential morphology of graft-versus-host disease in the rat radiation chimera. Clin Immunol Immunopathol 1982;22:203e24. [30] Snover DC. Biopsy interpretation in bone marrow transplantation. In: Rosen PP, Fechner RE, editors. Pathology Annual, Vol. 24. New York: Appleton-Century Crofts; 1989. p. 63. [31] Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172:603e6. [32] Hartwig UF, Robbers M, Wickenhauser C, et al. Murine acute graft-versus-host disease can be prevented by depletion of alloreactive T lymphocytes using activation-induced cell death. Blood 2002;99:3041e9. [33] Drobyski WR, Majewski D. Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHCmismatched donor marrow grafts. Blood 1996;87:5355e69. [34] Gorantla VS, Prabhune KA, Perez-Abadia G, et al. Composite tissue allotransplantation in chimeric hosts: Part I. Prevention of graft-versus-host disease. Transplantation 2003;75:922e32. [35] Neipp M, Exner BG, Maru D, et al. T-cell depletion of allogeneic bone marrow using anti-ab monoclonal antibody: Prevention of graft-versus-host disease without affecting engraftment potential in rats. Exp Hematol 1999;27:860e7. [36] Ge W, Jiang J, Arp J, et al. Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression. Transplantation 2010;90:1312e20. [37] Kong YY, Eto M, Omoto K, et al. Regulatory T cells in maintenance and reversal of peripheral tolerance in vivo. J Immunol 1996;157:5284e9. [38] Joffre O, Gorsse N, Romagnoli P, et al. Induction of antigenspecific tolerance to bone marrow allografts with CD4þCD25þ T lymphocytes. Blood 2004;103:4216e21. [39] Read S, Hogan TV, Zwar TD, et al. Prevention of autoimmune gastritis in mice requires extra-thymic T-cell deletion and suppression by regulatory T cells. Gastroenterology 2007;133: 547e58. [40] Kurtz J, Wekerle T, Sykes M. Tolerance in mixed chimerismdA role for regulatory cells? Trends Immunol 2004;10:518e23. [41] Yamazaki M, Pearson T, Brehm MA, et al. Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice. Am J Transplant 2007;7: 1710e21.
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Optimizing chimerism level through bone marrow transplantation and irradiation to induce long-term tolerance to composite tissue allotransplantation Jeng-Yee Lin, MD,a,b Feng-Chou Tsai, MD, PhD,b Christopher Glenn Wallace, MBChB, MS,c Wei-Chao Huang, MD, PhD,a,d Fu-Chan Wei, MD,a,c and Shuen-Kuei Liao, PhDa,e,* a
Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan Division of Plastic Surgery, Department of Surgery, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan c Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan d Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chiyi, Taiwan e Cancer Immunotherapy Program, Cancer Center, Taipei Medical University Hospital and Center of Excellence for Cancer Research, Taipei Medical University, Taipei, Taiwan b
article info
abstract
Article history:
Background: Mixed chimerism with long-term composite tissue allotransplant (CTA)
Received 29 November 2011
acceptance can be achieved through allogeneic bone marrow transplantation (BMT). The
Received in revised form
present study investigated the optimal chimerism level by giving different irradiation
28 February 2012
dosages to recipients to induce tolerance to CTA.
Accepted 29 February 2012
Methods: Chimera were prepared using Brown-Norway and Lewis rats with strong major
Available online 17 March 2012
histocompatibility complex incompatibility. The Lewis rats received 5 mg antilymphocyte
Keywords:
groups 1, 2, 3, 4, and 5 according to the day 1 irradiation dosage: 0, 200, 400, 600, and 950 cGy,
Graft-versus-host disease
respectively. The Lewis rats were then reconstituted with 100 106 T-celledepleted Brown-
Bone marrow transplantation
Norway bone marrow cells (day 0) and received vascularized Brown-Norway-CTA on day 28.
T-cell depletion
Chimerism was assessed monthly by flow cytometry starting on day 28 after BMT. Graft-
Composite tissue allotransplant
versus-host disease (GVHD) was assessed clinically and histologically.
globulin (day 1 and 10) and 16 mg/kg cyclosporine (day 0e10) and were separated into
Results: Chimerism, 4 weeks after BMT, averaged 0.2%, 9.2%, 30.7%, 58%, and 99.3% in groups 1 to 5, respectively. GVHD occurred as follows: groups 1 and 2, none; group 3, 1 case of GVHD; group 4, 7 cases of GVHD (of which 3 died); and group 5, 10 cases of GVHD (of which 6 died). The percentage of long-term CTA acceptance was 0%, 0%, 90%, 70%, and 40% in groups 1 to 5, respectively. The percentage of regulatory T cells was significantly lower in high-chimerism (20%, n ¼ 15) than in low-chimerism (<20%, n ¼ 5) rats that accepted CTA long-term . Conclusions: The chimerism level correlated positively with GVHD occurrence and longterm CTA acceptance but correlated negatively with regulatory T-cell levels. Optimal chimerism for CTA acceptance through pre-CTA BMT and irradiation occurs at 20e50% at day 28 after BMT in the rat model. ª 2012 Elsevier Inc. All rights reserved.
* Corresponding author. Cancer Center, Taipei Medical University Hospital, 252 Wu-Hsing Street, Taipei 110, Taiwan. Tel.: þ886 2 2739 8278. E-mail address: [email protected] (S.-K. Liao). 0022-4804/$ e see front matter ª 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jss.2012.02.064
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1.
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Introduction
Recent clinical application of composite tissue allotransplantation (CTA) has ushered in a new era for the restoration of massive tissue loss, including nerves [1], abdominal walls [2], tendons [3], bone and joints [4], larynx [5], hands [6e8], scalp and ear [9], and face [10]. However, the balance between the risks and benefits of reconstructive CTA remains controversial, especially regarding the problems of lifelong immunosuppressive therapy. First, current immunosuppression protocols do not completely protect transplants from acute rejection episodes. Second, long-term transplant function is still compromised by chronic rejection that is not responsive to current immunosuppressant agents [11,12]. Third, nonspecific lifelong immunosuppressive therapy is associated with the development of opportunistic infection, drug toxicity, and malignancy [13,14]. One approach that might overcome many of the disadvantages of immunosuppression is transplantation tolerance induction. Current research has focused on 3 main strategies to induce donor-specific tolerance in the recipient, including genetic matching, co-stimulatory blockade [15,16], and mixed chimerism [17,18] (usually by creating mixed allogeneic chimerism through bone marrow transplantation [BMT]). Chimerism refers to a state in which 2 genetically different hematopoietic systems coexist harmoniously in 1 organism. Mixed allogeneic chimerism has been shown to prevent chronic rejection in transplanted grafts while maintaining immunocompetence in the recipients [19,20]. Clinical application of mixed chimerism to induce tolerance was further encouraged by a pilot series of successful immunosuppression-free renal transplantations that were co-transplanted with donor bone marrow [21]. Although the level of chimerism that is needed to induce tolerance remains a topic of discussion, our previous work has demonstrated that the level of chimerism in the initial stage correlated with CTA acceptance in the cyclosporine-based vascularized allogeneic BMT (allo-BMT) rat model [22,23]. Moreover, a greater percentage of donor T lymphocytes circulating in recipients was associated with a greater percentages of graft-versus-host disease (GVHD) [22]. Regulatory T (Treg) cells play an important role in regulating the immune responses to self- and allogeneic antigens. The recent finding that Treg cells infiltrate the donor skin of long-term CTA in the mixed chimeric recipient implies a role in maintaining allograft survival [24]. Introducing Treg cells might, therefore, assist in tailoring immunosuppressive management as a part of the preconditioning regimen when creating mixed chimerism [25]. From these findings and inferences, our study aimed to investigate whether mixed allogeneic chimerism is related to GVHD occurrence, long-term allograft acceptance rate, and Treg cell level in the conventional allo-BMT rat model using differing radiation doses.
2.
Materials and methods
2.1.
Rats
Five- to eight-week-old (weight 200e350 g) male BrownNorway (RT1Ac) and Lewis (RT1Al) rats were used as donors and recipients for CTA. The rats were purchased from the
National Laboratory Animal Center (Taipei, Taiwan) and housed in a pathogen-free facility. The rats were cared for according to the Institutional Animal Care and Use Committee guidelines at the Laboratory Animal Care-approved Research and Resource Center of Chang Gung Memorial Hospital and University.
2.2.
Treatment groups
The Lewis recipients groups 1 to 5 (n ¼ 10, each group) received 0, 200, 400, 600, and 950 cGy of radiation, respectively (liner accelerator, 21EX, 6-MV X-ray; Varian Medical Systems, Palo Alto, CA). The myeloablative dose of total body irradiation in the rat is 950 cGy (group 5). The immunosuppression regimen included 5 mg of goat anti-rat lymphocyte serum (Accurate Chemical, Westbury, NY) by intraperitoneal injection 1 d before and 10 d after BMT. Cyclosporine (Sandimmune; Novartis Taiwan, Taipei, Taiwan) was administered at 16 mg/kg/d intramuscularly from days 0 to 10 after BMT.
2.3.
Bone marrow preparation
The bone marrow was harvested from the femurs, tibias, and humeri of Brown-Norway rats by flushing with Media 199 (Life Technologies, Rockville, MD) containing 10 mg/mL gentamycin using a 22-gauge needle. The cells were resuspended using an 18-gauge needle and filtered through sterile nylon mesh to produce a single cell suspension. The bone marrow cells were washed, centrifuged (500 g for 10 min at 4 C), resuspended, and counted before T-cell depletion (TCD).
2.4. Immunomagnetic bead depletion of T lymphocytes and BMT TCD before BMT was performed to reduce the incidence of GVHD. The bone marrow cells were incubated with purified anti-ab and anti-gd T-cell receptor (TCR) monoclonal antibodies (R73; mouse IgG1; Pharmingen, San Diego, CA) for 30 min at 4 C in the dark. Secondary antibody with coated beads (Dynabeads M-450; goat anti-mouse IgG; Dynal, Lake Success, NY) were then added to bind the primary monoclonal antibodies and incubated for 60 min at 4 C in the dark at a bead/T-cell ratio of 6:1. To negatively select T cells, tubes with bone marrow cell suspensions were placed in a magnetic cell separator (BioMag Separator; Advanced Magnetics, Cambridge, MA) for 5 min. The unbound cells were aspirated, and cell separation was repeated. T-lymphocyteedepleted bone marrow cells were washed, counted, and re-suspended in Dulbecco’s modified Eagle medium solution to achieve a concentration of 100 106 bone marrow cells/mL. The adequacy of TCD was confirmed by flow cytometry. The recipients were reconstituted within 24 h after total body irradiation with 100 106 T-cell-depleted bone marrow cells from Brown-Norway rats diluted in 1 mL of Dulbecco’s modified Eagle medium by way of the penile vein.
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2.5.
Composite tissue allotransplantation
At 28 d after BMT, hindlimb osteomyocutaneous flap CTAs from Brown-Norway donors were performed, as previously described [26]. Inhalational isoflurane (Halocarbon, Products Corporation, River Edge, NJ) provided continuous anesthesia for both donors and recipients. In brief, a longitudinal incision was made from thigh to ankle on the medial leg. A circumferential skin incision was made at the mid-thigh and the ankle joint to isolate a 3 5-cm skin paddle on the lateral leg. The femoral vessels were mobilized up to the inguinal ligament. A complete osteotomy was performed on the femur and tibia to harvest an osteomyocutaneous flap based solely on the femoral vessels. Bone marrow cells were aggressively flushed out of the femur and tibia cut ends before transplantation. The harvested flap was wrapped with wet gauze and placed on iced saline. The procedure on the recipient chimeric rat (Brown-Norway/Lewis) began with a transverse incision in the inguinal region to expose and mobilize the femoral artery and vein before they were cut and clamped for microanastomoses (10-0 nylon, using a standard microsurgical technique) with the donor femoral vessels. All rats were housed individually in filter-top cages and fed autoclaved rat chow to minimize the risk of opportunistic infection.
2.6. Characterization of chimerism and donor cell lineage by flow cytometry Engraftment of the allogeneic bone marrow was assessed 2 weeks after BMT with flow cytometry. Flow cytometry was also used monthly after BMT to determine the chimerism level, defined as the percentage of peripheral blood lymphocytes bearing donor (Brown-Norway) major histocompatibility complex class I antigen. In brief, whole blood aliquots of 100 mL were stained with antieBrown-Norway (RT1Ac-FITC [OX27, mouse IgG2a; Serotec, Raleigh, NC]), B-cell (CD45RA-PE [OX33]), mouse IgG1 (BD Pharmingen, San Diego, CA), T-cell (TCR-PerCP [R73], mouse IgG1; BD Pharmingen), natural killer cell (antiNKR-P1A-APC [10/78], mouse IgG1; Caltag Laboratories, Bangkok, Thailand), helper T-cell (CD4-APC [W3/25], mouse IgG1; BD Pharmigen), cytotoxic T cell (CD8a-PerCP:OX-8; mouse IgG1; Pharmingen), and macrophage-granulocyte (MAC1FITC:OX42; mouse IgG2a; Serotec, Baveria, Germany) lineagespecific fluorescein isothiocyanate-labeled monoclonal antibodies for 30 min. Flow cytometry was performed using a FASCort flow cytometer (Becton Dickinson, Franklin, NJ).
2.7.
Assessment of limb rejection
The transplanted flaps were examined daily for any signs of rejection, including edema, desquamation, epidermolysis, hair loss, exudation, and flap necrosis or shrinkage. Routine skin biopsies were performed monthly after flap transplantation. Standard histopathologic features were used to assess the degree of acute allograft rejection in skin biopsies using the following histopathologic grading classification [27]: grade 0, normal epidermal appearance without evidence of rejection; grade 1, focal basal cell layer vacuolization; grade 2, dyskeratosis of squamous cells in the epidermis or hair follicle epithelium; grade 3, subepidermal clefting or microvesiculation; and
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grade 4, complete separation at the epidermaledermal junction. Composite tissue allograft survival acceptance was defined as complete survival and growth of hair in the allotransplant without any rejection signs at the endpoint (150 d).
2.8.
Secondary skin grafting
To confirm whether donor-specific transplantation tolerance existed in CTA-accepting recipients, donor, and third-party skin grafts were performed in all CTA-accepting rats. Full-thickness skin grafts were harvested from the abdominal wall of the donor (Brown-Norway) and third party (ACI) rats. The grafts were sutured to the dorsal neck in anesthetized recipients with a 3-mm skin bridge between them and secured with a tie-over bolster dressing. The bolster was removed on the seventh day, and the grafts were inspected daily for signs of rejection.
2.9.
Assessment of GVHD
The rats were weighed at BMT and at varying intervals thereafter. The diagnosis of GVHD was determined from clinical signs, including weight loss, unkempt appearance, diarrhea, diffuse erythema, hyperkeratosis of foot pads, and/ or dermatitis [28,29]. GVHD was graded clinically as none, mild, moderate, or severe [30]. At necropsy, an ear wedge biopsy was performed to examine for lymphoid infiltration, subepidermal cleft formation, and/or epidermal necrosis.
2.10.
Treg cells identification
The cell population marker of Treg cells (CD4þCD25þFoxP3þ) in the peripheral lymphoid gate of recipients was confirmed with flow cytometry and intracytoplasmic staining.
2.11.
Statistical Analysis
Statistical analyses were performed using Fisher’s exact test.
3.
Results
3.1.
Efficacy of TCD
Predepletion donor bone marrow contained a mean of 1.63% ab-TCRepositive and 0.87% gd-TCRepositive T cells. After bead depletion, the ab-TCRepositive cell count was reduced to an average of 0.05% and the gd-TCRepositive cell count to 0.03% of the lymphoid gate (Fig. 1A). This demonstrated adequate depletion of donor T lymphocytes before BMT.
3.2. Mixed chimerism and donor multilineage hematopoietic cells The chimerism level refers to the percentage of donor cells in the peripheral blood leukocytes of the recipients. Multilineage chimerism of both lymphoid and myeloid lineages, including donor CD4þ cells, CD8þ cells, B cells, natural killer cells, and dendritic cells, was present in all groups except group 1, suggesting that engraftment of pluripotent hematopoietic
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Fig. 1 e (A) Most T cells express ab-TCR and 5% of T cells express gd-TCR on their cell surfaces. Immunomagnetic beads for binding both ab- and gd-T cell receptors were used to remove the T cell repertoire effectively. Histograms showed pre- and post-T cell-depletion results of donor bone marrow. Reductions of ab TCR T cells from 1.63% ± 0.03% to 0.05% ± 0.03% and gd TCR T-cells from 0.87% ± 0.1% to 0.03% ± 0.01% were routinely achieved. (B) Mutlilineage analysis of the recipient peripheral whole blood 2 weeks after BMT (group 3) showed evidence of donor bone marrow engraftment, as determined by dual expression of donor-specific (RT1Ac) and lineage-specific markers in flow cytometry. Engraftment of at least 6 different lineages of donor cells shown (CD4D [ CD4D T cells; CD8D [ CD8D T cells; B [ B cells; DC [ dendritic cells; Mac/Gran [ macrophages/granulocytes; NK [ natural killer cells).
stem cells from the donor was successful in all irradiated recipients from groups 2 to 5 (Fig. 1B). The average chimerism level in each group was as follows: group 1 (no irradiation), 0.2%; group 2 (radiation dose 200 cGy), 9.2%; group 3 (radiation dose 400 cGy), 30.7%; group 4 (radiation dose 600 cGy), 58%; and group 5 (radiation dose 950 cGy), 99.3%. The radiation dosage in group 5 caused complete myeloablation. Thus, full chimerism rather than mixed chimerism was observed in group 5. Flow cytometry 28 d after BMT demonstrated that
groups with a greater radiation dosage had a greater donor chimerism level in the engrafting recipient.
3.3. Occurrence of GVHD and survival of hindlimb transplantation The percentage of GVHD reflected the radiation dosage. The rats in groups 1 and 2 developed no signs of GVHD. One rat in group 3 had minor signs of acute GVHD from 7 d after BMT that
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continued until day 45. Also, 3 rats from group 4 died of acute GVHD with continued weight loss, ear and claw erythema, and severe hair loss, and 3 rats in group 4 developed moderate signs of GVHD with ear and paw erythema, edema, failure to increase body weight, and unkempt hair that continued through to the endpoint of the study (day 150). Another rat in group 4 developed minor signs of GVHD that persisted through the endpoint of the study. Group 5 had 6 rats that died of GVHD. Another 4 rats had moderate signs of GVHD that continued to the endpoint of the study. In the groups with greater total body irradiation, a greater percentage of acute GVHD was observed (Fig. 2). In groups 1 (nonirradiated recipients) and 2 (200 cGy radiation), none of the CTAs survived beyond 29 and 45 d, respectively, after CTA. In group 3 (400 cGy radiation), 90% (9 of 10) of the rats accepted the CTA until the endpoint of study (day 150). The CTA acceptance rate for group 4 and 5 was 70% (7 of 10) and 40% (4 of 10), respectively.
3.4.
Secondary skin grafting
Mixed allogeneic chimeras (Brown-Norway/Lewis) permanently accepted donor (Brown-Norway) but rejected (mean survival 14 d) third-party (ACI) skin grafts.
3.5. Correlation of mixed chimerism level to GVHD, CTA acceptance, and Treg level To determine the relationship between chimerism level with GVHD and CTA acceptance, all chimera rats were classified according to their level of chimerism into groups of 10%
Fig. 2 e Chimerism level, GVHD rate, and CTA acceptance rate in each radiation group. Diagram shows that chimerism level, GVHD, and CTA rate correlated positively with total body radiation dosage. Note, however, that CTA acceptance rates deceased with greater radiation, groups 4 (600 cGy) and 5 (950 cGy), owing to GVHD-related deaths of recipient chimera. This showed that group 3 (400 cGy) had the greatest CTA acceptance rate with an acceptable GVHD rate. (Inset A) Long-term CTA acceptance in recipient chimera rats. (Inset B) Histologic findings of biopsy taken from accepted CTA shows allograft skin free of lymphocyte infiltration. (Color version of figure is available online.)
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increments. The results showed that the optimal chimerism level was about 20e50%, with this range producing a 100% CTA acceptance rate and no GVHD (Fig. 3). The percentage of Treg cells (CD4þ CD25þ) in the CD4þ cell population was significantly lower in the CTA acceptance recipients with greater chimerism (20%; n ¼ 15) at the endpoint of this study (day 150) compared with the CTA acceptance recipients with lower chimerism (<20%; n ¼ 5; P < 0.01; Fig. 4).
4.
Discussion
CTA has provided a solution to massive tissue loss such as major limb amputations and extensive face or tissue injuries. The long-term functional results of CTA might not be completely maintained because of chronic rejection occurring despite control of acute rejection episodes. Donor-specific tolerance that results from mixed chimerism has shown some promise in the search to solve this problem in CTA. The concept of mixed allogeneic chimerism has gained increasing popularity since the 1950s when Billingham et al. [31] first successfully performed skin grafting between dizygotic cattle. Durable mixed allogeneic chimerism through allo-BMT has been shown to induce tolerance to allogeneic transplantation of several tissues and organs in animal and human recipients. However, 2 facts could explain why it is considered more difficult to induce tolerance for CTA compared with solid organ transplantation. First, the skin tissue is thought to be more immunogenic than other tissues. Second, the engrafted lymphoid tissue (bone or lymph nodes) engrafted provides a constant source of donor antigens. Efforts using a variety of aforementioned methods independently have obtained good results to date. Our study, which combined the available methods and adjusted the radiation dosage to increase CTA success through the conventional allo-BMT model, revealed
Fig. 3 e Relationship of chimerism level to GVHD and CTA acceptance rates. Chimerism level correlated positively with GVHD and CTA acceptance rates. Note, CTA acceptance rate decreased in higher chimerism groups owing to GVHD-related deaths of chimera after BMT, suggesting that optimal chimerism level was about 20e50% with 100% CTA acceptance and 0% GVHD. (Color version of figure is available online.)
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Fig. 4 e Comparison of percentage of Treg (CD4DCD25 DFoxP3D) cells/CD4D cells in high-chimerism (‡20%, n [ 15) and low-chimerism (<20%, n [ 5) chimera recipients with long-term CTA acceptance. This histogram showed that the percentage of Treg cells/CD4D cells was significantly greater in rats with low chimerism that accepted CTAs (63.9% versus 31.3%, *P < 0.001).
(20%) group (n ¼ 15) that accepted allotransplants was observed to have a lower Treg cell percentage in the CD4þ cell population than the low-chimerism (<20%) group (n ¼ 5) that accepted allotransplants (31.3% versus 63.9%, P < 0.001). One explanation was that the acceptance model with a high chimerism level ensured the continuous contribution of donor and host cells to antigen-presenting cells that induced intrathymic deletion. In contrast, although in acceptance models in which only low levels of donor chimerism persist, Treg cells might have a significant role because the deletion of donor-reactive T cells appears to be less complete. The emergence of donor-reactive T cells from the thymus might then permit the expansion of regulatory mechanisms. However, this finding also implied augmentation of Treg cells might help tolerance induction in low levels of mixed chimerism. However, more studies are needed to investigate the mechanism that Treg cells play in low-level mixed chimerism. Although the preconditioning regimens are not yet clinically practical owing to possible host toxicity, graft failure, GVHD, and a waiting period for chimerism to develop, ongoing active research is bringing tolerance induction closer to clinical reality.
5. the relationship among GVHD, acceptance rate, Treg cell level, and the optimal radiation dose. It has been reported that TCD using anti-ab plus anti-gd TCR monodispersed antibodies and immunomagnetic beads effectively removed T cells from the donor bone marrow and effectively prevented GVHD in major histocompatibility complex-mismatched mice [32,33] but only partially prevented GVHD in rats [34,35]. That study confirmed that although adequate TCD before BMT was performed, an unacceptably high GVHD rate was observed in the chimeric recipient rats with a chimerism level of more than 50% at 28 d after BMT. Obviously, more sophisticated immune systems play a role in the rat model in which TCD could not prevent GVHD completely after allo-BMT. In our study, the rats with a mixed chimerism level of more than 20% at 28 d after BMT maintained a long-term CTA, suggesting that a threshold donor contribution to the hematopoietic stem cell pool might be required to ensure the constant presence of donor-derived thymic dendritic cells to contribute to negative selection. Therefore, it seems that a harmonious balance between GVHD and allotransplant acceptance can be achieved through titration of chimerism to its optimal level. Thus, to keep the chimerism level as low as possible to avoid GVHD but still greater than the threshold level to ensure allotransplant acceptance. Our chimeric rat model suggested that an optimal chimerism level of about 20e50% at 28 d after BMT resulted in a low GVHD rate and high CTA acceptance. A considerable body of data implicating CD25þCD4þ Treg cells in the maintenance of peripheral tolerance to organ-specific self-antigens has been described [36e39]. Although all published studies on mixed chimerism suggested only a limited role for Treg cells in tolerance induction [40,41], they did not preclude a role for suppression of donor reactive cells in protocols involving BMT in which only low levels of chimerism were established. In our study, the high-chimerism
Conclusions
Our experiments have offered a suitable rat model with durable mixed chimerism through BMT for studying CTA. The data showed that the chimerism level positively correlated with GVHD occurrence and long-term allograft acceptance. Titration of mixed chimerism to its optimal level (20e50%) at day 28 through pre-CTA BMT, and irradiation, therefore, provides an approach to reducing GVHD and increasing rejection-free CTA survival in the rat model.
Acknowledgments The present report was supported by grant 100-TMU-TMUH11 from the: Taipei Medical University Hospital.
references
[1] Bain JR. Peripheral nerve and neuromuscular allotransplantation: Current status. Microsurgery 2000;20: 384e8. [2] Levi DM, Tzakis AG, Kato T, et al. Transplantation of the abdominal wall. Lancet 2003;361:2173e6. [3] Guimberteau JC, Baudet J, Panconi B, et al. Human allotransplant of a digital flexion system vascularized on the ulnar pedicle: A preliminary report and 1-year follow-up of two cases. Plast Reconstr Surg 1992;89:1135e47. [4] Hofmann GO, Kirschner MH, Brauns L, et al. Vascularized knee joint transplantation in man: A report on the first cases. Transpl Int 1998;11(Suppl):S487e90. [5] Strome M, Stein J, Escalmado R, et al. Laryngeal transplantation and 40-month follow-up. N Engl J Med 2001; 344:1676e9. [6] Dubernard JM, Owen E, Herzberg G, et al. Human hand allograft: Report on first 6 months. Lancet 1999;353:1315e20.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 7 8 ( 2 0 1 2 ) 4 8 7 e4 9 3
[7] Jones JW, Gruber SA, Barker JH, et al. Successful hand transplantation: One-year follow-up. Louisveille Hand Transplant Team. N Engl J Med 2000;343:468e73. [8] Cooney WP, Hentz VR. Successful hand transplantation: One year follow-up. N Engl J Med 2001;344:65. [9] Jiang HQ, Wang Y, Hu XB, et al. Composite tissue allograft transplantation of cephalocervical skin flap and two ears. Plast Reconstr Surg 2005;115:31ee5e. [10] Dubernard JM, Devauchelle B. Face transplantation. Lancet 2008;372:603e4. [11] Colson YL, Zadach K, Nalesnik M, et al. Mixed allogeneic chimerism in the rat: Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allografts. Transplantation 1995;60: 971e80. [12] Orloff MS, DeMara EM, Coppage ML, et al. Prevention of chronic rejection and graft arteriosclerosis by tolerance induction. Transplantation 1995;59:282e8. [13] Penn I. Depressed immunity and the development of cancer. Clin Exp Immunol 1981;46:459e74. [14] Bach FH, Sachs DH. Current concepts: Immunology, transplantation immunology. N Engl J Med 1987;317:489e92. [15] Zhu ZX, Fan LY, Wang Q. Simultaneous blockade of costimulatory signals CD28-CD80 and CD40-CD154 combined with monoclonal antibody against CD25 induced a stable chimerism and tolerance without graft-versus-host disease in rat. Eur Surg Res 2011;46:109e17. [16] Foster RD, Pham S, Li S, et al. Long-term acceptance of composite tissue allografts through mixed chimerism and CD28 blockade. Transplantation 2003;76:988e94. [17] Foster RD, Fan L, Neipp M, et al. Donor-specific tolerance induction in composite tissue allografts. Am J Surg 1998;176: 418e21. erratum in Am J Surg 1999;177:529. [18] Sharabi Y, Sachs DH. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen. J Exp Med 1989;169:493e502. [19] Blom D, Morrissey NJ, Mesonero C, et al. Induction of specific tolerance through mixed hematopoietic chimerism prevents chronic renal allograft rejection in a rat model. Surgery 1996; 120:213e9. [20] Colson YL, Zadach K, Nalesnik M, Ildstad ST. Mixed allogeneic chimerism in the rat: Donor-specific transplantation tolerance without chronic rejection for primarily vascularized cardiac allograft. Transplantation 1995;60:971e80. [21] Kawai T, Cosimi B, Spitzer TR, et al. HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med 2008;358:353e61. [22] Huang WC, Lin JY, Wallace CG, et al. Vascularized bone grafts within composite tissue allotransplants can autocreate tolerance through mixed chimerism with partial myeloablative conditioning: An experimental study in rats. Plast Reconstr Surg 2010;125:1095e103. [23] Huang WC, Liao SK, Wallace CG, et al. Greater efficacy of tolerance induction with cyclosporine versus tacrolimus in composite tissue allotransplants with less myeloablative conditioning. Plast Reconstr Surg 2011;127:1141e8. [24] Bozulic LD, Wen Y, Xu H, et al. Evidence that FoxP3þ regulatory T cells may play a role in promoting long-term acceptance of composite tissue allotransplants. Transplantation 2011;91:908e15.
493
[25] Pilat N, Baranyi U, Klaus C, et al. Treg therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning. Am J Tansplant 2010;10:751e62. [26] Adamson LA, Huang WC, Breidenbach WC, et al. A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts. Microsurgery 2007; 27:630e6. [27] Saurat JH, Gluckman E, Bussel A, et al. The lichen planus-like eruption after bone marrow transplantation. Br J Dermatol 1975;93:675e9. [28] Foster RD, Ascher NL, McCalmont TH, et al. Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers. Transplantation 2001;72:791e9. [29] Beschorner WE, Tutschka PJ, Santos GW. Sequential morphology of graft-versus-host disease in the rat radiation chimera. Clin Immunol Immunopathol 1982;22:203e24. [30] Snover DC. Biopsy interpretation in bone marrow transplantation. In: Rosen PP, Fechner RE, editors. Pathology Annual, Vol. 24. New York: Appleton-Century Crofts; 1989. p. 63. [31] Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953;172:603e6. [32] Hartwig UF, Robbers M, Wickenhauser C, et al. Murine acute graft-versus-host disease can be prevented by depletion of alloreactive T lymphocytes using activation-induced cell death. Blood 2002;99:3041e9. [33] Drobyski WR, Majewski D. Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHCmismatched donor marrow grafts. Blood 1996;87:5355e69. [34] Gorantla VS, Prabhune KA, Perez-Abadia G, et al. Composite tissue allotransplantation in chimeric hosts: Part I. Prevention of graft-versus-host disease. Transplantation 2003;75:922e32. [35] Neipp M, Exner BG, Maru D, et al. T-cell depletion of allogeneic bone marrow using anti-ab monoclonal antibody: Prevention of graft-versus-host disease without affecting engraftment potential in rats. Exp Hematol 1999;27:860e7. [36] Ge W, Jiang J, Arp J, et al. Regulatory T-cell generation and kidney allograft tolerance induced by mesenchymal stem cells associated with indoleamine 2,3-dioxygenase expression. Transplantation 2010;90:1312e20. [37] Kong YY, Eto M, Omoto K, et al. Regulatory T cells in maintenance and reversal of peripheral tolerance in vivo. J Immunol 1996;157:5284e9. [38] Joffre O, Gorsse N, Romagnoli P, et al. Induction of antigenspecific tolerance to bone marrow allografts with CD4þCD25þ T lymphocytes. Blood 2004;103:4216e21. [39] Read S, Hogan TV, Zwar TD, et al. Prevention of autoimmune gastritis in mice requires extra-thymic T-cell deletion and suppression by regulatory T cells. Gastroenterology 2007;133: 547e58. [40] Kurtz J, Wekerle T, Sykes M. Tolerance in mixed chimerismdA role for regulatory cells? Trends Immunol 2004;10:518e23. [41] Yamazaki M, Pearson T, Brehm MA, et al. Different mechanisms control peripheral and central tolerance in hematopoietic chimeric mice. Am J Transplant 2007;7: 1710e21.