Chimerism studies as an approach for the induction of tolerance to extremity allografts

Journal of Plastic, Reconstructive & Aesthetic Surgery (2008) 61, 1009e1015

REVIEW

Chimerism studies as an approach for the induction of tolerance to extremity allografts* Keiichi Muramatsu*, Ryutaro Kuriyama, Song You-Xin, Takahiro Hashimoto, Tsunemitsu Matsunaga, Toshihiko Taguchi Department of Orthopedic Surgery, Yamaguchi University School of Medicine, Yamaguchi, Japan Received 12 June 2007; accepted 14 December 2007

KEYWORDS Chimerism; Composite tissue allograft; Extremity allograft; Immunosuppression; Tolerance; Graft-versus-host disease

Summary Recent advances in the field of transplant immunology and reconstructive surgery have resulted in an increased interest in extremity allograft. Until now, more than 20 hand transplants have been performed in humans. Rejection is well controlled by currently available immunosuppressive drugs. The hand transplant, however, is not a life-supporting organ transplant and these drugs are unlikely to represent the final solution for hand transplantation due to serious adverse effects. The ultimate goal of extremity allograft is the induction of donorspecific immunotolerance. The major strategies for tolerance induction are: (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells. Amongst these, the establishment of a high level of chimerism may be the most stable strategy for donor-specific tolerance, and our laboratory has been investigating the induction of macrochimerism following extremity allotransplantation. Recently, some studies demonstrated that macrochimerism induces immunotolerance for extremity allograft in the rodent model. We made a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Limb allografting could function as a vascularised carrier for bone marrow transplantation, providing a continuous source of donor cells and contributing to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolong the survival of limb allografts, but frequently cause chronic graft-versus-host disease in the recipients.

*

This work was supported by the Japanese Society for the Promotion of Science, Grant-in-Aid for Scientific Research #15591578. * Corresponding author. Address: Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. Tel.: þ81 836 22 2268; fax: þ81 836 22 2267. E-mail address: [email protected] (K. Muramatsu). 1748-6815/$ - see front matter ª 2008 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2007.12.082

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K. Muramatsu et al. In this review, recent experimental chimerism studies are presented for tolerance induction and we review the prospect of clinical applicability in extremity allograft. ª 2008 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

The transplantation of composite tissue allograft (CTA) is a challenging new area.1 To date, more than 50 CTA transplants have been performed worldwide in humans, including 25 hands,2e10 nine abdominal walls,11 eight vascularised bones,12,13 seven peripheral nerves,14 two vascularissed tendons,15 two tracheas,16,17 one larynx,18 one isolated muscle,19 one tongue,20 and two ears.21e23 Amongst them, the hand transplants performed in Europe and the United States have generated much public interest and debate in the surgical community. Over the past three decades, nonspecific immunosuppressive drugs have contributed to the enormous success in the field of transplantation. Immunosuppressive protocols applied for CTA are based on solid-organ immunosuppressive regimens and a combined therapy that includes tacrolimus (FK506),24,25 mycophenolate mofetil 26,27 (MMF), and steroids has been commonly used for hand transplant patients. Several case reports showed excellent rejection control and functional recovery of the hand allografts.3e7 However, major obstacles that limit the future of extremity allotransplantation are the complications associated with chronic use of immunosuppressants.26e34 These include direct side effects of the drug, opportunistic infections, and malignancies. The hand transplant is not a lifesupporting organ transplant, and these serious adverse effects are not acceptable in CTA. Currently available drugs are unlikely to represent the final solution for hand transplantation and the ultimate goal is the induction of transplantation tolerance. Recently, several approaches have been demonstrated to induce immunotolerance in the rodent hindlimb or nonhuman primate allograft model, including (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells.35e47 Our laboratory has focused on chimerism and here we further investigate this approach for inducing tolerance to extremity allografts. This paper presents results of recent experimental studies on chimerism based on the rodent model for tolerance induction, and reviews the prospect of clinical applicability in extremity allograft.

Prolonged rat hindlimb graft survival with a limited course of immunosuppression in MHC major mismatched pair There are three ways to overcome the immune system at transplantation: (1) major histocompatibility complex (MHC) matching, (2) non-specific immunosuppression, and (3) the induction of donor-specific tolerance. MHC matching is clearly the simplest path to success, but most transplants are performed from MHC-unmatched donors. Current strong immunosuppressive agents, such as FK506,24,25 cyclosporine A (CsA),48e50 deoxyspergualin (DSG),51,52 and MMF,26,27,53e56 act by randomly blocking T-cell activation.57

Unfortunately, these drugs are associated with significant side effects, including nephrotoxicity and diabetes, and an increased risk of opportunistic infections and malignancies.28e34,58 Our laboratory focused on inducing immunotolerance using a limited course (30 d) of immunosuppressive therapy. Combined therapy using different types of immunosuppressive agents might have a synergistic effect in preventing rejection.53,54 Based on previous reports of limb allografts,58e61 the dosage of immunosuppressant given was 70e 80% of the lethal dose. Our results and those of others have shown that the most potent immunosuppressant in the rodent limb allograft model was FK506 and a synergistic effect was apparent in combination with MMF.51,52,62,63 However, despite improvements in immunosuppressive drugs, there have been no reports of short-course therapy that induce immunotolerance and therefore allow permanent survival of limb allografts (Table 1). Interestingly and as previously demonstrated by several research groups, the deeper tissue components such as muscle, bone, cartilage, nerve, and tendon showed no rejection even after the skin component was rejected by the recipient.64 This rejection is a unique phenomenon of extremity allotransplantation and differs to that of visceral organ allografts. The extremity consists of many tissues (of ectodermal, mesodermal, and haematopoietic origin) that are highly antigenic and capable of differential tissue rejection. The differing immunogenicity of different organs and tissues in the body is a well-described phenomenon and the order of decreasing antigenicity is: skin > bone marrow > small bowel > lung > pancreas > islets > kidney > liver > bone > cornea.65 Combining the results from many recent studies, the tissue hierarchy of antigenicity in extremities can be summarised as: skin > subcutaneous tissue/bone marrow > vascular endothelium > muscle > periostenum > nerve > bone > tendon > cartilage.66 The skin component is clearly the most antigenic tissue and as such becomes the first target of rejection.

Mixed chimerism and tolerance induction The ultimate goal of transplant surgery is the induction of donor-specific immunotolerance. Tolerance can be defined as the acceptance of antigenic donor tissue by a recipient without the need for continuous immunosuppression. However, immune responses to all other antigens, including bacteria and viruses, must remain intact. Recent advances in our understanding of the immune system following transplantation have resulted in several approaches to transplant tolerance that have proved to be successful in the rodent model. The major strategies for tolerance induction are: (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells.35e47 Amongst

Chimerism studies for extremity allografts Table 1

Prolonged rat hindlimb graft survival with 30 days of immunosuppression

Immunosuppressant 1

Rejection control FK506 CsA DSG MMF FTY FK506 FK506 FK506 CsA

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Dosage (mg per kg per day) 1 15 2.5 15 30 1.5 3 1 1 1 15

Immunosuppressant 2

DSG MMF MMF DSG

these, our laboratory has been investigating the induction of chimerism following extremity allotransplantation.40,41,67e71 Mixed chimerism is defined as the existence of two genetically disparate cell populations in the same organism.72,73 It is one of the oldest and best-studied approaches for establishing tolerance in organ transplantation.74 Two different types of chimerism have been described: microchimerism and macrochimerism.75 The main players in microchimerism are passenger leucocytes or dendritic cells. The interactions between passenger leucocytes from the transplanted allograft and the recipient’s own leucocytes may lead to the induction of donor-specific tolerance. During the condition of microchimerism, donor cell levels are usually present at <1% frequency in the recipient’s peripheral blood and <2% in the bone marrow, thus requiring detection by polymerase chain reaction.69,70,76 Preconditioning of the recipient before allotransplantation is not necessary. However, it remains controversial as to whether tolerance is induced by the microchimerism or whether the latter is simply a result of the former. Recent reports have demonstrated allograft rejection even in the presence of microchimerism, suggesting that this condition may simply be a result of tolerance.77,78 Macrochimerism results after transplantation of donor bone marrow pluripotent stem cells into a preconditioned recipient. Grafted bone marrow cells are acutely engrafted into the recipient lymphoid tissues and thymus, leading to deletion of reactive T cells. Once the donor stem cells are engrafted in the recipient, their frequency in the lymphoid tissue is >10%, allowing detection by flow cytometric analysis.47,74,79 The concept of immunologic cytoreduction followed by haematopoietic reconstitution was first demonstrated in 1953 by Billingham and Medawar80 They infused replicating donor haematopoietic cells into immunologically incompetent neonates to induce tolerance (Figure 1). In the 1960s, Monaco et al.81 demonstrated experimentally in mice that addition of donor bone marrow to an immunosuppressive regimen resulted in long-lasting survival of skin allografts without the use of ongoing immunosuppressants. To induce macrochimerism, some type of recipient pretransplant conditioning is required. This can include total body irradiation or cytoreductive chemotherapy

Dosage (mg per kg per day)

Graft survival (days  S.D.) 41 65  16 36  2 44  6 14  7 45  5 61 82 76  6 132  8 134  7 37  3

2.5 15 30 2.5

combined with pharmacologic immunosuppressive therapy.81e85 The aim is to create a ‘clean space’ for engraftment of the donor haematopoietic cells in the recipient bone marrow. Once the donor chimeric cells have begun to reconstitute, tolerance is primarily induced and maintained by central deletion of potential donor-reactive T cells, although peripheral mechanisms are also likely to contribute to the process.66

Macrochimerism and tolerance induction in the rat hindlimb allograft The concept of macrochimerism inducing immunotolerance has enjoyed increased popularity in the past 10 years. Colson et al.86 first achieved reliable, mixed allogeneic chimerism by transplanting a mixture of T-cell-depleted bone marrow cells into pretreated recipients in a rat cardiac allograft model. Chimerism ranging between 12% and e93% was achieved in 91% of the cases across several strongly antigenic, MHCedisparate strain combinations. In a rat hindlimb transplant model, Foster et al.47 developed reliable tolerance across a strong MHC barrier by inducing a state of mixed chimerism with T-cell-depleted bone marrow transplantation (BMT) (Figure 2). There were no signs of rejection of the limb allografts after more than 100 days in 6 out of 10 animals in which the donor chimerism level was >60%. All three animals with <20% chimerism rejected the limb transplant. The delay period required between the induction of macrochimerism and transplantation is not likely to be an issue in selected cases of living, solid-organ transplantation. However, this delay is not possible for CTA, where, for example, a hand allograft Irradiation

BMT

Organ TX

Chimerism

Definite Survival

Figure 1 Classical technique to induce chimerism at BMT (Medawar et al.).

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K. Muramatsu et al.

Irradiation BMT ATS

Check chimerism level

Transplant

Irradiation fludarabine

Transplant IBM-BMT

FK506; 10 days Day -5

Day 0

12 months

Day 10

Day -1

Figure 2 TBI, ATS, BMT, and FK506 protocol. TBI; total body irradiation, ATS; antilymphocyte serum (Foster et al.).

is always procured from a cadaveric donor and preconditioning of the recipient is therefore impossible. Prabhume et al.45 transplanted the limb allograft and BMT simultaneously in order to cover the delay period. Following 28 days of immunosuppression with FK506 and MMF, the limb allografts survived without rejection (Figure 3). This may be the most reasonable protocol to consider for clinical CTA. Interestingly, Esumi et al.87 demonstrated successful allogeneic rat limb allotransplantation using combined pretreatments of fludarabine injection, low-dose irradiation and BMT directly into the bone marrow cavity. These limb allografts survived for >1 year without signs of rejection (Figure 4). The conclusion from these studies is that development of high-level, mixed chimerism allows long-term survival of limb allotransplants similar to that observed for other visceral organ allotransplants.

Vascularised tibial bone and hindlimb allograft model as a carrier of vascularised BMT Tolerance induced by mixed chimerism became one of the most promising approaches for extremity allotransplantation. Chimerism is frequently observed in successful organ transplants in animal models. In tolerant animals, however, the degree of chimerism as well as the tissue distribution is highly variable and dependent in part upon the type of organ transplanted.88 For example, microchimerism was detected in 66% of non-immunosuppressed recipients and 100% of FK506-treated rats at 60 days post-liver transplantation. No microchimerism was seen in a similar study involving heart transplant recipients.89 Hindlimb allografts are vascularised carriers of bone marrow, thus serving as a constant source of donor haematopoietic stem cells capable of inducing chimerism and donor-specific tolerance. Because vascularised bone marrow graft contains many

Transplant Irradiation BMT

Check Chimerism level Check graft survival FK 506+MMF; 28 days

Day 0

Figure 3 et al.).

Check Chimerism level Check graft survival

Day 28

TBI, BMT, FK506, and MMF protocol (Prabune

Day 0

12 months

Figure 4 TBI, fludarabine, intra-bone marrow-BMT protocol (Esumi et al.).

undifferentiated haematopoietic cells, chimerism would likely be more frequent than following heart transplantation.46 Recent studies have demonstrated donor cell migration following limb transplants. Hewitt et al.44,46 first investigated rat hindlimb allografts for their ability to induce chimerism and tolerance or graft-versus-host disease (GVHD). Their study showed that low-level mixed lymphocyte chimerism (18%  3%) was associated with the induction of tolerance following limb transplantation, but a high level of chimerism (60%  14%) was associated with the development of GVHD. Ajiki et al.90 developed a green fluorescent protein transgenic rat for marking the donor cells and found that the frequency of donor cells in recipient bone marrow was about 1% at 48 weeks post-transplantation. Previously, we examined the level of chimerism after vascularised tibial bone and whole hindlimb allograft in the rat and following the chronic low-dose FK506 treatment. Our results demonstrated low-level microchimerism (1e10%) in peripheral blood, bone marrow, and lymphoid tissues following vascularised bone transplantation.40,41 These levels are similar to those observed following liver transplants. The rat hindlimb allograft was shown to act as a vascularised BMT and to repopulate the recipient with a donor chimerism level of between 1% and 10%. However, this level of mixed chimerism does not allow the induction of tolerance and clinical signs of rejection developed once the immunosuppressants are withdrawn.

Raising the level of chimerism following limb allograft/vascularised bone marrow transplant Recent clinical studies and the use of larger animal models indicate that the level of chimerism following extremity allotransplantation is unexpectedly low and results in microchimerism. Mathes et al.,91 using a miniature swine model and flow cytometry, found no evidence of donorcell engraftment in a recipient animal. Similarly, Granger et al.92 reported that donor cell microchimerism in clinical hand transplant patients was barely detectable in the early post-transplantation specimens and was undetectable thereafter. It is not surprising that peripheral microchimerism could not be detected in recipients of a hand allograft since the mass of bone marrow engrafted with the composite allograft is very low. The recipient’s cells were more likely to be present in the allograft than were donor cells in the recipient’s blood. To our knowledge, no study to date has attempted to raise the level of chimerism following limb allograft/

Chimerism studies for extremity allografts vascularised BMT. Recently, we experimented with a new protocol using CYP, G-CSF93 and FK506 to induce high-level chimerism following rat whole-limb allotransplantation across an MHC major barrier.67,68 CYP was injected at day 2 for cytoreduction and G-CSF was given from days 0e3 to stimulate donor chimeric cell migration. FK506 was used for 28 days after transplant (Figure 5). Limb survival was significantly prolonged with 30% of hindlimb allografts surviving more than 300 days. Non-lethal chronic GVHD occurred in 24% of recipients. A high level of chimerism was maintained when limb allografts were not rejected by recipients. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolonged the survival of limb allografts but frequently caused chronic GVHD in the recipients. GVHD remains a major and devastating complication of BMT. GVHD occurs when donor immunocompetent cells within the graft attack the recipient patient tissues. Wick et al.94 proposed three factors necessary for the development of GVHD: (1) a sufficient number of immunocompetent cells within the graft, (2) major immunogenic differences between recipient and donor, and (3) the inability of the recipient’s immune system to mount an effective response against the graft. Because recipients of hand allografts fulfil these three criteria, they were considered in principle to be at high risk for GVHD. To prevent GVHD in chimeric recipients, Gorantla et al.79 transplanted irradiated limb allografts into pretreated recipients with T-celldepleted BMT. All limb allografts survived up to 5 months without clinical signs of GVHD. To prevent GVHD after limb allograft, regulation of the chimeric cell may be necessary. Lethal GVHD after conventional BMT has not been demonstrated because T cells were depleted in vitro and the bone marrow cell count was adjusted to 10e100 million. Following limb allograft/vascularised bone marrow allograft, all donor cell types including haematopoietic stem cells and stromal cells can migrate into the recipient. We have no data on the total number of bone marrow cells that migrate into the limb graft. CYP/GeCSF/FK506 combination therapy resulted in the prolonged survival of limb allografts, but more studies aimed at the control of chronic GVHD are necessary.

Future Composite tissue transplantation offers immense potential, but the riskebenefit balance must be carefully considered.95 At present, macrochimerism is one of the most promising strategies for the induction of immunotolerance. However, preconditioning of the recipient must be a serious LimbTx (V. BMT) G-CSF Pretreatment Cyclophosphamide

4 days Check Chimerism level Check graft survival FK506 ; 28 days

Day -2

Day 0

Day 3

Day 28

Figure 5 Cyclophosphamide, G-CSF and FK506 protocol (Muramatsu et al.).

1013 treatment for the non-vital organ transplant. Total body irradiation is the most toxic form of ablation. Recently, less toxic non-radiation-based protocols have been developed using nonlethal cytoreduction, such as fractionated or thymus irradiation, with additional immunotherapy.96 These nonmyeloablative techniques lead to mixed chimerism and a low incidence of GVHD. There seems to be a direct relationship between the dose of bone marrow cells, the level of chimerism achieved, and the tolerogenic efficacy.

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