Minimizing immunosuppression in hand transplantation

Abstract

Hand transplantation, despite all initial skepticism, has developed from myth to reality over the past decade and has shown highly encouraging immunological and functional outcomes. However, the requirement of life-long, multidrug immunosuppression bearing the risk of serious side effects still remains the limiting factor for widespread clinical application of this novel reconstructive modality. Recent advances in immunosuppressive drug development and the design of novel cell-based therapeutic strategies that take into consideration the unique immunological and biological aspects of vascularized composite allografts have shown favorable results with regard to minimization of immunosuppressive medication and tolerance induction in both translational animal studies and first clinical trials in reconstructive transplantation. This review provides an overview of the current available conventional treatment protocols and novel immunosuppression minimization concepts for hand transplantation, which ultimately could significantly favor the risk–benefit ratio for this life-changing type of transplant.

Keywords::

• hand transplantation
• immune regulation
• immunosuppression minimization
• stem cells
• tolerance induction

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Release date: 17 October 2012; Expiration date: 17 October 2013

Learning objectives

• Upon completion of this activity, participants will be able to:

• • Distinguish the most antigenic tissue in hand transplantation

• • Evaluate the issue of acute rejection after hand transplantation

• • Identify elements of common immunosuppressive therapy after hand transplantation

• • Analyze emerging treatments for immune therapy after hand transplantation

Financial & competing interests disclosure

Editor

Elisa Manzotti

Publisher, Future Science Group, London, UK

Disclosure: Elisa Manzotti has disclosed no relevant financial relationships.

CME Author

Charles P Vega, MD

Health Sciences Clinical Professor; Residency Director, Department of Family Medicine, University of California, Irvine, USA

Disclosure: Charles P Vega, MD, has disclosed no relevant financial relationships.

Authors and Credentials

Gerald Brandacher, MD

Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Disclosure: Gerald Brandacher, MD, has disclosed no relevant financial relationships.

WP Andrew Lee, MD

Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Disclosure: WP Andrew Lee, MD, has disclosed no relevant financial relationships.

Stefan Schneeberger, MD

Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA;

Center for Operative Medicine, Department of Visceral, Thoracic and Transplant Surgery, Innsbruck Medical University, Innsbruck, Austria

Disclosure: Stefan Schneeberger, MD, has disclosed no relevant financial relationships.

Activity in hand transplantation worldwide

The clinical realization of limb transplantation has been desired since the myth of Cosmos and Damian and attempted first in modern history in 1964 in Ecuador [1]. Failure of this first hand transplant and the substantial immunological challenges preventing skin to be transplanted successfully between genetically nonidentical individuals led to the belief that transplantation of skin-bearing body parts could not be achieved.

In 1991, Lee et al. provided data indicating that in the context of combined or ‘composite’ tissue transplantation, the skin would not be rejected as stringently as when skin or other tissues were transplanted individually [2]. Furthermore, results from large animal trials have indicated that with the newer generation of immunosuppressive drugs established for use in solid organ transplantation, rejection of a limb could be prevented or significantly delayed [3]. When human hand transplantation was eventually revisited in 1998, the first clinical cases clearly indicated that with multidrug immunosuppression similar to that used in solid organ transplantation graft survival could be achieved [4–10]. Close to 80 hand transplantations have been performed since then and the overall outcome with regards to graft survival and function has been satisfactory [11–13]. It is fair to say that transplantation of a hand at wrist level has been established as a suitable therapeutic option for hand loss and a meaningful alternative to prostheses. The major limiting factor towards large-scale performance of hand transplantation, however, is the need for long-term high-dose immunosuppression. Concurrent with the advancements in solid organ transplantation, minimization or elimination of the need for long-term high-dose and/or multidrug immunosuppression is the primary goal of global research in this novel field.

Hand allografts may be considered as immunologically challenging, and it appears that both the skin as well as the vasculature are specific targets for an alloimmune response. Conventional immunosuppressive treatment applied in hand transplantation currently includes either poly- or mono-clonal antibody induction followed by a tacrolimus, mycophenolate mofetil (MMF) and triple steroid drug combination for maintenance therapy [11,12]. Graft loss can be prevented in patients compliant with such a protocol but episodes of skin rejection occur in the majority of cases. Two patients have died following combined hand and face transplantation and a combined upper and lower extremity transplantation [12,14]. In addition to several unconfirmed cases of hand loss in China, one patient in the USA has lost a hand allograft as a consequence of arterial intimal hyperplasia on day 275 and one patient in France requested amputation following progressive rejection of the skin after stopping immunosuppression at 29 months [15,16]. However, in patients compliant with immunosuppression, all episodes of skin rejections were reversible and no grafts were lost due to immunological complications [11].

Cell-based protocols including donor bone marrow (BM) and/or stem cells are considered a suitable tool to establish macro-, micro- or mixed-chimerism, which remains the most reliable approach for tolerance induction following organ transplantation [17–22]. Donor BM cell infusion has been successfully used in both solid organ transplants and vascularized composite allotransplantation (VCA) and has resulted in a reduction or elimination of long-term immunosuppression in solid organ transplantation [23–30]. For development of innovative immunomodulatory treatment protocols (detailed later), VCA bares the advantage of continuous and sensitive monitoring and targeted biopsy sampling guided by visual inspection of the skin.

Appearance & mechanisms of acute skin rejection after hand transplantation

The ability to visually monitor the procedure and the ease of performing protocol biopsies allows for close observation of skin rejection in hand transplantation. Rejection of the skin appears clinically as focal or diffuse maculopapulous lesions with diverse dissemination and color intensity [7–10,31]. Skin lesions can either be scattered over the allograft or present in a confluent pattern. Apart from a skin rash, rejection of the palm and nailbeds was described as an atypical manifestation of acute rejection in patients exposed to mechanical stress to the palms or due to heavy manual use of the allograft [32].

For histopathological characterization of skin rejection, a specialized Banff classification system has been established [33]. The histological features of Grade I rejection include mainly lymphocytic perivascular aggregates in the dermis. In mild rejection stages, the inflammatory infiltrate is found in the interstitium and interphase between dermis and epidermis and/or adnexal structures [33]. Moderate rejection is characterized by a cellular infiltrate within the epidermis. When progressing further, necrosis of single keratinocytes can be observed in advanced stages of rejection, resulting in focal dermal–epidermal separation and finally necrosis and loss of the epidermis [33].

Immunohistochemically, the infiltrate in acute skin rejection is comprised predominantly by CD3⁺ T cells spreading, with progression of rejection, from the perivascular space towards the dermis and then the epidermis. Among the CD3⁺ cells, CD8⁺ cells are more prominent than CD4⁺ cells. Depending on the grade of rejection, 10–50% of infiltrating leukocytes stain positive for CD68. A total of 0.5–5% of cells are positive for CD20 [34].

While cellular rejection has been well characterized, the understanding of antibody-mediated rejection (AMR) in hand transplantation is very limited. The assessment of C4d complement depositions in protocol skin biopsies has been performed routinely by some but not all hand transplant centers in an attempt to monitor for AMR. Thereby, deposits of C4d were found in approximately 50% of all skin biopsy samples investigated and no correlation with graft function or cellular rejection could be established [35–37].

However, donor-specific antibodies (DSAs) have been found in the serum of some hand transplant recipients but did not necessarily correlate with C4d deposition in skin biopsies [36]. A recent study using a rat osteomyocutaneous transplant model for VCA showed that these grafts are rejected in an accelerated fashion but not hyperacutely in the presence of allosensitization and preformed antidonor antibody. The rejection of a vascularized composite allograft in sensitized recipients was mainly cell mediated and differed mechanistically from that for solid organ transplants [38]. At this point, little evidence supports the relevance of AMR in hand transplantation, however, the observation that DSA occur after hand transplantation in some patients requires further investigation into the long-term effect of these antibodies.

Regarding the mechanism of skin rejection in hand transplantation, adhesion molecules on the endothelium of the graft vasculature have been demonstrated to be associated with the presence and severity of rejection. ICAM-1 and E-selectin and also leukocyte functional antigen-1 have been shown to be upregulated upon rejection. Inhibition of E- and P-selectin by, for example, Efomycine M administered in the subcutaneous compartment of the graft in a rat hindlimb transplantation model enabled allograft acceptance, indicating that adhesion molecules promote skin rejection in VCA [34].

Current immunosuppression used in hand transplantation

Since vascularized composite allografts are derived from genetically disparate cadaveric donors, recipients require life-long immunosuppression to prevent rejection of the transplant. Thus, routine clinical application of hand transplantation has been dampened by the necessity for long-term, high-dose multidrug maintenance immunosuppressive therapy. This also led to considerable concern regarding the risk–benefit balance for hand transplantation due to potential adverse effects and toxicities of those immunosuppressive drugs including infection, diabetes, hypertension, nephrotoxicity or even malignancy.

Black et al. in 1985 were the first to demonstrate that long-term graft survival in a rodent hindlimb transplantation model can be achieved across major histocompatibility barriers using the calcineurin inhibitor cyclosporine A, which subsequently paved the way for clinical VCA [39]. Additional developments in immunosuppressive drug therapy and the introduction of new and potent agents such as tacrolimus and MMF in the 1990s provided further evidence that skin-bearing grafts can be transplanted in large animal translational models with reproducible results and ultimately initiated the modern era of hand transplantation. Immunosuppressive strategies applied to hand and upper extremity transplantation today are largely extrapolated from regimens used in solid organ transplantation. However, there is no standard immunosuppressive protocol established for reconstructive transplantation. The overall amount of immunosuppressive medication required to ensure graft survival is comparable or even slightly higher than it is for kidney transplantation [40]. However, such protocols for hand transplantation have resulted in a 100% patient and 96% graft survival at 1 year after transplantation (in patients compliant with immunosuppressive medication) – an outcome that has not been achieved in any other field of transplantation [12].

Conventional immunosuppressive strategies rely, for the most part, on agents that halt the robust immunological attack on the graft in a nonspecific manner. According to the International Registry for Hand and Composite Tissue Transplantation, the majority of hand transplant patients received either polyclonal (antithymocyte globulins [ATGs]) or monoclonal (alemtuzumab, basiliximab) antibody preparations as an induction agent followed by a high-dose triple-drug combination for maintenance therapy comprising of tacrolimus, MMF and steroids [11,12]. The tacrolimus trough levels were adjusted to 10–15 ng/ml during the first 1–3 months and 5–10 ng/ml thereafter by most centers. With regard to steroid management, prednisone doses were rapidly tapered in the early post-transplant period and then maintained at lowered doses (5–15 mg/day) for 6–12 months in the majority of the patients. Of the 33 patients included in the most recent report of the international registry – all recipients received tacrolimus as the baseline immunosuppressive agent – 26 received MMF and 27 received steroids. During the follow-up period, eight patients were converted from tacrolimus to the mTOR inhibitor sirolimus with the rationale to minimize renal side effects, improve glycemic control and to potentially avoid chronic vascular changes (myointimal hyperproliferation) and neurotoxicity [41]. In five cases steroids were withdrawn, two recipients received steroids and low doses of tacrolimus and everolimus and two patients received sirolimus and MMF [11,40]. In those patients who did not receive induction therapy (n = 2) or were not started on triple immunosuppressive regimens (n = 2), topical steroid and tacrolimus ointments were applied in addition to systemic immunosuppressive medication. As of now, induction therapy followed by maintenance immunosuppression with at least a dual drug combination at optimum dose can be considered the most widely used treatment regimen for hand transplantation.

Overall such conventional regimens have proven sufficient to prevent early immunological graft loss, but have not been able to prevent acute rejection. Of all hand transplant recipients, 85–90% experienced at least one acute rejection episode within the first year after transplantation regardless of their induction or maintenance immunosuppressive treatment protocol. This incidence is significantly higher than what is currently seen in solid organ transplantation where acute rejection rates are less than 10% in the first year after renal transplantation [42]. However, this is probably partly due to the fact that a hand transplant represents a visible graft, which enables immediate diagnosis of rejection based on even minor changes in appearance. Therefore, all episodes of skin rejection encountered and diagnosed clinically were confirmed with skin punch biopsies.

Similar to maintenance immunosuppressive regimens, treatment of acute rejection in hand transplantation, mirrors therapies currently applied in solid organ transplantation. According to the International Registry for Hand and Composite Tissue Transplantation, treatment of acute skin rejection episodes included high-dose boluses of intravenous steroids in 60% of all reported cases. In 30% of the recipients, however, increasing oral steroids was sufficient to reverse skin rejection. In only 7.5% of patients, in which rejection episodes were steroid-resistant, the treatment consisted of aARGs, basiliximab or alemtuzumab [11]. In addition to systemic treatment, topical application of immunosuppressive ointments (tacrolimus, corticosteroid or flumix) was performed taking advantage of the unique possibility of treating skin rejection locally. As underimmunosuppression was thought to be the most important cause for rejection maintenance, immunosuppression was increased at the same time in most cases.

When a second rejection episode was encountered, steroids were given intravenously with or without topical ointment in several patients. Alternatively, ATG, basiliximab or topical drugs were only given in a few patients. In one case of a steroid- and ATG-resistant rejection, alemtuzumab was administered and resulted in full restoration of normal skin histology [9]. Thus far, no graft has been lost due to acute irreversible rejection or immunological sequelae in patients compliant with their medication in the Global experience of hand transplantation. Only in one case, namely that of the first hand transplant performed in France in 1998, did progressive acute rejection following noncompliance with immunosuppression lead to lichen-like lesions of the skin and progressive loss of function and prompted the patient to request amputation of the hand [15].

Experimental & clinical data on minimization of immunosuppression in hand transplantation

A specific feature of a hand transplant is that such grafts, unlike solid organ transplants, consist of various heterogeneous tissue types and components of different antigenicity including skin, muscle, cartilage, tendon, nerve, blood vessels, bone and vascularized BM. Owing to the fact that the skin is thought to be most antigenic, historically, hand transplantation has been regarded as an immunological challenge [43,44]. This high immunogenicity of the skin has been mainly attributed to skin-specific antigens in the epidermis as well as the presence and high frequency of skin-specific APCs such as Langerhans cells and dermal dendritic cells, which are extremely efficient at initiating innate immune responses to trigger sensitization and priming of recipient T cells [45]. Following skin, the muscle, bone cartilage, blood vessels and nerve predictably induce a relatively lower immune response [46]. Considering the obvious downsides of multidrug immunosuppression and its various, sometimes severe, side effects, there is evident need for novel concepts of systemic immunosuppression in hand transplantation to prevent rejection.

Along these lines, several specific features of VCA might be advantageous to achieve this goal such as, for example, the unique ability to constantly monitor the graft by simple visual inspection that allows for early diagnosis and individualized treatment of rejection. In addition, although the long-term follow-up after hand transplantation is still limited, it appears that if skin rejection is treated sufficiently, such rejection episodes do not impart graft function and survival. In a previous study performed by the authors, individual vascularized limb tissues (skin, subcutaneous tissue, muscle, bone and blood vessels) or an entire hindlimb were transplanted across a stringent histocompatibility barrier utilizing a rat model [2]. No single tissue predominated in the elicited immune response, and the entire limb allograft actually revealed a weaker alloimmune response as compared with its individual components. Rather, the various tissue components interacted with the host immune system in a complex but predictable pattern with differing timing and intensity in this model [2]. In addition, certain types of VCA contain immunocompetent elements such as BM and a vascularized BM niche that may either hasten or slow down the rejection processes or potentially even result in a graft versus host disease (GvHD). If these factors observed in experimental animal model can be ultimately verified in the clinical setting, then they will not only ultimately govern the immunogenicity of these allogeneic tissues, but also define and dictate potential immunomodulating/minimization strategies that might require different approaches to those currently used in solid organ transplantation.

Certain types of VCAs can include varying amounts of vascularized bone and BM and might thereby function as a vascularized BM transplant by itself [47–50]. Such a graft represents a continuous source of donor-derived stem cells, which have been demonstrated in animal models to favorably modulate the host immune response [51]. In such experimental settings, induction of donor-specific tolerance was attributed to the BM component and to specific immunomodulatory protocols (myeloablative and non-myeloablative strategies) permissive for BM engraftment [23]. In particular for hand transplantation, representing a non-life-saving procedure, non-myeloablative regimens are critical since intensive cytotoxic myeloablative protocols bearing the risk of significant morbidity and mortality are contraindicated.

BM-based therapeutic principles have consistently shown a beneficial effect of supportive cellular therapy on organ as well as VCA survival [52–54]. Underlying mechanisms include, for example, effects such as macro- and micro-chimerism, and exhaustion and deletion of the recipient’s alloreactive T-cell clones. These insights can now help to refine treatment protocols aiming to support long-term graft survival on minimal maintenance immunosuppression [54]. However, one potential disadvantage of transplanting a graft with functional immune effector cells and renewable sources of donor-derived stem cells is the potential for these cells to mature into allogeneic T cells and NK cells and to attack the host, resulting in GvHD.

This recently led to an intense search of optimal cell types and stem cell-based protocols with nonmyeloablative induction to prolong hand allograft survival and to take advantage of the biological features of VCAs to induce donor antigen-specific immune tolerance.

As outlined above, routine therapy after hand transplantation has been a nonspecific suppressive multidrug regimen, administered to dampen the recipient’s immune system bearing an increased risk for opportunistic infections, metabolic disorders or malignancies [41]. For this reason, transplant-related immunological research currently focuses on developing new drugs and biologic agents, which aim to specifically target key pathways and receptors during allogeneic T-cell activation, as to avoid the unwanted negative effect on other immune lineages. This selective and targeted approach significantly reduces undesirable complications while still enabling graft survival [55].

Furthermore, various modifications have been applied over the years to the established immunosuppressive protocols used in hand transplantation (Box 1). Such modifications include, but are not limited to, steroid sparing/avoidance attempts, alemtuzumab induction, cell-based immunomodulatory protocols, conversion from tacrolimus to the mTOR inhibitor sirolimus for long-term therapy or the use of topical steroid and tacrolimus ointments to reduce the overall amount of required systemic immunosuppression [56].

Of note, a small number of solid organ transplant recipients have been documented to tolerate their grafts and allow minimization or even withdrawal of immunosuppression without promoting rejection [57]. This phenomenon of spontaneous donor antigen-specific immunological tolerance, also referred to as operational tolerance, has given rise to the realization that there is a feasible way to overcome histocompatibility barriers, not by means of immunosuppression but rather by immunomodulation or immunoregulation. However, complete withdrawal of immunosuppressive maintenance therapy even in the most favorable scenario of liver transplantation is only successful in about 20% of recipients in prospective studies to date [58]. This is most likely due to well-described mechanisms of calcineurin inhibitors to inhibit immunoregulation and an unfortunate lack of availability of well-defined and specific immune-monitoring assays to detect immunoregulation and to predict unresponsive, tolerogenic clinical phenotypes. Spontaneous tolerance, achieved in experimental organ transplant models, is often the result of an exhausted host-versus-graft immune response mediated by donor-derived hematolymphopoietic cells. Such a state of operational tolerance is achieved when the immune response towards the transplant is exhausted because the specific cell clones mediating rejection are deleted or controlled in the circulation by activation-induced or programmed cell-death, or the suppressive action of Tregs and specific cytokines. Furthermore, maintenance of immunosuppression-free engraftment is facilitated when a small number of donor leukocytes persist in the recipient (microchimerism) long term [59]. The concept here is that the induction of mixed donor/recipient chimerism could provide a new T-cell repertoire tolerant to both recipient and donor and might subsequently facilitate earlier removal of immunosuppressive medication. Studies have also demonstrated the possible regulatory effects of poly- or mono-clonal antibodies (e.g., thymoglobulin, alemtuzumab) in deleting alloreactive effector cells while preserving Tregs and their components thereby favoring operational tolerance [60]. However, the limited existing data suggest that induction approaches may not actually increase the ability of immunosuppression withdrawal but rather might favor a state in which minimized doses of immunosuppressive medication can be achieved [58,61].

Nevertheless, all these strategies ultimately aim to facilitate the development of central and peripheral tolerance mechanisms, which result from clonal deletion of donor-reactive T cells during development in the thymus (central tolerance) and the induction of Tregs that suppress alloreactive T cells that escape intrathymic deletion (peripheral tolerance). This concept of operational tolerance would be particularly appealing for the setting of hand transplantation since those transplants even though significantly improving the quality of life are not considered life saving and have inherent unique biological and immunological features that might favor especially cell-based minimization/tolerance-inducing strategies.

To date, several small and large animal studies show highly encouraging results when transplanting vascularized composite allografts without the need for long-term maintenance immunosuppression [62,63]. Strategies applied in these experimental models include: the use of total lymphoid irradiation; costimulatory blockade (CD28/B7 and CD154/CD40 pathways); selective depletion of alloreactive recipient T and B cells (e.g., αβ-T-cell and CD20-specific monoclonal antibodies); inhibition of lymphocyte trafficking; infusion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and tolerogeneic APCs; as well as donor BM infusion and chimerism induction [34,64–70]. Indefinite graft survival without the need for long-term systemic immunosuppressive treatment was achieved with the use of an immunomodulatory concept employing donor BM cells in complete MHC-mismatched swine heterotopic hindlimb transplant models [71,72]. This concept has been recently translated into a novel clinical cell-based treatment protocol for upper extremity transplantation by a joint team at Johns Hopkins University School of Medicine and the University of Pittsburgh [73]. Patients were pretreated (1–2 h prior to transplantation) with an intravenous dose of 30 mg alemtuzumab for lymphocyte depletion plus 250 mg methylprednisolone. Tacrolimus monotherapy was commenced with target trough levels of 10–15 ng/ml (first month), 8–10 ng/ml (2–3 months), 5–10 ng/ ml (4–12 months) and 3–7 ng/ml thereafter. In addition, all patients received an unmodified donor BM cell infusion (5–10 × 10⁸/kg bodyweight) 2 weeks post-transplantation. This study is currently ongoing but has demonstrated thus far that the protocol is safe, efficacious and well tolerated and has allowed reconstructive transplantation in upper extremity amputees with low-dose tacrolimus monotherapy. The underlying hypothesis of this novel protocol for upper extremity transplantation is that removal of circulating T cells achieved by the administration of a monoclonal antibody (alemtuzumab) followed by low-dose immunosuppression and donor BM cell augmentation in the early post-transplant period would increase the intrinsic tolerogenic properties of the allograft and lead to a state in which both graft- and recipient-derived immune cell clones accommodate each other [61].

Mathes et al. have developed a translational protocol that combines a non-myeloablative induction approach and VCA in a canine model that has led to tolerance to all components of the VCA, including skin, without the need for long-term maintenance immunosuppression [74].

The notion that VCA are different from traditional solid organ transplants due to the fact that they represent the only type of transplants including donor BM and a vascularized BM microenvironment and niche make cell-based approaches that aim to minimize or even entirely avoid maintenance immunosuppression particularly appealing following hand transplantation. This is also encouraged by recent data from living related kidney-transplant recipients, where BM and stem-cell based therapies enabled reduction and elimination of immunosuppressive medication [75–77].

Ever since the seminal experiments of Billingham, Brent and Medawar more than 50 years ago, such cellular transplants have been a premier tool for immunomodulation and the establishment of donor antigen-specific tolerance in the field of solid organ transplantation [78]. With the insights gained in the pathophysiology of vascularized composite allografts over the past decade, there is great interest in utilizing BM-based protocols to prolong survival of vascularized composite allografts and to minimize or even avoid maintenance immunosuppression. The reasons being that: donor-specific blood transfusion has been successfully used as part of induction regimens for solid organ transplants [79,80]; donor BM promotes the opportunity to reduce/avoid maintenance immunosuppression required for graft survival; BM is critical to establish macro-, micro- or mixed chimerism following organ transplantation a known prerequisite for donor-antigen specific tolerance induction; and hematopoietic stem cells and cell types with regulatory properties have been identified to possess tolerogenic properties and have become the backbone of a number of protocols aiming for tolerance induction in transplantation [81].

Along these lines, Barth et al. developed a nonhuman VCA primate model utilizing selectively mismatched cynomolgus macaques of facial segment allotransplantation to elucidate the unique immunogenicity and immunosuppressive requirements of VCA with the addition of concomitant vascularized BM [82]. Heterotopically transplanted facial segment VCA with vascularized BM treated only with tacrolimus and MMF but no radiation or T-cell depletion for induction demonstrated prolonged rejection-free survival, as compared with VCA without vascularized BM that experienced early rejection episodes and graft loss by postoperative day 7–15 [82]. While VCA with vascularized BM demonstrated sporadic low-level macrochimerism, acute and chronic rejection and graft loss occurred after discontinuation of maintenance immunosuppressive therapy. However, radiologic, phenotypic, histologic and genotypic evidence supported the persistence of viable donor BM within the transplanted vascularized BM component. Data from this study further substantiate an important immunoregulatory role of the vascularized BM component in VCA with the potential to minimize the need for immunosuppressive medication. Despite the use of different types and numbers of cells from various different resources – unmanipulated, manipulated, fractionated or not – in combination with established conventional as well as novel immunosuppressive agents and biological, GvHD remains a concern. The implementation of cell-based therapies including most recent developments such as Tregs and tolerogenic dendritic cells might further enable immunomodulation subsequent to hand transplantation and thus optimize outcomes [83,84].

Future research

Although various approaches to tolerance induction focusing on peripheral mechanisms have been shown to be effective in experimental models and clinical renal transplant studies, the establishment of stable mixed chimerism remains the only strategy to have achieved tolerance of skin transplants in preclinical large animal models. However, whether stable chimerism will also be a requirement for tolerance induction after hand transplantation still needs to be determined, particularly as in the setting of living related renal transplantation, in the Massachusetts General Hospital experience, some of the recipients lost their donor-cell chimerism, yet continued to be tolerant and maintain allograft function and survival [75,85]. To confirm the role of donor BM cells for durable chimerism induction in VCA, Siemionow et al. developed an experimental model of bilateral vascularized BM transplantation. This model, which contains only intact bone with BM, confirmed the migratory potential, engraftment and repopulation of donor BM cells leading to the maintenance of chimerism [86].

Mesenchymal stem cells (MSCs) are another potential source of cells with immunoregulatory and tolerogenic properties that have currently much attention [86,87]. As one of their immunomodulatory mechanisms, MSCs are thought to downregulate overshooting immune responses in various different clinical conditions. In addition, MSCs are currently applied to prevent and treat GvHD after BM transplantation and to prevent acute rejection episodes after both cell and solid organ transplantation [88,89]. Most recently, Kuo et al. have shown prolonged allograft survival of vascularized composite tissue grafts by treatment with BM-derived MSCs that were correlated with a significant increase in the percentage of CD4⁺/CD25⁺ and CD4⁺/FoxP3⁺ T cells in both peripheral blood and intragraft tissues in a swine hindlimb model [90].

Despite all these advances to minimize maintenance immunosuppression, overcoming chronic rejection still seems to be a formidable task in most transplant cases for solid organs and continues to limit the best possible long-term outcomes. However, whether similar concerns are warranted for hand transplantation still needs to be determined. Thus far, only a few patients have a follow-up beyond the 10-year mark and the incidence of chronic rejection in hand transplantation as compared with solid organ transplantation seems to be exceedingly low [91,92]. There is only one report in the world series of upper extremity transplantation of graft loss in a patient compliant with immunosuppression that showed vascular lesions such as intima hyperproliferation and luminal occlusion that are reminiscent of chronic rejection in solid organ transplantation. However, in experimental rat hindlimb transplantation models, changes such as intimal hyperproliferation and luminal narrowing/occlusion consistent with chronic rejection or allograft vasculopathy have been shown after repeated episodes of acute skin rejection and frequent lapses in maintenance immunosuppression [93]. Most recently, Kaufman et al. reported some level of vasculopathy in all six of their hand transplant recipients with aggressive and severe intimal hyperplasia observed early post-transplant in two patients [94]. Of concern, in four of their patients standard techniques used for surveillance of rejection (protocol skin biopsies, DSA and conventional vascular imaging modalities) were obviously inadequate for detecting early, potentially reversible, stages of allograft vasculopathy. A high degree of vasculopathy was also reported by Diefenbeck et al. in patients receiving an allogeneic vascularized knee transplant [95].

This underscores the importance of close long-term surveillance and standardized follow-up protocols in hand transplantation in particular with more and more emerging experimental immunosuppression minimization and tolerance-inducing protocols.

Expert commentary

When planning to design novel immunosuppressive treatment protocols for reconstructive transplantation utilizing minimal maintenance, both the medication-specific requirements and the unique biologic features of a hand transplant need to be taken into consideration. Despite the fact that great advances have been made in choosing the most favorable combination of medications from the multitude of available agents to prevent solid organ allograft rejection, the same regimen may not always be optimal when used or attempted in reconstructive transplantation. In addition, immunosuppression minimization strategies designed for solid organ transplantation should be carefully reviewed and applied with due diligence to hand transplantation. However, many questions concerning the concept of immunosuppression minimization strategies in hand transplantation remain, including patient selection, availability of suitable immunomonitoring assays, pretransplant immune activation status and sensitization and donor-recipient HLA compatibility.

As we continue to develop and implement minimization and tolerance-inducing concepts to the novel field of reconstructive transplantation, these attempts must safeguard highest scientific and ethical standards and demonstrate in basic and translational model systems for VCA that such protocols pit neither transplants nor patients at risk. In any case, it will be an exciting but long and winding road for the reconstructive transplant community to ultimately favor the risk–benefit balance of these life-changing type of transplants by minimizing the requirement for maintenance immunosuppression.

Five-year view

Hand and upper extremity transplantation has become a clinical reality with excellent short and intermediate immunological and functional outcomes that have exceeded all initial expectations. Even though the field is still in its infancy, the patient numbers and the trend seen over the past decade with regard to hand transplants performed worldwide clearly indicate the dawn of a new era in reconstructive transplantation. Despite all these achievements and the highly encouraging results, there are still several remaining challenges for upper extremity transplantation that will need to be addressed to make such procedures become the future standard of care. One of the most obvious remaining challenges, as discussed in this review, is the requirement for long-term, high-dose multidrug maintenance immunosuppressive therapy. How new and promising strategies to minimize, or even avoid, the requirement for maintenance immunosuppression will ultimately change the risk–benefit balance for upper extremity transplantation, due to potentially improved outcomes and less adverse effects and toxicities related to immunosuppressive medications, needs to be determined. Another remaining challenge apart from minimizing immunosuppression is to develop novel strategies to enhance nerve regeneration in particular when considering expanding the indications for upper extremity transplantation and performing proximal humerus level or full arm transplants in the future. Such advances will be critical to improve and optimize functional outcomes in this field. The world experience with hand transplantation has demonstrated that excellent short and intermediate outcomes can be achieved, but the question regarding long-term results and the role of graft vasculopathy and chronic rejection remains unanswered. A most recent report by Kaufman et al. has shown vasculopathy in six out of six hand transplant recipients. Aggressive and severe intima hyperproliferation was observed early post-transplant in two out of six of the recipients in their patient series. Of concern, standard monitoring techniques were inadequate to detect early potentially reversible stages of allograft vasculopathy in four out of the six recipients [94]. These data show that chronic rejection will be a topic in the next 5 years that requires due diligence and intense further investigation as we are expanding the number of transplants performed and about to develop novel treatment protocols for hand transplantation.

Although more and more centers are embarking on reconstructive transplantation and implementing hand transplant programs, the number of procedures performed to date is still too small to conduct any randomized clinical trials. Therefore, establishing standardized outcome measures such as uniform criteria and assessment tools for motor and sensory recovery or improvement in quality of life that will allow the comparison of data across institutions and across protocols will be an important task within the next years. Along these lines, the implementation of regulatory oversight by professional societies and governing agencies will also be important to further streamline any issues related to organ donation, allocation and data management unique to hand transplantation.

Box 1. Current clinical immunosuppression minimization protocols.

• • Steroid minimization/avoidance

• • Calcineurin inhibitor sparing – switch to mTOR inhibitors long term

• • mTOR or mycophenolate mofetil-based maintenance regimens

• • Alemtuzumab induction

• • Cell-based immunomodulatory protocols followed by long-term immunosuppression minimization/weaning

• • Non-myeloablative induction strategies

• • Biologic agents (costimulatory blockers, belatacept)

Key issues

• • Hand transplantation has become a clinical reality with highly encouraging immunological and functional outcomes.

• • Conventional immunosuppressive protocols as used in solid organ transplantation have proven successful to enable graft-survival and to prevent graft-loss in hand transplantation but bear the risk of significant side effects and toxicities.

• • Balancing risks and benefits remains the prime task for hand and upper extremity transplantation.

• • Small and large animal studies have demonstrated that vascularized composite allografts have unique biological and immunological features that might favor minimization of maintenance immunosuppression and implementation of tolerance-inducing strategies.

• • Several novel clinical protocols are currently implemented for hand transplantation to minimize immunosuppressive medications.

• • Standardized outcome measures and long-term follow up data will be critical to advance this novel field.

References

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Flares of systemic lupus erythematosus during pregnancy and the puerperium: prevention, diagnosis and management

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Activity Evaluation: Where 1 is strongly disagree and 5 is strongly agree

	1	2	3	4	5
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2. The material was organized clearly for learning to occur.
3. The content learned from this activity will impact my practice.
4. The activity was presented objectively and free of commercial bias.

You are caring for a 26-year-old man who has just received a hand transplant. What is the most antigenic component of hand transplants?

• □ A Blood vessels

• □ B Skin

• □ C Nerves

• □ D Bone

The patient is concerned regarding rejection of his new graft. Which of the following statements regarding acute rejection of hand transplants is most accurate?

• □ A Skin rejection always appears in a diffuse pattern across the transplant area

• □ B C4d deposits are universally found in graft skin samples

• □ C 85% to 90% of all hand transplant recipients experience at least one acute rejection episode in their first year after transplantation

• □ D Graft survival is less than 50% after one year

You initiate treatment to prevent graft rejection. Which of the following immunosuppressive medications is part of the standard maintenance regimen after hand transplant?

• □ A Tacrolimus

• □ B Cyclosporine

• □ C Azathioprine

• □ D Alemtuzumab

4. The patient has an acute rejection episode. Which of the following statements regarding emerging immunological concerns following hand transplant is most accurate?

• □ A Hand transplant does not raise the possibility of graft versus host disease

• □ B Donor-derived stem cells may favorably modulate the host immune response

• □ C Cell-based therapies are particularly ill-suited to hand transplants

• □ D The most common response to skin rejection is initiation of treatment with basiliximab