The clinical era of composite tissue allotransplantation (CTA) was inaugurated by the first successful hand transplantation performed in 1998. Ever since 53 recipients have received an upper extremity allotransplantation and 14 recipients a face allotransplantation (data presented at the 10th Meeting of the International Society of Hand and Composite Tissue Allotransplantation, Atlanta, April 7–9, 2011).
During this pioneer period, we have learned that immunosuppressive drugs used in solid organ transplantation are able to ensure survival of heterologous tissues transplanted for the treatment of complex soft tissue defects, and that we are also able to detect and treat acute rejection (AR) episodes, which occur frequently after transplantation (1). However, the insufficient long-term follow-up and the small number of transplants prevent us from knowing the evolution of this type of transplants and the features of chronic rejection. Indeed, CTA is a new field of transplantation and many questions still remain unanswered.
Herein, we will review the different aspects of composite tissue allograft rejection, immunosuppression management and the possible mechanisms involved in the long-term acceptance of these grafts.
Composite tissue allografts are made of histogenetically different tissues including skin, connective tissue, muscle, bone, bone marrow, nerves, and blood vessels. Skin was considered the most antigenic tissue as proposed by Murray (2) in his relative scale of antigenicity of tissues and organs. However, on the basis of their experimental studies, some authors concluded that no single tissue is dominant in primarily vascularized limb allografts; moreover, they demonstrated that a whole limb allograft elicits a less intense immune response than each individual component of the composite tissue allografts (3).
Clinical experience seems to confirm the contention that skin is the main target of AR. Indeed, the first clinical signs of AR manifest on the skin: the suspicion is based on visual inspection, and then confirmed by histological examination. Whether the dominant immune response is really directed toward the skin is uncertain, because information on the involvement of the other components of composite tissue allografts is difficult to obtain. Indeed, much fewer data are available on the pathologic findings of deeper tissues during AR. It seems, however, that even during severe rejection, the changes found in underlying tissues (muscles, nerves, bones, and tendons) are less severe than those present in the skin (4, 5).
AR reactions manifest clinically as erythematous macules, diffuse redness (Fig. 1), or asymptomatic papules over the allografted skin (4–6). Microscopically, they show characteristic, although nonspecific, changes involving mainly the dermis and the epidermis, that may extend to the hypodermis in the case of severe rejection (Fig. 2). The earliest changes consist in a perivascular lymphocytic infiltrate in the superficial and mid dermis, predominantly made of CD3+/CD4+ T-cells, with smaller proportions of CD8+ and TIA-1+ cytotoxic T-cells, FoxP3+ T-regulatory cells and occasional CD68+ histiomonocytic cells of recipient’s origin (7). In more severe rejection, this infiltrate may fill the dermis and invade the epidermis (exocytosis). The epidermis is initially spared in the early phase of AR; at later stages it shows exocytosis and keratinocyte necrosis or apoptosis associated with basal keratinocyte vacuolization. More rarely the epidermis shows spongiosis (intercellular edema) or lichenoid changes (orthokeratotic hyperkeratosis, hypergranulosis, acanthosis, and band-like subepidermal infiltrate), similar to those observed in (lichenoid) graft-versus-host disease. In the case of very severe rejection, the epidermis (and its appendages, hair follicles and sweat glands) may show extensive necrosis. In those severe cases, the infiltrate may extend to the hypodermis and contain also eosinophils (4). On the basis of these changes, a specific score (Banff score 2007) has been established to assess the severity of AR (8). This system comprises the following five severity grades (8):
- Grade 0 (no rejection): no or rare inflammatory infiltrates;
- Grade I (mild rejection): mild perivascular infiltration. No involvement of the overlying epidermis;
- Grade II (moderate rejection): moderate-to-severe perivascular inflammation with or without mild epidermal and adnexal involvement (limited to spongiosis and exocytosis). No epidermal dyskeratosis or apoptosis;
- Grade III (severe rejection): dense dermal inflammation associated with and epidermal involvement (basal keratinocyte vacuolization, keratinocyte apoptosis, necrosis);
- Grade IV (necrotizing AR): frank necrosis of epidermis or other skin structures.
In the case of facial allotransplantation, biopsies obtained from the allografted oral mucosa show qualitatively similar changes, which are as a rule more pronounced than those found concomitantly on the skin (9–11). The explanation for this discrepancy is unclear; it could include a higher density of vessels and antigen-presenting cells (dendritic and endothelial cells) in mucosa versus skin.
It should be reminded here that the above pathological changes are not specific for AR as they can be found in a number of inflammatory, infectious, or proliferative dermatoses (12). Ancillary techniques have been applied in an attempt to increase the specificity of AR diagnosis, such as immunophenotyping of infiltrating cells or detection of C4d in the allografted skin. The composition of the cell infiltrate is not very discriminative, because it is similar to that found in most inflammatory cutaneous reactions. The presence of FoxP3+ T-regulatory cells, detectable in the allograft several years postgraft (13), is interesting, although its prognostic significance remains unclear. The presence of C4d deposits in the skin and their significance is somewhat controversial. In our experience such deposits are exceptionally, if ever, detected in the allografted skin and mucosae (14). In some studies such deposits have been reported in the skin with and without signs of AR, albeit in the absence of concomitant donor-specific antibodies (15, 16). On the other hand, vascular C4d deposits may be found in inflammatory dermatoses unrelated to rejection. It seems therefore that humoral rejection does not play a significant role in CTA (17) and that the usefulness of C4d detection in diagnosing CTA rejection is questionable. In support of this no clear evidence of an antibody-mediated alloresponse in experimental limb transplantation has been demonstrated (18).
It is interesting to note that, as for most cutaneous tissue inflammatory reactions, the immune response is essentially T-cell mediated and the cytotoxic activity of these T cells is donor-specific, as shown in the first face transplantation recipient (19). The predominance of the T-cell response during CTA rejection probably explains the efficacy of conventional immunosuppression therapy, which primarily targets the T-cell response, to prevent and treat cutaneous AR.
Although the immunosuppressive drugs currently used in solid organ transplantation usually ensure CTA viability, the majority of patients experience at least one episode of AR in the first year after transplantation. According to the International Hand and Composite Tissue Transplantation Registry (1) 85% of the hand-grafted patients and 54.5% of the face grafted patients presented at least one episode of AR in the first posttransplant year. On the other side, the high rate of AR episodes reported in this field of transplantation might be due to the easy diagnosis of AR, as the corresponding lesions are easily seen and confirmed histologically on biopsy specimens taken from them under direct inspection. In addition, despite the high incidence of AR episodes, graft survival rate reaches 96% at 1 year and no clear evidence of chronic rejection has been found in compliant recipients on long-term follow-up (1).
The late AR episodes often occurred when patients were not adherent to the immunosuppressive treatment or when the latter was decreased for various reasons (i.e., side effects or team decision) or after a viral infection, above all cytomegalovirus infection. It is interesting to note that herpes simplex virus preceded the occurrence of an AR episode in face transplant patients (1, 19), suggesting that viral infection might activate antigen-presenting cells during an inflammatory process leading to AR. All AR episodes were reversible in compliant patients, provided they were promptly diagnosed and treated. The choice of AR treatment (1) is based on the Banff score grade, the frequency of the episodes, and the sensibility to steroid treatment. In the majority of cases the episodes were reversed by increasing oral steroid dose (12.9%) or by using intravenous steroids (87%), or with administration of polyclonal (Thymoglobulin) or monoclonal antibodies (Campath-1H) in the other cases. In addition, local immunosuppressants were often used although their efficacy remains unproven; these do not seem to be sufficient to reverse episodes of severe AR without additional systemic immunosuppressive treatment. For the first time in CTA, extracorporeal photochemotherapy was used in few cases of face allotransplantation, although at present it is difficult to propose it as antirejection therapy for CTA (20).
At present, there is no convincing evidence that subclinical rejection, defined by microscopic skin infiltration in the absence of clinical signs of rejection, should be treated to reduce the risk of long-term chronic rejection, but these patients should be carefully monitored.
Despite a high incidence of AR, the occurrence of chronic rejection in CTA might be much rarer than in solid organ transplantation. At present, insufficient data are available to define specific changes of chronic rejection in CTA. The Banff 2007 classification has not yet included features of chronic rejection (8). Clinicopathologic features suggestive of chronic rejection could include myointimal proliferation of arterioles, loss of adnexa, nail changes, skin and muscular atrophy, and fibrosis of deep tissues (8).
We have recently investigated (17) all allograft structures by histology, magnetic resonance imaging, ultrasonography, and high resolution peripheral quantitative computed tomography scan in four bilateral hand-grafted patients and one facial allotransplantation without founding any evidence of lesions that could suggest chronic rejection, namely dermal fibrosis or vascular stenosis. The absence of circulating antihuman leukocyte antigen antibodies might explain the absence of C4d deposition and of graft vasculopathy. All patients had a follow-up longer than 1 year and were treated with triple-immunosuppressive therapy combining tacrolimus, mycophenolate mofetil (MMF), and low-dose steroids. The use of a triple-immunosuppressive therapy in these patients and their adherence to the treatment may have also contributed to these findings.
An acute arterial thrombosis in a unilateral hand-grafted recipient was reported to occur 275 days after transplantation (1), but its cause is not clearly established; therefore, it is difficult to affirm that acute ischemia of the grafted upper extremity in the absence of other previous signs, is a clinical manifestation of chronic graft rejection. On the other hand, although a recent experimental murine study (21) showed that graft vasculopathy is the last lesion to occur during the chronic rejection process, pathological examination of the removed hand showed proliferative vasculopathy, consistent with the possibility that the patient, who was receiving only tacrolimus as immunosuppressive treatment and had presented several episodes of AR, which had not been treated with systemic therapy, was developing chronic rejection.
Current data thus suggest that composite tissue allografts are relatively resistant to chronic rejection, suggesting that in this type of transplantation the mechanisms involved in chronic rejection might differ from those involved in solid organ transplantation.
One possibility is that composite tissue allografts (particularly upper extremities and face when the mandible is grafted) function as a vascularized bone marrow transplant (VBMT). Experimental studies in rats (22, 23) have clearly demonstrated that limb composite tissue allografts function as VBMT with the development of a stable mixed chimerism. However, in clinical experience, peripheral blood microchimerism was detected for a very short period in merely two hand recipients in Louisville (24), in two face recipients and in one hand recipient in Lyon. The absence of a stable microchimerism might be explained by the insufficient number of hematopoietic stem cells contained in the adult upper extremities and in the mandible, or by the rejection of the donor bone marrow by the recipient’s immune system. Thus, the very promising results obtained in rodent VBMT model cannot be transferred to the clinical practice without recipient conditioning.
Our group documented in the first bilateral hand transplantation that epidermal Langerhans cells (LC) remain of donor origin up to 10 years after graft (25, 26), although an occasional LC of recipient’s origin was found in the epidermis of 1 patient shortly after an episode of AR in the first posttransplant year. The maintenance of donor epidermal LC in the allografted skin might have contributed to prevent other significant rejection episodes to develop. Studies in hand-allografted patients have demonstrated the presence of CD24+/CD25+/FoxP3+T-regulatory cells in donor skin at different time points of follow-up (13, 27) in several patients. Other authors did not detect such cells in skin biopsy samples in the absence of rejection, and found that FoxP3 expression was increased on rejection episodes at later time points after transplantation (28). T-regulatory cells seem to play a crucial role in immunoregulation modulating the recipient’s immune response to donor-specific antigens by establishing a regulatory feedback loop. T-regulatory cells in the recipient lymphoid tissue may protect the allograft from an initial attack, while these cells when present in the allograft may help to downregulate the effector cells that have infiltrated it. Actually, the role of CD24+/CD25+/FoxP3+T cells in the skin of composite tissue allografts remains unclear, including their clinical relevance in terms of immunoregulation and tolerance induction.
The absence of chronic rejection in CTA might also be related to the ability of the skin graft to heal without fibrosis thanks to the prompt diagnosis and treatment of AR episodes. Moreover, whether nonimmunological factors exist (29) that could contribute to chronic graft dysfunction in CTAs as in renal transplantation, remain unknown.
Immunosuppression and Complications in CTA
The large majority of recipients have been maintained on immunosuppression therapy similar to that used in solid organ transplantation, consisting of tacrolimus, steroids, and MMF. The induction therapy included antithymocyte globulin, basiliximab, and more recently Campath-1H.
Although steroid-sparing maintenance and switch from tacrolimus to sirolimus have recently been used in CTA, in the early postoperative period all recipients received tacrolimus because of the stimulatory effect of this drug on the synthesis of axotomy-induced growth-associated protein (GAP-43) that seems to promote nerve regeneration (30).
Use of conventional immunosuppression in CTA is associated with the complications usually reported in solid organ transplantation. The main complications reported in the International Registry of Hand and Composite Tissue Transplantation (1) are metabolic ones (above all hyperglycemia, increased creatinine values with several cases of renal function deterioration, arterial hypertension), infections (above all cytomegalovirus reactivation or infection, Herpes virus infection, and bacterial infection) and malignancies (two lymphoproliferative diseases and one nasal basal cell carcinoma). These complications prompted different strategies aimed to minimize the maintenance immunosuppression or to induce donor-specific tolerance.
At present, it is difficult to demonstrate the superiority of one immunosuppressive regimen over another due to the lack of prospective randomized studies and to the limited number of grafted patients. Indeed, the superiority of the regimen based on low dose steroids, tacrolimus, and MMF, which has been successfully used by the majority of the teams, over that based on donor cell infusion associated to depleting induction therapy followed by calcineurin inhibitor monotherapy, has not been demonstrated; similarly, the superiority of an induction therapy, based on alemtuzumab instead of antithymocyte globulins has not been shown.
The first attempt to improve long-term acceptance was made in the first patient with face allotransplantation (19), who was administered bone marrow cells on days 4 and 11 after transplantation in addition to induction therapy (antithymocyte globulins) and the conventional triple-maintenance therapy. In this case, the absence of durable donor chimerism and tolerance might be explained by an insufficient hematopoietic engraftment due to poor hematopoietic stem cell quality or an insufficient conditioning regimen. Moreover, it did not influence the patient’s immunological “profile” so that 5 years after transplantation, the maintenance therapy is based on low steroid dose, sirolimus, and MMF. Donor cell infusion associated to depleting induction therapy followed by calcineurin inhibitor monotherapy is considered an interesting approach in CTA, although up to now the induction of adequate tolerance has not been achieved (data presented at the 10th Meeting of the International Society of Hand and Composite Tissue Allotransplantation, Atlanta, April 7–9, 2011).
At present, no CTA recipient proved to be spontaneously tolerant; indeed, it was noted in the first hand allotransplantation and in all recipients who discontinued the immunosuppressive therapy that consequent rejection of the graft inevitably occurs (1, 4).
Mixed allogenic chimerism induces donor-specific tolerance in a wide spectrum of allografts (31, 32), including some experimental composite tissue allografts (33), but the major obstacle to widespread application of bone marrow infusion to achieve mixed chimerism is graft versus host disease, which would be unacceptable in a nonlife saving transplantation (34).
We have learned that (i) an immunosuppressive treatment similar to that used in solid organ transplantation allows composite tissue allograft survival and function, despite a high rate of AR episodes; (ii) adherence to the immunosuppressive therapy is essential; (iii) the graft can be easily monitored and the AR episodes promptly treated do not seem to adversely influence graft survival and function; and (iv) the relevance of donor-specific antibodies seems less than in solid organ transplantation as humoral rejection does not seem to play an important role in CTA.
We have to resolve the following issues: (i) to what extent we can taper the immunosuppressive load in the long term; (ii) the incidence and the features of chronic rejection in CTAs; and (iii) the effect of donor marrow infusion in chronic rejection and consequently in graft survival. Hopefully, the increasing number of CTA patients and the longer follow-up will permit to answer these questions.
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