Long-Term Follow-Up in Composite Tissue Allotransplantation: In-Depth Study of Five (Hand and Face) Recipients

Introduction

Composite tissue allotransplants involve transplantation of various tissues including vessels, nerves, skin, bones, and immune cells and entail a significant antigenic load. Different immunosuppressive protocols are used for experimental and clinical composite tissue allotransplantation (CTA) that allow survival of the different components of composite tissue allografts.

Although chronic rejection is difficult to induce in rodent models of CTA (1,2), a recent study using a rat hind-limb allotransplantation model showed pathological changes secondary to multiple acute rejection episodes, involving skin, muscles, bones and finally vessels (3).

On the basis of clinical experience (4) CTAs have shown so far little evidence of chronic rejection and in the ‘Chronic Rejection’ section of the Banff 2007 working classification of skin-containing CTA pathology (5), it is stated that ‘currently, insufficient data are available to define specific changes of chronic rejection in a CTA’.

In solid organ transplantation chronic rejection is characterized by a gradual deterioration of graft function months to years after transplantation accompanied by typical pathological features (6,7). The remarkable pathological feature of the chronic rejection process is intimal hyperplasia accompanied by organ specific lesions. The development of this transplant vasculopathy, referred as transplant arteriosclerosis, is a multifactorial process but its precise pathogenetic mechanisms remain undefined (8).

In order to search for changes related to chronic rejection we have investigated all tissues composing CTAs including skin, bones, joints, nerves, muscles, tendons and vessels as well as graft function. This study was performed in four bilateral hand transplantations and one partial facial allotransplantation, which did not show any apparent clinical signs of chronic rejection.

Material and Methods

Results

Histological aspect of the grafts

Acute rejection episodes were characterized mainly by the presence of a dermal perivascular lymphoid infiltrate made predominantly of CD3+/CD4+ T cells occasionally reaching and penetrating the epidermis. Some FoxP3+Treg cells and TIA-1+ cytotoxic cells were found within the perivascular dermal infiltrate (Fig. 1)

Apart from episodes of acute rejection during the protocol follow-up, skin biopsies did not show significant changes, or showed only minimal ones. Figure 2 shows the pathological aspects of skin biopsies of each bilateral hand-grafted patient taken at two time points of the follow-up. Immunohistochemically, the skin also showed a normal structure, and contained all its normal cell components, including keratin+ keratinocytes, CD1a/CD207 epidermal Langerhans cells, MART-1+ melanocytes, keratin 20+ Merkel cells, factor XIIIa+ dermal dendrocytes, CD34+ blood endothelial cells, S100+ Schwann cells and neurofilament+ axons, suggesting that the skin was both immuno- and melano-competent. Several basal keratinocytes of the epidermis and its appendages (hair follicles and sweat glands) expressed the proliferation antigen Ki67. Deep biopsies showed a sparse interstitial lymphocytic infiltrate in patients 1, 2 and 4, while in patient 3 a mild dermal perivascular lymphocytic infiltrate coming occasionally in contact with the epidermis and a mild interstitial lymphocytic infiltrate between muscle fibers were found.

The sentinel skin flap of the partial face transplantation looked histologically normal (Figure 3).

HR-pQCT system

The presence of metallic plates and screws caused some artifacts in the median part of forearms, particularly in patients 1 and 4.

As reported in Figure 4, all male recipients showed alterations at level of distal tibia and distal radius: a slight decrease in trabecular density and microarchitecture at the distal tibia level, and in patient 2 also at cortices level; these alterations were more pronounced at the distal radius level. Patient 3, a woman, showed normal bone structure at level of distal tibia and radius.

MRI

In the bilateral hand transplantations the presence of metallic plates and screws caused some artifacts in the median part of forearms. The results achieved in these patients are summarized in Table 2. They showed normal structure of bones, nerves and tendons. The muscles (particularly the intrinsic ones) showed a slight hypotrophy and some degree of fatty degeneration (Figure 5).

Table 2. Summary of MRI results in the recipients of bilateral hand transplantation

	Patient 1	Patient 2	Patient 3	Patient 4
Bones	Normal	Normal	Normal	Normal
Tendons	Normal	Normal	Normal	Normal
Muscles	Discrete hypotrophy of intrinsic muscles with fatty degeneration	Trophysm of muscles on left side. Signs of fatty degeneration on right side	Discrete trophysm but with signs of fatty degeneration	Hypotrophy of intrinsic muscles with fatty degeneration
Nerves	Normal	Normal	Normal	Normal
Vessels	Tortuous aspect of ulnary arteries	Normal	Normal	Normal (? Many artifacts)

The vessels were not well appreciable in patients 1 and 4 because of the metallic material; patient 1 showed a slight tortuosity of the ulnar arteries.

In the facial transplant all structures were normal without significant difference between recipient and graft tissues (Figure 3); the vessels looked normal.

Discussion

In the present study the histological and clinical features of chronic graft rejection have been investigated in two types of CTAs, four bilateral hand allografts and a partial facial transplantation, for a period ranging from 2 to 10 years.

The term CTA indicates simultaneous transplantation of skin, muscles, nerves, tendons, bones, vessels and other tissues. These multiple tissues are of ectodermal and mesodermal origin and represent a different antigenic load. The skin has been considered the main target of rejection process and for this reason it was strictly studied during the whole period of follow-up; moreover skin can be easily observed macroscopically and biopsied, thus allowing early detection and consequent prompt treatment of the acute rejection episodes. Apart from them, all patients showed a normal skin structure at level of epidermis, dermis and hypodermis. The main skin cell types (keratinocytes, melanocytes, Langerhans cells, Merkel cells, endothelial cells, dermal dendrocytes, Schwann cells, axons, fat cells) were detectable by routine staining and/or immunohistochemistry. The presence of cycling (Ki67+) basal cells in the epidermis and its appendages (hair follicles and sweat glands) shows that these structures are viable and have the potential of regeneration. The presence of melanocytes and dendritic cells (Langerhans cells, dermal dendrocytes) shows that the skin is able to protect itself from UV light and antigenic challenges, respectively. The presence of normal vasculature and innervation accounts for the normal trophicity of the skin (noted clinically) and its ability to perceive sensitive stimuli. In some patients, skin biopsies occasionally disclosed a mild dermal perivascular lymphocytic infiltrate; however, neither significant vascular changes nor dermal fibrosis were observed. Muscle biopsies disclosed a sparse perivascular lymphocytic infiltrate.

On the basis of these results we also studied all the other tissues of the CTAs at the last time point of the follow-up.

In the bilateral hand-grafted patients MRI and HR-pQCT system were used to study some tissues of CTAs although in hand transplantation these exams showed some limits due to the artifacts caused by the presence of metallic materials.

MRI allowed a qualitative study of the bones and did not show any difference between recipient and graft bones, while the HR-pQCT, which allowed a quantitative study of the bones, showed in male patients a decrease in trabecular density in the recipient's tibia as well as in graft bones, even though in graft radius the alterations involved also the cortices. The reported alterations of bone structure seem to be correlated with several factors, such as nutrition and recipient BMI, immunosuppressive treatment (steroid dose), recipient and donor age.

MRI and ultrasonography were used to study nerves and tendons and did not show any specific alteration in any hand-grafted patient. In addition, electromyography was performed each year after the transplantation showing a reinnervation process at the level of median and ulnar nerves, which improved significantly during the first years after transplantation (11).

Muscles were the only tissue that showed some modifications. Indeed, MRI evidenced a variable degree of muscular hypotrophy, particularly of intrinsic muscles, accompanied by fatty degeneration. These alterations were not considered as signs of chronic rejection as it is known that after denervation rapid degeneration of muscles occurs (12); biochemical (enzyme activities) and morphological changes in muscle fibers are permanent after the first months (13). Changes in muscle activation pattern also lead to muscular atrophy. In addition, ischemia/reperfusion injury could contribute to muscle hypotrophy and fatty degeneration since oxidative damage can induce muscle cell death and degeneration.

The major histomorphological feature of chronic rejection in solid organ transplantation is endoarteritis. During the last meeting of the International Society of Hand and Composite Tissue Allotransplantation (Valencia, Spain, September 2009) an acute arterial thrombosis in a unilateral hand-grafted recipient was reported to occur 275 days after transplantation but its cause was not clearly established: was it a case of chronic graft rejection? At present it is not possible to affirm that acute ischemia of the grafted upper extremity without any other previous signs is a clinical manifestation of chronic graft rejection; however, this case prompted us to investigate particularly the vessels of CTAs. Although a recent experimental study (3) showed that graft vasculopathy is the last lesion to occur during the chronic rejection process, in this study macro- and micro-circulation were investigated by Duplex ultrasounds, MRI and histology. Moreover, all patients underwent nailfold capillaroscopy, which did not detect any changes (data not shown), and at the last time point of the follow-up an angiography in patient 1 at 1 and 10 years after transplantation (data not shown). These exams showed that the main vessels were patent without any stenosis or significant alterations of arterial silhouette, except for some tortuosity of ulnar arteries in patient 1. Histological examination of deep biopsies did not show any arteriolar thrombosis. Despite these reassuring results in the future an angiography will be performed in all recipients at 1 year and each 5 years after transplantation. In addition, on the basis of the results achieved using intravascular ultrasounds in heart transplantation, this interventional procedure might be used to study intimal thickening in CTAs.

Patient 2 developed transiently anti-HLA class II antibodies without showing any vascular alteration and his muscular degeneration was inferior compared with the other hand-grafted patients. We did not detect C4d deposition in CTA specimens (14) including the biopsies of this patient. This is consistent with the fact that during acute rejection, the cellular infiltrate consists mainly of CD4 and CD8 lymphocytes, with only sparse B lymphocytes, and that the rejection process is mediated more by the cellular than the humoral arm of the immune response. Whether clinically significant humoral rejection occurs in CTAs is still unknown.

In these four bilateral hand transplantations neither fibrosis, intimal hyperplasia nor alterations of other tissues, which could be considered as signs of chronic rejection, were detected. In addition, no deterioration of graft function but instead an increase of their ability, at least during the first 5 years after transplantation, was noted.

In the face transplantation no differences between recipient and graft tissues were found upon MRI and ultrasonographic examination. The patient experienced continuous sensorimotor recovery during the follow-up period (15).

Up to now the authors did not detect any sign that could suggest a process of chronic graft rejection by any of the reported procedures. Thus, despite a high incidence of acute rejection, the occurrence of chronic rejection in CTA might be much lower than that in solid organ transplantation, suggesting that mechanisms of chronic rejection might differ from that of solid organ transplantation. Some features of the CTA graft might explain this discrepancy: (i) the graft contains hematopoietic stem cells in the transplanted bones that might be involved in the acceptance of the graft, as reported in rodent models of vascularized bone marrow transplantation, where mixed chimerism is associated with tolerance to CTA (16). However, we did not detect any chimerism in the peripheral blood of our hand transplanted patients; (ii) CD4+CD25+ T-regulatory cells were found in the skin graft of the first bilateral transplant patient (1) 6 years after the graft (17). However, this high percentage of T-regulatory cells was only transient in this patient and was not found in the other patients; (iii) the absence of chronic rejection of the skin might also be related to the ability of the skin graft to heal without fibrosis after the prompt diagnosis and treatment of acute rejection; (iv) the absence of circulating anti-HLA antibodies might explain the lack of evidence of transplant vasculopathy; (v) the persistence of donor derived immunocompetent cells (Langerhans cells) in the grafted skin could also favor graft nonrejection (18).

The use of a triple immunosuppressive therapy in these patients and their adherence to the treatment may have also contributed to these findings. The low incidence of chronic rejection in our CTA cohort prompted us to maintain our patients on triple immunosuppression and not to wean it off in the absence of complications.

In conclusion, a close continuous follow-up and an appropriate study of all tissues composing the CTAs (above all the skin that proved to be the main target of the rejection process) are of paramount importance.

Disclosure

The manuscript was not prepared in any part and was not funded in any part by a commercial organization, including educational grant. All the authors of the manuscript AJT-0-10-01004 have no conflicts of interest to disclose as described by the American Journal of Transplantation.