Steroid- and ATG-Resistant Rejection After Double Forearm Transplantation Responds to Campath-1H

Authors


*Corresponding author: Raimund Margreiter, raimund.margreiter@uibk.ac.at

Abstract

We herein report on immunological and histological observations in the first bilateral forearm transplant recipient. The last of three rejection episodes occurring on day 95 after transplantation was resistant to steroid and antithymocyte globulin (ATG) treatment. Histology demonstrated lymphocytic infiltrates, apoptotic and necrotic keratinocytes and focal desquamation of the epidermis. Therapy with Campath-1H, however, resulted in complete restitution of normal skin. This is the first report on a successful rescue therapy with Campath-1H in a severe, steroid- and ATG-resistant rejection. Hence, Campath-1H treatment might be a novel and powerful therapeutic option for multiresistant allograft rejection.

Introduction

As of today a total of 24 hand transplants have been performed in eight centers. Despite recovery of sensomotoric function exceeding initial expectations, composite tissue allotransplantation (CTA) is discussed controversially because hand transplantation is not a life-saving procedure, but recipients have to face short- and long-term risks of immunosuppression. We herein report on immunological and histological aspects of the first double forearm transplant. Our observations provide novel insights into immunological aspects of CTA and demonstrate the response to Campath-1H of an otherwise therapy-resistant high-grade rejection.

Methods

A 41-year-old sportive male lost both hands and distal forearms in an accident involving a high-voltage electric current in March 2000. The use of myoelectrical prostheses allowed him to reach a fair level of satisfaction, but, encouraged by the favorable results of our first hand recipient, he asked to have a bilateral transplant himself. The complex surgical procedure was performed on 17 February 2003. The operation included transposition and fixation of forearm muscles to the recipients humerus in addition to reconstruction of bones, nerves, vessels and skin. There was a four HLA-antigen donor/recipient mismatch and the lymphocytotoxic crossmatch was negative. Total ischemia time was 155 min and 153 min for the right and left transplant, respectively.

Antithymocyte globulin (ATG, Fresenius Medical Care AG, Bad Homburg, Germany) was given at 5 mg/kg for days 0–3. Prophylactic immunosuppression consisted of methylprednisolone (750, 250, 125 mg on days 0–2), which was then switched to prednisolone with rapid taper to 25 mg on day 9, FK506 at a dosage to achieve trough levels of 20–25 ng/mL and mycophenolatmophetil at 2 × 1g.

Previously, a scoring for skin rejection in a CTA has been described in a swine model (1). For definition of rejection severity in humans, samples were investigated with particular regard to the epidermal lymphocytic interphase reaction, keratinocyte necrosis as well as dermal/epidermal separation. The proposed grading system is based on own observations in more than 80 skin biopsy samples of four transplanted limbs and published data (Table 1) (2,3). To our knowledge, this grading represents the first scheme for rejection of the skin component in a CTA in humans.

Table 1.  Grading applied for determination of limb allograft rejection severity
DescriptionGradeHistology
  1. *Observed in animal models only.

Minimal rejectionGrade IPerivascular lymphocytic and eosinophilic infiltrates
Mild rejectionGrade IIAdditional interphase reaction in epidermis and/or adnexal structures
Moderate rejectionGrade IIIDiffuse lymphocytic infiltration of epidermis and dermis
Severe rejectionGrade IVaNecrosis of single keratinocytes and focal dermal-epidermal separation
 Grade IVb*Necrosis and loss of the epidermis

Results

Apart from a small necrotic area at the left proximal forearm requiring a skin autograft there were no surgical complications. Maculopapular cutaneous lesions, however, occurred as early as day 10 after transplantation. In punch-biopsies a lymphocytic perivascular infiltrate was indicative for mild rejection (grade I, Table 1). Lesions were scattered over both grafts and biopsies taken at unaffected sites revealed normal skin. The rejection promptly responded to 3 × 500 mg of methylprednisolone and topical steroids. Lesions recurring on both forearms on day 46 with biopsy showing low-grade rejection (grade I, Table 1) were treated with 2 × 20 mg of the chimeric IgG1κ monoclonal CD25 Ab Basiliximab. Following this therapy, lesions disappeared completely. On day 95 a third rejection episode occurred. Systemic (500, 250, 250, 250 mg) and local steroid treatment failed to reverse rejection. ATG (ATG Thymoglobuline, Imtix-Sangstat, Lyon, France) administered at 2 mg/kg for 4 days caused lymphocytopenia, but had no effect on progression of this process. Histology described extensive lymphocytic infiltrates in the dermis/epidermis together with apoptotic and necrotic keratinocytes on both transplants. Blisters occurring in superficial epidermal layers and in particular at the basal membrane resulted in epidermal desquamation (grade IVa, Table 1; 1A, 2A in Figure 1). Immunohistochemistry identified the majority of infiltrating cells as CD3-positive T lymphocytes with less than 5% CD20 and CD79a-positive B lymphocytes. The infiltrates did not change after ATG therapy. As the rejection process was rapidly progressing despite high-dose steroids and ATG, the possibility as well as the potential risks of treatment with Campath-1H were discussed in detail with the patient. After informed consent was given, Campath-1H was administered at 20 mg i.v. on days 109 and 110 after pretreatment with 250 mg of methylprednisolone. Simultaneously, MMF was discontinued and FK506 reduced to achieve trough levels of 10–15 ng/mL. In response to this treatment, most of the lesions disappeared within 2 days and skin normalised completely within 2 weeks. White blood cell count at 2 weeks after administration of Campath-1H was 6700 G/l, lymphocytes dropped from 22.5% to 3% after ATG and to 0.5% after Campath-1H. Follow-up skin biopsies confirmed disappearance of lymphocytes and finally total restitution of normal skin histology. Five months later the patient was still free of rejection. Immunosuppression at that timepoint consisted of FK506 (trough level 8–10 ng/mL) and 10 mg of prednisolone.

Figure 1.

Steroid- and Antithymocyte globulin (ATG)-resistant rejection at day 109 after transplantation (1A). Histology demonstrated excessive lymphocytic infiltrates and desquamation of the epidermis (→) as signs of sever CTA rejection (2A). After treatment with Campath-1H the lesions disappeared completely within 2 weeks and complete restitution of normal skin histology was observed (1B, 2B).

Cytomegalovirus (CMV) prophylaxis resulted in neutropenia, therefore valganciclovir was withdrawn and granulocyte colony stimulating factor was started. When the patient developed CMV infection, cidofovir was given and a sustained response was achieved. Under trimethoprim/sulfamethoxazol coverage no other infectious complications occurred.

Discussion

This case demonstrates that exceptional amounts of immunosuppression may not be able to prevent rejection and thus the high immunogenicity of CTAs. In contrast to previous hand transplants, forearm muscles here are of donor origin. The skin closure was performed at the elbow level, giving a transplanted skin area of approximately 2030 cm2 (Dubois equation, approximation of 11% of total surface). In contrast to solid organs, the skin as the main target of CTA rejection is of little relevance for its function, nor does it influence hemodynamic or metabolic stability. However, loss of the immune barrier must be avoided in order to prevent local and systemic infection as well as the development of chronic rejection.

Rejection in this patient showed a heterogeneous pattern of affected skin. Biopsies taken close to the lesions but not exactly from the affected areas showed normal histology. Therefore, the exact site of skin sampling is of utmost importance. Morphological changes of severe CTA rejection included destructive processes such as cell death and desquamation of the epidermis. Observations made upon treatment, however, provide first evidence that even such an advanced degree of skin lesions is reversible.

Steroid-resistant rejections occur in 4–8% of kidney transplants, of which 65–85% respond to antilymphocyte preparations such as ATG or OKT3 (4). Long-term outcomes with this therapeutic approach, however, are disappointing. Recently, a strong association between steroid resistance and a CD20+ B-cell infiltrate was described for renal allograft rejection, calling for a treatment targeting B-lymphocytes (5). The Campath-1H antibody is directed against the CD52 epitope expressed on T cells, B cells, monocytes and NK cells. Promising results have been reported for the use of Campath-1H induction therapy in kidney, liver, intestinal and recently abdominal wall transplantations, which led us to use this antibody for treatment of an otherwise lost CTA (6,7).

To our knowledge, this is the first report on a successful rescue therapy of a steroid- and ATG-resistant allograft rejection with Campath-1H. This case proves complete reversibility of a high-grade rejection. Apart from a CMV infection which was controlled with Cidofovir, the patient experienced neither infectious complications nor severe drug-related side-effects. Therefore, treatment with Campath-1H might be a powerful therapeutic option for multiresistant CTA as well as solid organ transplant rejection.

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