Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression

Abstract

Objective: To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure.

Background: Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol").

Methods: Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring.

Results: All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates.

Conclusions: Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Trial registration: ClinicalTrials.gov NCT00722280.

Conflict of interest statement

Disclosure: The authors declare no conflicts of interest.

Figures

FIGURE 1

Immunosuppression, kidney function, and peripheral blood WBC/lymphocyte/eosinophile counts are summarized for patients 1–5 (A–E). Tacrolimus dose was adjusted in individual patients to achieve target serum trough levels. The frequency of acute rejection episodes was 3 (patient 1), 1 (patient 2), 3 (patient 3), 1 (patient 4), and 1 (patient 5) (A–E). Patients received bolus steroids in the event of high-grade acute rejection that was nonresponsive to topical therapy. Rejection episodes resolved in all cases without requirement for a second maintenance immunosuppressive drug. In contrast to previous reports (1), no steroid-resistant episodes of acute rejection were observed with this regimen. All 5 patients are currently being maintained on tacrolimus monotherapy. Trough levels range between 4 and 6 ng/mL in patient 1 (A), 8 and 10 ng/mL in patient 2 (B) and patient 3 (C), and 10 and 12 ng/mL in patient 4 (D) and patient 5 (E). Side effects included transient increases in serum creatinine and hyperglycemia after transplant that initially required insulin but was then managed with glipizide 5 mg BID (patient 2, B), and a deep-vein thrombosis in the left lower extremity requiring coumadin treatment as well as a single episode of hyperuricemia that was treated with colchicine (patient 3, C). Patients 1 and 2 (A and B, respectively) required isoniazid prophylaxis with 300 mg QD after incidental and unanticipated exposure to a tuberculous patient while in the hospital.

FIGURE 2

Rejections manifested as mild lymphohistiocytic and eosinophilic perivascular inflammation primarily involving the superficial dermal capillaries, and secondarily, the adnexal structures. acute rejection was associated with eosinophilic predominant infiltrates in 1 recipient (patient 1). The inflammation extended variably into the epidermis and/or adnexa, causing epithelial cell necrosis/apoptosis. Angiography and high-resolution ultrasound biomicroscopy did not reveal any vascular alterations. Adnexal and dermal fat capillaries were positive for C4d in patients 2, 3, and 5. Strong diffuse capillary C4d was noted in patient 5. Tissue and capillary endothelial C4d deposits were minimal (patient 2) and weak (patient 3), and negative (patients 1 and 4). C4d deposits were not accompanied by margination of monocytes and/or neutrophils in the microvasculature. None of the patients demonstrated evidence of luminal narrowing secondary to myointimal proliferation or increased intima medial thickness on high-resolution ultrasound biomicroscopy of radial and ulnar arteries at 1 year. Blood flow parameters remained unchanged. Patient 1: right ulnar artery luminal diameter 2.677 mm, intimal wall thickness 0.261 mm (January 18, 2011). Patient 2: right radial artery luminal diameter 1.96 mm, intimal wall thickness 0.22 mm (June 7, 2010). Patient 3: right radial artery luminal diameter 2.049 mm, intimal wall thickness 0.226 mm (February 3, 2011). Patient 4: right radial artery luminal diameter 1.642 mm, intimal wall thickness 0.326 mm (January 5, 2011). Patient 5: right radial artery luminal diameter 2.1 mm, intimal wall thickness 0.16 mm (January 5, 2011).

FIGURE 3

A, Both human leukocyte antigen class I and class II donor-specific alloantibodies were first observed within the first month after transplant. Changes in donor-specific alloantibodies were limited to specific haplotypes in each patient, except for patient 1, who initially showed donor-specific alloantibodies against DQ7, which resolved with treatment but later recurred together with DR4 and DQ5 donor-specific alloantibodies. Such changes coincided with admitted noncompliance with immunosuppression. The donor-specific alloantibodies significantly subsided after compliance was re-established. B, Global CD4+ T-cell immune response were measured using the Immuknow™ assay and categorized into low (<225 ng/mL ATP), moderate (226–525 ng/mL ATP), and high (>525 ng/mL ATP). Patients 1, 2, and 4 demonstrated moderate responses over a majority of time points. In patients 2 and 3, the assay indicated a stronger cellular immune reactivity early after transplantation, with a dip during 12–18 months (patient 2) and 3 to 6 months (patient 3). However, recent values have trended to pretransplant levels. In patient 5, the response has varied between low and moderate despite stable target troughs on low-dose tacrolimus monotherapy. C, One-way carboxyfluorescein diacetate succinimidyl ester-MLR to assess T-cell allospecific proliferation. Proliferation of alloreactive CD3+ T cells was measured by carboxyfluorescein diacetate succinimidyl ester dilution (% of carboxyfluorescein diacetate succinimidyl ester -low cells) of CD4+ and CD8+ T cells after 5 days of in vitro stimulation of patient or healthy control peripheral blood mononuclear cells with donor (D) or third (3rd)-party peripheral blood mononuclear at 1:1 ratio. FACS analysis revealed that patient 1 displayed a significant CD4+ T-cell proliferation to donor antigens as opposed to third-party stimulation (donor-specific reactivity). Patient 3 demonstrated CD4+ and CD8+ T cells with normal memory subset distribution, a robust CD4+ and CD8+ T-cell proliferation to third-party stimulation, but lower levels of CD4+ and CD8+ T-cell proliferation to donor stimulation, denoting a quiescent status (donor-specific hyporesponsiveness).