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Evidence for Allograft Rejection in an Islet Transplant Recipient and Effect on ß-Cell Secretory Capacity

Departments of Medicine, Division of Endocrinology, Diabetes, and Metabolism (M.R.R.), Pathology and Laboratory Medicine (M.K., J.K.), and Surgery, Division of Transplantation (J.F.M., A.N.), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6149

Address all correspondence and requests for reprints to: Michael R. Rickels, M.D., University of Pennsylvania School of Medicine, Division of Endocrinology, Diabetes, and Metabolism, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104-6149. E-mail: rickels{at}mail.med.upenn.edu.

	Abstract

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Context: The majority of islet transplant recipients experience a gradual decline in islet graft function, but the identification of islet-specific immune responses remains uncommon.

Objectives: The aim was to present a case in which decline in islet graft function was accompanied by the appearance of islet donor-specific alloantibodies and demonstrate the effect on ß-cell secretory capacity, an estimate of functional ß-cell mass.

Setting: The study was conducted at the Transplant Center and General Clinical Research Center of the University of Pennsylvania.

Results: A 42-yr-old woman with type 1 diabetes who had a living-related kidney transplant received two intraportal islet infusions of a total 17,525 islet equivalents per kg body weight under daclizumab, prednisone, tacrolimus, and rapamycin immunosuppression. She became insulin independent, but 4 months later, the rapamycin was discontinued for associated colitis. She remained normoglycemic for another 6 months before manifesting impaired fasting glucose and requiring 5–10 U insulin daily. The decline in clinical islet graft function coincided with the detection of islet donor-specific human leukocyte antigen class I antibodies. ß-Cell function and secretory capacity were assessed by the insulin secretory responses to iv glucose, arginine (AIR_arg), and glucose-potentiated arginine (AIR_pot) before and at alloantibody detection. The acute insulin response to glucose was almost entirely lost, whereas the AIR_arg and AIR_pot both decreased by approximately 50%.

Conclusions: Because the AIR_pot, a measure of ß-cell secretory capacity, provides an estimate of functional ß-cell mass, this case documents that islet graft loss can coincide with donor human leukocyte antigen sensitization and that the effect on ß-cell mass may be best estimated from the AIR_arg or AIR_pot.

	Introduction

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ALLOGENEIC ISLET transplantation can restore insulin secretion for patients with type 1 diabetes; however, the majority of islet transplant recipients experience a gradual decline in graft function (1, 2). Loss of islet graft function is presumably mediated in part by alloimmune or recurrent autoimmune mechanisms (3), but it is often difficult to delineate a precise mechanism in any individual patient. Measures of islet graft function can be derived from the insulin or C-peptide secretory responses to the iv administration of glucose, arginine, or glucose-potentiated arginine, and used to estimate the functional ß-cell mass (4, 5). In the present report, we document the appearance of donor-specific human leukocyte antigen (HLA) antibodies in an islet transplant recipient that was temporally associated with a decline in functional ß-cell mass.

	Subject and Methods

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Case

A 42-yr-old woman had developed type 1 diabetes at 9 yr and received a living-related kidney transplant at 40 yr with prednisone, tacrolimus, and rapamycin as immunosuppression. She experienced frequent severe episodes of hypoglycemia (6) that were complicated by impaired symptom recognition (unawareness) despite receiving lispro insulin (30 U daily) via a continuous sc infusion "pump" for the previous 7 yr.

On examination, the height was 62 in., and the weight was 66 kg (body mass index 24 kg/m²); there was decreased vibratory and 10 g monofilament sensation in both feet. The glycosylated hemoglobin (HbA_1c) was 7.7%, and there was no evidence of C-peptide secretion after ingestion of Boost (Novartis Nutrition Corp., Minneapolis, MN) (Table 1). The islet transplantation protocol was approved by the institutional review board of the University of Pennsylvania, and the patient provided written informed consent to participate.

From 4–5 months after islet transplantation, the patient experienced a protracted diarrheal illness with colonic ulcerations attributed to rapamycin toxicity when no other etiology could be identified. The rapamycin was tapered, and she was continued on prednisone and tacrolimus for immunosuppression (Table 1). The colitis resolved, and she remained metabolically stable for the next 6 months.

In the 11th month after transplant, the fasting serum glucose had increased to 114 mg/dl, and Neutral Protamine Hagedorn insulin was started at 5 U daily (Table 1). At 12 months after transplant, with insulin held overnight, the serum glucose was 132 mg/dl fasting and 267 mg/dl 90 min after ingestion of Boost with a stimulated C-peptide of 3.6 ng/ml; the HbA_1c remained 6.1%. The patient changed to 10 U glargine insulin daily, but her insulin requirement gradually increased, and by 21 months after transplant lispro insulin was added at meal times. At 30 months after transplant, she had returned to her pre-transplant insulin dose of approximately 30 U daily, the HbA_1c had increased to 7.4%, and the C-peptide level was at the lower limit of detection, indicating that the islet graft had clinically failed. She experienced her first severe hypoglycemic episode since undergoing islet transplantation over 21/2 yr earlier and was reinitiated on insulin "pump" therapy.

Immunological studies

HLA typing was performed on the patient and living kidney donor using DNA-based methods: sequence-specific oligonucleotide probes for class I and sequence-specific primers for class II. HLA class I confirmation on the first islet donor was performed by serological typing using the microcytotoxicity assay. Class II typing information was obtained from the United Network for Organ Sharing. DNA-based typing was performed on the second islet donor for class I and II antigens. Standard three-color flow cytometric T and B lymphocyte crossmatches were performed to assess HLA compatibility (9).

HLA alloantibodies were evaluated using a combination of assays, including flow panel reactive (allo-) antibodies (PRAs), flow specificity, flow single antigen bead assays, and/or Luminex specificity and single antigen bead assays [One , Inc., Canoga Park, CA (10)]. Islet autoantibodies against GAD65 and IA-2 and insulin autoantibody were assayed retrospectively from frozen plasma collected at 3, 9, 12, 13, and 15 months after transplant by immunoprecipitation of radioactively labeled molecules at the Barbara Davis Center (Aurora, CO).

Metabolic studies

Metabolic studies were performed in the General Clinical Research Center as previously described (5). The patient underwent testing in the third and 12th months after islet transplantation, with the iv glucose and arginine tests conducted on consecutive days. The same tests were performed in healthy nondiabetic control subjects (n = 10) (5). The metabolic studies protocol was approved by the institutional review board of the University of Pennsylvania, and all participants gave their written informed consent to participate.

The iv glucose tolerance test involved baseline blood sampling at –15, –10, and –5 min before the injection of 0.3 g/kg of 50% glucose over a 1-min period starting at t = 0. Additional blood samples were collected at t = 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20 min after injection. The arginine stimulation test entailed baseline blood sampling at –5 and –1 min before the injection of 5 g of 10% arginine hydrochloride over a 1-min period starting at t = 0. Additional blood samples were collected at t = 2, 3, 4, and 5 min after injection. After the baseline arginine stimulation test, a hyperglycemic clamp technique (11) using variable rates of 20 and 10% glucose solutions was used to achieve a plasma glucose concentration of 230 mg/dl. After 45 min of the glucose infusion, a 5-g arginine pulse was injected again with identical blood sampling.

Plasma insulin and C-peptide were measured in duplicate by double-antibody RIAs (Linco Research Inc., St. Charles, MO). The acute insulin response to glucose (AIR_glu) was calculated as the mean of the insulin levels obtained at 3, 4, and 5 min after the glucose injection minus the mean of the baseline levels (12). The acute insulin response to arginine (AIR_arg) was calculated as the mean of the 2-, 3-, 4-, and 5-min values minus the mean of the baseline values (11). The acute insulin response to glucose-potentiated arginine (AIR_pot) was similarly calculated as the mean of the post-arginine values minus the mean of the pre-stimulus values (11).

	Results

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Immunological studies

The HLA type of the patient, the living-related kidney donor, and the two deceased islet donors are presented in Table 2. All donors were major blood group compatible with the patient. Also, the patient’s serum crossmatches against donor T and B lymphocytes were negative before each transplantation. The PRA was negative (0%) for both class I and class II HLA before and after the kidney transplant, as well as before, and at 3 and 9 months after the islet transplants (Table 1). In the 12th month after transplant, the PRA became weakly positive against both class I (19%) and class II (16%) HLA. Further analysis by flow specificity beads revealed antibodies against class I antigens A24,25 present on the transplanted islets and against class II antigen DQ2 present on the transplanted kidney (Tables 2 and 3). Luminex techniques showed that the antibodies against the islet donor antigens A24,25 were absent at 9 months and present at 12 and 15 months after transplant, whereas the antibody against the kidney donor DQ2 was present at 9, 12, and 15 months (Table 3). Despite the continued presence of alloantibodies detected by the more sensitive Luminex assays at 15 months, the flow PRA had become negative (0%). The identification of the islet-specific alloantibodies corresponded with a worsening of glycemic control and the requirement for low-dose insulin therapy. Immunosuppression was intensified by increasing the dose of tacrolimus, as reflected in the increase in trough levels (Table 1). No islet autoantibodies against GAD65 or IA-2 were detected; however, insulin autoantibodies were consistently positive between 3 and 15 months after transplant.

At 3 months after transplant, the AIR_glu was 97% of the control mean (Fig. 1, left), AIR_arg was 89% (Fig. 1, right), and the AIR_pot was 39% (Fig. 1, right). At 12 months after transplant, the AIR_glu decreased dramatically to 2% of the control mean (Fig. 1, left). In contrast, the AIR_arg and AIR_pot were both reduced by approximately 50% of their respective value at 3 months, the AIR_arg to 44% of the control mean, and the AIR_pot to 18% (Fig. 1, right). These measured declines in ß-cell function and secretory capacity occurred at the time low-dose insulin therapy was required to maintain glycemic control and coincided with the detection of alloantibodies directed against donor islet antigens.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Plasma insulin levels in response to bolus injections of glucose (left) and arginine (right). The arginine was administered under both fasting and 230 mg/dl hyperglycemic clamp conditions.

	Discussion

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We have previously reported a significant linear correlation between the fasting glucose and ß-cell secretory capacity in islet transplant recipients (5). Consistent with this, our patient’s secretory capacity likely began to decrease at 10–11 months after transplant as the fasting glucose increased from a mean of 96 mg/dl over the previous 9 months to 106 and 114 mg/dl, respectively. By 12 months, when the alloantibodies were detected, the fasting glucose had increased to 132 mg/dl, and after that continued to deteriorate gradually over the next 12 months despite the intensification of immunosuppression and reinitiation of insulin therapy.

There is also a relationship between fasting glucose and AIR_glu, such that AIR_glu is nearly absent when the fasting glucose is more than 115 mg/dl (13). This relationship can be interpreted to mean either that fasting hyperglycemia results from a reduction in the insulin secretory response, such as from a decrease in ß-cell mass, or that hyperglycemia results in refractoriness to glucose-stimulated insulin secretion. In this report, glycemia was normal with the use of 5 U of daily insulin as indicated by an HbA_1c of 6.1% when near absent AIR_glu was demonstrated; furthermore, the fasting glucose was 82 mg/dl on the morning of the iv glucose tolerance test, so glucose toxicity cannot explain the defect in AIR_glu reported here. More likely, a decrease in functional ß-cell mass indicated by the decline in ß-cell secretory capacity was responsible for both the development of mild hyperglycemia and absent AIR_glu. Because the AIR_glu is lost earlier than the response to arginine during the progressive ß-cell loss that accompanies type 1 diabetes (14), as well as after islet transplantation (5), the AIR_arg may provide a better estimate of functional ß-cell mass than AIR_glu in the setting of a marked reduction (<20% of normal) in which the AIR_glu is often absent, a conclusion consistent with a study previously reported (12). Indeed, in our patient the AIR_arg similarly predicted a 50% loss in ß-cell mass as estimated by the decline in secretory capacity (AIR_pot).

The sensitization to donor antigens in our patient was likely a consequence of insufficient immunosuppression with prednisone and tacrolimus because the rapamycin had been discontinued approximately 7 months earlier for associated colitis. Antibodies were directed against class I antigens from both islet donors and against a class II antigen from the donor kidney. This allogeneic immune response seems to explain the initial 50% reduction in functional ß-cell mass and subsequent gradual loss of the islet graft. Although the function of the transplanted kidney has remained clinically stable, the slight increase in serum creatinine over the 21/2 yr of follow-up (Table 1) may be due to chronic rejection, in which kidney graft dysfunction may sometimes not appear until several years after the appearance of alloantibodies (15, 16).

The loss of the islets but not the haplotype-matched kidney graft in this report may be explained by the multiple HLA mismatches on the islet grafts. A previous report documented sharp declines in islet graft function that occurred simultaneously with the detection of multiple islet donor-specific class I antibodies in three transplant recipients who also had a previous stable functioning kidney graft; two of these subjects each developed one class I antibody against the kidney graft, but only one experienced a temporally associated increase in serum creatinine (17). In another report, immediate allograft rejection occurred in three islet recipients who received a combination of cultured and cryopreserved islets from multiple donors mismatched with at least 14 class I antigens (3).

In the case reported here, allograft rejection can explain the islet graft loss; however, the majority of islet recipients experience a gradual decline in graft function that is not understood. The detection of PRA has been associated with decreased graft function at 1 yr after transplantation (1), but new antidonor antibodies were not detected in a recent multicenter trial in which subjects experienced a similar rate of decline in graft function, except in cases in which immunosuppression was withdrawn for already clinically failed grafts (2). Other mechanisms that may be at play in islet graft loss include recurrent autoimmunity (2, 18, 19, 20) or, possibly, nonimmune mechanisms related to the intrahepatic site of engraftment (21, 22) and exposure to potentially toxic orally administered immunosuppressive drugs that reach increased concentrations in the portal circulation (23). A role for recurrent islet autoimmunity in the case presented here is unlikely because antibodies against GAD65 and IA-2 did not develop at the time of graft loss, nor was any increase in the already present insulin autoantibodies detected. It remains possible that alloimmune processes may more often evade our conventional flow PRA methods of detection and that more sensitive approaches, such as using Luminex beads, may be required.

In conclusion, islet graft loss can coincide with the appearance of donor-specific HLA antibodies and the effect on ß-cell mass may be best estimated from the acute secretory responses to the iv administration of arginine or glucose-potentiated arginine.

	Acknowledgments

 
We thank the patient for her participation in the Juvenile Diabetes Research Foundation-Penn trial of islet transplantation and the metabolic studies protocol; Eileen Markmann and Maral Palanjian for coordination of the clinical trial; Rebecca Mueller for coordination of the metabolic studies; the nursing staff of the University of Pennsylvania General Clinical Research Center for their subject care and technical assistance; Dr. Heather Collins of the University of Pennsylvania Diabetes Endocrinology Research Center for performance of the RIAs; Drs. Liping Yu and George Eisenbarth of the University of Colorado Barbara Davis Center for Childhood Diabetes for conducting the autoantibody assays; and Dr. Eline Luning-Prak for conducting a critical review of the manuscript.

	Footnotes

 
This work was supported by the Juvenile Diabetes Research Foundation and by the Public Health Services Research Grants M01-RR00040 (General Clinical Research Center), P30-DK19525 (DERC), U42-RR016600 (Penn Islet Cell Resources Center), U01-DK070430 (to A.N.), and K12-RR017625 (to M.R.R.) from the National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 8, 2007

Abbreviations: AIR_arg, Acute insulin response to arginine; AIR_glu, acute insulin response to glucose; AIR_pot, acute insulin response to glucose-potentiated arginine; HbA_1c, glycosylated hemoglobin; HLA, human leukocyte antigen; PRA, panel reactive (allo-) antibody.

Received January 24, 2007.

Accepted April 26, 2007.

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