Calcineurin Inhibitor–Free CD28 Blockade-Based Protocol Protects Allogeneic Islets in Nonhuman Primates

Captive bred adolescent rhesus monkeys (Macaca mulatta) (∼3–18 kg) were used as recipients and donors. The absence of preformed donor-specific antibodies in the recipient was confirmed before transplant. All potential donors and recipients were tested for anti-cytomeglovirus antibodies, and sero-negative recipients were not paired with sero-positive donors.

The donor pancreatectomy was performed under general anesthesia 1 day before transplantation. The splenorenal and splenocolic ligaments were divided so as to mobilize the spleen and tail of the pancreas. The head of the pancreas and second portion of the duodenum were mobilized after Kocher maneuver. After administration of heparin (200 units/kg), the aorta was cannulated just above its bifurcation and the animal was exsanguinated. Cold slush was immediately placed in the lesser sac and behind the body of the pancreas. The body and neck of the pancreas were carefully excised, taking care not to violate the pancreatic capsule.

Rhesus monkey islet isolation was completed via minor modifications of the automated method for human islet isolation (23,24) by using Liberase (Boehringer Mannheim, Indianapolis, IN) at a concentration of 0.47–0.71 mg/ml. A three-layer discontinuos Euroficoll gradient (densities 1.108, 1.097, and 1.037; Mediatech, Herndon, VA) and a Cobe 2991 blood cell processor (Gambro, Lakewood, CO) were used for purification of islets from the pancreatic digest. Samples of the final islet preparation were stained with dithizone (Sigma, St. Louis, MO), and the preparation was assessed by counting the number of islets in each of the following size ranges: 50–100, 100–150, 150–200, 200–250, 250–300, 300–350, and 350–400 μm. The data were mathematically converted to determine the number of islets with an average diameter of 150 μm and were expressed as islet equivalents (IEQ) (25).

Total pancreatectomy without duodenectomy or splenectomy was performed at least 1 week before transplant. The splenic, inferior and superior mesenteric, middle colic, and portal veins were identified and preserved during dissection of the body of the pancreas. The duodenum was mobilized, and branches of the pancreaticoduodenal vessels that entered the pancreas were ligated and divided, leaving the duodenal branches intact. The common bile duct was identified and preserved. The main and accessory pancreatic ducts were ligated and divided, and the pancreas was removed from the abdominal cavity. The procedure was well tolerated, and two forms of pancreatic enzyme replacement were administered postoperatively: Pancrease (pancrelipase enteric coated microspheres; Ortho-McNeil, Raritan, NJ) and Viokase V pancreatic enzyme supplement powder (Fort Dodge Animal Health, Fort Dodge, IA).

Overnight-cultured islets were washed in media and counted to determine the number of IEQ. Islets were then resuspended in 20 ml of media supplemented with 200 units of heparin. Intra-hepatic islet transplantation was performed via gravity drainage of islets into a sigmoid or branch of the left colic vein through a 22-gauge intravenous catheter.

Fasting and postprandial blood glucose levels were monitored (Glucometer Elite; Bayer, Elkhart, IN) twice daily (prebreakfast and postlunch) via ear-stick. Insulin (NPH, Ultralente; Eli Lilly, Indianapolis, IN) was administered three times daily in attempt to maintain fasting blood glucose <300 mg/dl in pretransplant pancreatectomized animals or in those who had rejected their allografts.

Two protocols were tested: LEA29Y-based protocol, rapamycin, and anti–IL-2R monoclonal antibody (mAb) (1) and control protocol treated with rapamycin and anti–IL-2R alone (2). LEA29Y was administered intravenously intra-operatively (10 mg/kg) and on postoperative day (POD) 4 (15 mg/kg). Additional doses of 20 mg/kg were given on POD 14 and every 2 weeks until POD 154. The drug was well tolerated, and there was never any evidence of gross toxicity. The chimeric anti-human IL-2R mAb (0.3 mg/kg i.v.) was administered intra-operatively and on POD 4. Rapamycin was administered orally in a Prima-burger (Bio-Serv, Frenchtown, NJ) treat at 1.25 mg/kg bid POD 0–50 (average plasma drug levels 8–12 ng/ml), 1 mg/kg q.d. POD 50–100, and then tapered to terminate dosing by POD 121. The LEA29Y used in these experiments was provided by Bristol-Myers Squibb (Princeton, NJ). Rapamycin (Rapamune) and anti-human IL-2R mAb (Simulect) were purchased from the Emory University Hospital Pharmacy. Survival of the islet grafts among experimental groups was compared using the Mann-Whitney U test.

The presence of detectable donor-specific allo-antibody was determined using flow cytometry. Peripheral blood leukocytes served as the target cells for the pretransplant analysis. Leukocytes isolated from mesenteric lymph nodes obtained at the time of transplant were the target cells for the posttransplant assays.

Responses were measured by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay using peripheral blood leukocytes obtained from the recipient and donor animals. An equal number of irradiated stimulators (donor leukocytes) and responders (recipient leukocytes) were added to ester cellulose bottom plates (Millipore, Bedford, MA) coated with the capture antibody, mouse anti-human IFN-γ (clone GZ-4; Mabtech, Sweden). After 14–16 h incubation, biotinylated mouse anti-human IFN-γ (clone 7-B6-1; Mabtech, Sweden) was added, unbound antibody was removed, and horseradish peroxidase-Avidin D (Vector, Burlingame, CA) was added. Spots were developed with the substrate 3-amino-9-ethyl-carbazole (Sigma). Each spot represents an IFN-γ-secreting cell; the frequency of these cells can be determined by dividing the number of spots generated by the total number of responder cells plated.

Based on the current success of a protocol using the combination of tacrolimus, rapamycin, and anti-IL-2R mAb (5), we sought to develop a steroid-free, calcineurin inhibitor-free regimen. LEA29Y was used in combination with rapamycin and a chimeric antibody against IL-2 receptor α-chain. The dose and schedule of administration were selected based on previous pharmacokinetic studies of LEA29Y in primates and humans. Using these data, we selected a regimen designed to maintain serum trough LEA29Y concentrations ≥20 μg/ml. Diabetes was induced by surgical pancreatectomy of recipient animals and confirmed by pretransplant intravenous glucose tolerance test. Donor-recipient pairings were defined based on molecular typing using a panel of previously defined major histocompatibility alleles (8 class I and 12 class II) (26–28). Pairings maximized disparity at both class I and II loci. Rejection was defined as two consecutive fasting blood glucose values >125 mg/dl on subsequent days. Intra-portal infusion of allogeneic islets (>10,000 IEQ/kg) resulted in initial restoration of euglycemia and insulin independence in diabetic monkeys in both groups (Fig. 1A and B).

Treatment of pancreatectomized macaques with the LEA29Y-based regimen significantly prolonged islet allograft survival (204, 190, 216, >220, and 56 days, respectively). In contrast, the control regimen (rapamycin and anti-IL-2R) did little to alter the tempo of rejection (both at 7 days) (Fig. 1C). Four of five animals receiving the LEA29Y regimen enjoyed rejection-free survival for the duration of the treatment period (Table 1). Intravenous glucose tolerance test with measurement of insulin and glucose levels confirmed islet function posttransplant (representative animal,Fig. 1D).

At 100 days posttransplant, the dosing of rapamycin was decreased and tapered to zero by day 121. Animals continued to remain insulin-independent while receiving LEA29Y monotherapy. At ∼150 days posttransplant, the remaining islet recipients received their final dose of LEA29Y, ceasing any additional immunosuppressive therapy. As expected, ∼1–2 months after discontinuation of therapy, recipients became hyperglycemic and required exogenous insulin therapy. Histological analysis revealed a mononuclear infiltrate, strongly suggesting rejection as the etiology of the loss of glucose control (Fig. 2B).

The frequency of primed alloreactive T-cells can effectively be detected by using the ELISpot assay, which can discriminate production of IFN-γ at the single-cell level. Peripheral blood samples from islet recipients were analyzed at various time points both pre- and posttransplant for their ability to generate IFN-γ in response to donor antigen. Animals treated with the base regimen alone quickly developed a measurable anti-donor response that coincided with rejection ∼1 week after transplant. In contrast, the frequency of anti-donor IFN-γ-producing cells in animals receiving the LEA29Y-containing regimen was undetectable until therapy was withdrawn (representative animals,Fig. 3A and B). Thus, the LEA29Y regimen effectively blocked the generation of anti-donor T-cell responses as measured by the ability to produce IFN-γ.

Flow cytometry was used to examine the development of anti-donor antibody responses. One animal within the control group generated a strong anti-donor Ab response, whereas the other failed to develop a detectable response, presumably because it was euthanized before the antibody response could be measured (Fig. 3C). In contrast, four of five animals failed to generate an antibody response while receiving LEA29Y therapy. This is consistent with previously reported results using CTLA4-Ig in an islet transplant model (19) as well as our experience in a renal allograft model where recipients failed to generate anti-donor antibodies (20). One animal of five recipients underwent a rejection episode while receiving the LEA29Y therapy and subsequently developed an anti-donor antibody response. As expected, the remaining four animals receiving the LEA29Y regimen consistently developed anti-donor antibody responses around the time of rejection (∼200 days posttransplant, 50 days after the final dose of LEA29Y).

Islet transplantation is quickly becoming a viable treatment option for patients with brittle type 1 diabetes. Recent reports describing steroid-free immunosuppressive regimens, which result in successful insulin independence after islet transplantation, have ushered in renewed optimism for the practical application of islet transplantation. Whereas the elimination of glucocorticoids from immunosuppressive regimens represents a major step forward in the effort to treat type 1 diabetes, the reliance on calcineurin inhibitor therapy for primary immunosuppression may limit the application of this approach. Calcineurin inhibitors have numerous unwanted side effects, including nephrotoxicity, diabetes, hypertension, impaired lipid metabolism, and hirusitism (6–8). Even when drug levels are kept low, significant side effects may develop. This is particularly true in the diabetic patient population where renal function may already be impaired. Indeed, in the most recent reports from Edmonton, two patients with mildly elevated pretransplant creatinine levels had significant decreases in renal function while on calcineurin inhibitor therapy and ultimately required withdrawal of this drug (9). In the same report, two-thirds of recipients developed some degree of glucose intolerance, with one- quarter developing frank posttransplant diabetes thought to be related to the use of tacrolimus. This underscores the appealing and essential nature of a calcineurin inhibitor-free immunosuppressive regimen, particularly for islet transplantation.

Blockade of T-cell costimulatory pathways is a promising strategy for the development of nontoxic immunosuppressive and potentially tolerogenic regimens. This approach targets those T-cells that receive “signal 1” during the period of drug administration. For example, treatment during the peritransplant period is thought to render allo-specific T-cells impotent upon encounter with the new organ or tissue, whereas other T-cells are left unimpaired (29). Blockade of the CD28/B7 pathway has demonstrated remarkable promise in experimental models of autoimmunity and transplantation, making it a particularly appealing immunosuppressive target in islet transplantation, where presumably both auto- and allo-immune obstacles exist. Bluestone and colleagues were the first to describe the potential of CD28 blockade in a large animal transplant model (19). Treatment with CTLA4-Ig was found to significantly, although modestly, prolong islet allograft survival in nonhuman primates (19). Similarly, we have shown that CTLA4-Ig monotherapy does little to prolong renal allograft survival (20). Recently, there have been several reports of long-term survival of islet allografts in nonhuman primate models. Anti-CD40L mAb therapy has shown the most impressive results thus far; however, similar to experiments using a renal transplant model, tolerance was not achieved, as withdrawal of therapy eventually resulted in rejection (30,31). In another encouraging report, Thomas et al. (32) recently described the use of an anti-CD3 immunotoxin and the immune modulatory agent DSG (15 deoxyspergualin) to dramatically prolong islet survival in streptozotocin-induced diabetic primates. Although promising, these reports used therapeutics whose clinical potential at the present time is still uncertain.

Our data using LEA29Y, a novel mutant form of CTLA4-Ig, in the Rhesus islet allograft model is consistent with in vitro evidence indicating that this second generation molecule is a more potent inhibitor of T-cell responses than the parent molecule. Given that CTLA4-Ig has already shown efficacy in a clinical trial of psoriasis patients (22), there is significant enthusiasm for the trials using LEA29Y as the primary immunosuppresant. It is clearly compatible, if not synergistic, with clinically approved immunosuppressive agents (anti-IL-2R mAb and rapamycin) facilitating the design of clinical trials (current study and T.C.P., unpublished observations). Initial human trials with LEA29Y are already underway in patients afflicted with rheumatoid arthritis and those undergoing renal transplant. Although a direct comparison of a tacrolimus-based protocol and the LEA29Y regimen was not attempted because of reported intolerable toxicities in nonhuman primates (33), our results suggest that LEA29Y has the potential to be at least as effective as tacrolimus as a primary immunosuppressant.

In summary, we have identified a novel calcineurin inhibitor/steroid-free immunosuppressive regimen that provides significant protection from rejection and prolongs the survival of islet allografts in nonhuman primates. Together, the encouraging results of clinical trials using the lower affinity parent molecule, CTLA4-Ig, in psoriasis and the results described here provide a strong rationale for clinical trials to test these strategies in human islet transplantation.

This research was supported by grants from the National Institutes of Health (award #U19 AI44644-04) with additional support from the ERC Program of the National Science Foundation (award #EEC-9731643), the Carlos and Marguerite Mason Trust, and the Livingston Foundation.

We are indebted to the excellent support from the Yerkes veterinary staff, in particular, Jack Orkin, Rachel Fest, Gray Simmons, Rachel Black, and Amy Souder.