Norma S. Kenyon

Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope

In autoimmune type 1 diabetes, pathogenic T lymphocytes are associated with the specific destruction of insulin-producing β-islet cells. Identification of the autoantigens involved in triggering this process is a central question. Here we examined T cells from pancreatic draining lymph nodes, the site of islet-cell-specific self-antigen presentation. We cloned single T cells in a non-biased manner from pancreatic draining lymph nodes of subjects with type 1 diabetes and from non-diabetic controls. A high degree of T-cell clonal expansion was observed in pancreatic lymph nodes from long-term diabetic patients but not from control subjects. The oligoclonally expanded T cells from diabetic subjects with DR4, a susceptibility allele for type 1 diabetes, recognized the insulin A 1–15 epitope restricted by DR4. These results identify insulin-reactive, clonally expanded T cells from the site of autoinflammatory drainage in long-term type 1 diabetics, indicating that insulin may indeed be the target antigen causing autoimmune diabetes.

Islet Transplantation in Type 1 Diabetes Mellitus Using Cultured Islets and Steroid-Free Immunosuppression: Miami Experience

Following the success obtained with transplantation of fresh human islets under steroid‐free immunosuppression, this trial evaluated the transplantation of islets that had undergone a period of in vitro culture and the potential of tumor necrosis factor (TNF‐α) blockade to improve islet engraftment. Subjects included 16 patients with type 1 diabetes mellitus (T1DM); half were randomly assigned to receive Infliximab immediately preceding initial infusion. Immunosuppression consisted of daclizumab induction and sirolimus/tacrolimus maintenance. Out of 16 subjects 14 achieved insulin independence with one or two islet infusions; adverse events precluded completion in two. Without supplemental infusions, 11/14 (79%) subjects were insulin independent at 1 year, 6/14 (43%) at 18 months; these same subjects remain insulin independent at 33 ± 6 months. While on immunosuppression, all patients maintained graft function. Out of 14 patients, 8 suffered chronic partial graft loss, likely immunological in nature, 5 of these received supplemental infusions. Currently, 11 subjects remain on immunosuppression, 8 (73%) are insulin independent, two with supplemental infusions. Insulin independent subjects demonstrated normalization of HbA1c, fructosamine and Mean Amplitude of Glycemic Excursions (MAGE) values. No clinical benefit of infliximab was identified. These results demonstrate that transplantation of cultured human islet allografts results in reproducible insulin independence in all subjects under this immunosuppressive regimen, comparable to that of freshly transplanted islets (Edmonton protocol).

Development of a Chimeric Anti-CD40 Monoclonal Antibody That Synergizes with LEA29Y to Prolong Islet Allograft Survival

In recent years, reagents have been developed that specifically target signals critical for effective T cell activation and function. Manipulation of the CD28/CD80/86 and CD40/CD154 pathways has exhibited extraordinary efficacy, particularly when the pathways are blocked simultaneously. Despite the reported efficacy of anti-CD154 in rodents and higher models, its future clinical use is uncertain due to reported thromboembolic events in clinical trials. To circumvent this potential complication, we developed and evaluated a chimeric Ab targeting CD40 (Chi220, BMS-224819) as an alternative to CD154. Although Chi220 blocks CD154 binding, it also possesses partial agonist properties and weak stimulatory potential. The anti-CD40 was tested alone and in combination with a rationally designed, high affinity variant of CTLA4-Ig, LEA29Y (belatacept), in a nonhuman primate model of islet transplantation. Although either agent alone only modestly prolonged islet survival (Chi220 alone: 14, 16, and 84 days; LEA29Y alone: 58 and 60 days), their combination (LEA29Y and Chi220) dramatically facilitated long term survival (237, 237, 220, >185, and 172 days). We found that the effects of Chi220 treatment were not mediated solely through deletion of CD20-bearing cells and that the combined therapy did not significantly impair established antiviral immunity.

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.

Current advances and travails in islet transplantation.

The successful demonstration that insulin-producing β-cells can be isolated (in the form of cell clusters called islets containing β and other endocrine and nonendocrine cells) from a recently deceased donors pancreas, then transplanted into subjects with type 1 diabetes, and thereby restore, at least temporarily, insulin-independent normoglycemia has firmly established the important “proof of concept.” Even so, worldwide efforts to advance the therapy for widespread applicability have served to focus attention on the hurdles yet to clear. This review will briefly describe the present state of the art and succinctly define the research problems being attacked along with some recent advances that demonstrate significant progress. Since Paul Lacys early rodent experiments in the 1960s established that pancreatic islets could be isolated from one animal and transplanted into a diabetic recipient to restore normoglycemia (1), investigators have pursued efforts to develop the therapy for clinical use. After years of development in various animal models and efforts to improve human islet isolation techniques (see [2–4] for reviews with a historical perspective), the first patient achieving short-term insulin independence was reported by the group at Washington University in St. Louis. That advance was based on new islet isolation technology utilizing islets pooled from several donors, intensive insulin treatment in the peritransplant period, and induction immunosuppression with antithymocyte globulin (ATG) to avoid glucocorticoid therapy (5). The development of new immunosuppressive drugs that allowed patients to remain off glucocorticoid therapy while awaiting subsequent islet infusions (because most recipients require islets from two or more donors) enabled the group in Edmonton to optimize the clinical islet transplantation procedure (6). The approach allowed the group to conclude that about 12,000 islet equivalents per recipient body weight (in kilograms) was required to restore insulin-independent normoglycemia (6) and sparked intense international interest and effort. Current …

Glutamate is a positive autocrine signal for glucagon release.

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.

Long‐Term Survival of Nonhuman Primate Islets Implanted in an Omental Pouch on a Biodegradable Scaffold

The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and five allogeneic diabetic monkey transplants under the cover of steroid‐free immune suppression (SFIS) were undertaken. Fasting blood glucose (FBG) and C‐peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/mL following transplant, a 66–92% posttransplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well‐granulated and well‐vascularized, insulin‐positive islets, surrounded by T‐cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n = 20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human β cells sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca2+ concentration, [Ca2+]i, and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X3 receptors in the β-cell plasma membrane, resulting in increased [Ca2+]i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cells secretory machinery. This may explain how the human pancreatic β cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Islet allograft survival in nonhuman primates immunosuppressed with basiliximab, RAD, and FTY720

Objective. In a preclinical, nonhuman primate islet allotransplant model, the authors evaluated a novel immunosuppressive combination of basiliximab for induction and of RAD and FTY720 for maintenance. Methods. Five ABO-compatible and mixed lymphocyte reactivity-mismatched streptozotocin-induced diabetic juvenile cynomolgus monkeys underwent transplantation intraportally with 48-hr cultured 10,000 islet equivalents per kilogram. Induction immunosuppression was with intravenous basiliximab (10 mg on postoperative days 0 and 4). Maintenance immunosuppression was with RAD (everolimus) (0.075 mg/kg per day administered subcutaneously) and FTY720 (0.3 mg/kg per day administered orally), both administered on day −2 through day 180 posttransplant. Results. All five recipients tolerated their transplants and immunosuppressive therapy well, without adverse events or infectious complications. Insulin requirements pretransplant were 2.6 to 4.0 U/kg per day. All recipients became normoglycemic and insulin-independent posttransplant. Posttransplant serum C-peptide levels averaged 2.7 ng/mL (range, 0.6–6.2 ng/mL). Morning blood glucose levels ranged from less than 100 mg/dL to 150 mg/dL. Posttransplant acute C-peptide response to intravenous arginine averaged 1.3 ng/mL (range, 0.23–2.72 ng/mL). In one recipient with subtherapeutic RAD blood levels on day 7 posttransplant, exogenous insulin was resumed 100 days posttransplant; basal C-peptide levels remained positive in this recipient and averaged 2.6 ng/mL. The other four recipients remained insulin-independent for more than 6 months. Conclusions. This study provides preliminary evidence of the safety and efficacy of corticosteroid- and calcineurin inhibitor-free immunosuppression in a relevant preclinical transplant model. These findings provide a strong rationale for evaluating this nondiabetogenic regimen in a clinical trial of islet transplants in type 1 diabetic recipients.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

Background. Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. Methods. Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 μg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10–25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. Results. Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. Conclusions. These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.