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Objective: The T helper 17 (Th17) population, a subset of CD4-positive T-cells that secrete interleukin (IL)-17, has been implicated in autoimmune diseases, including multiple sclerosis and lupus. Therapeutic agents that target the Th17 effector molecule IL-17 or directly inhibit the Th17 population (IL-25) have shown promise in animal models of autoimmunity. The role of Th17 cells in type 1 diabetes has been less clear. The effect of neutralizing anti-IL-17 and recombinant IL-25 on the development of diabetes in NOD mice, a model of spontaneous autoimmune diabetes, was investigated in this study., Research Design and Methods and Results: Although treatment with either anti-IL-17 or IL-25 had no effect on diabetes development in young (<5 weeks) NOD mice, either intervention prevented diabetes when treatment was started at 10 weeks of age (P < 0.001). Insulitis scoring and immunofluorescence staining revealed that both anti-IL-17 and IL-25 significantly reduced peri-islet T-cell infiltrates. Both treatments also decreased GAD65 autoantibody levels. Analysis of pancreatic lymph nodes revealed that both treatments increased the frequency of regulatory T-cells. Further investigation demonstrated that IL-25 therapy was superior to anti-IL-17 during mature diabetes because it promoted a period of remission from new-onset diabetes in 90% of treated animals. Similarly, IL-25 delayed recurrent autoimmunity after syngeneic islet transplantation, whereas anti-IL-17 was of no benefit. GAD65-specific ELISpot and CD4-positive adoptive transfer studies showed that IL-25 treatment resulted in a T-cell-mediated dominant protective effect against autoimmunity., Conclusions: These studies suggest that Th17 cells are involved in the pathogenesis of autoimmune diabetes. Further development of Th17-targeted therapeutic agents may be of benefit in this disease.

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine beta-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Xenotransplantation of porcine tissue to human recipients promises to alleviate the organ shortage. Human antibody-mediated and cell-mediated immune responses against porcine grafts, however, represent barriers to successful xenotransplantation. We compared neonatal porcine islet cells (NPICs) and neonatal porcine splenocytes for the ability to stimulate proliferation of human peripheral blood lymphocytes (PBLs), and for their susceptibility to human natural killer (NK) and cytotoxic T-lymphocyte (CTL)-mediated lysis. Human peripheral blood CD4+ lymphocytes showed strong proliferation in response to NPICs, likely because of occasional swine leukocyte antigen (SLA) class II+ cells in the NPIC preparations. In contrast, human peripheral blood CD8+ lymphocytes did not proliferate in response to NPICs, although they showed clear responses to both porcine splenocytes and endothelial cells. Both human CTL-raised-against-porcine splenocytes and endogenous NK cells lysed porcine splenocytes, but the same cells showed little or no lytic activity against NPICs. Lysis of porcine splenocyte targets was completely abrogated by pretreatment of the human NK or CTL populations with concana-mycin A, suggesting a perforin-dependent effector mechanism. Pretreatment of the NPIC targets with proinflammatory porcine cytokines to upregulate SLA class I expression failed to enhance human CTL-mediated lysis. However, lysis of NPICs by human CTLs could be elicited when a lectin was added to form stable effector:target cell conjugates. It appears that NPICs do not express sufficiently high levels of co-stimulatory and/or adhesion molecules to either activate human CD8+ T-cells or to be effective targets for activated human CTLs. These data suggest that NPICs may not be destroyed by NK- or CTL-mediated lytic mechanisms after transplantation into humans.

The 65KD isoform of GAD is considered to be a major target autoantigen in many humans with autoimmune prediabetes or diabetes. The major histocompatibility complex class II allele DQA1*0301, DQB1*0302, which encodes HLA-DQ8, confers susceptibility to type 1 diabetes and occurs in up to 80% of affected individuals. To map T-cell epitopes for GAD65 restricted to the diabetes-associated DQ8 heterodimer, we generated transgenic NOD mice expressing HLA-DQ8 and human CD4 while having the mouse class II gene (IA(beta)) deleted. These mice were immunized with full-length purified recombinant GAD65, and the fine specificity of T-cell responses was mapped by examining recall responses of bulk splenocytes to an overlapping set of 20-mer peptides encompassing the entire GAD65 protein. Four different peptides (P121-140, P201-220, P231-250, and P471-490) gave significant T-cell recall responses. P201-220 and P231-250 have been shown previously to bind DQ8, whereas the other two peptides had been classified as nonbinders. Interestingly, the peptide giving the greatest response (P201-220) encompasses residues 206-220 of GAD65, a region that has been shown to be a dominant T-cell epitope in wild-type IA(g7) NOD mice. Overlap in this T-cell epitope likely reflects structural similarities between DQ8 and IA(g7). The fine specificity of antibody responses in the GAD65-immunized mice was also examined by testing the antisera by enzyme-linked immunosorbent assay (ELISA) against the same overlapping set of peptides. The two dominant B-cell epitopes were P361-380 and P381-400; P121-140 and P471-490 appeared to correspond to both B- and T-cell epitopes. Although the NOD human CD4, DQ8, IA(null) transgenic mice generated in these studies do not develop autoimmune diabetes either spontaneously or after cyclophosphamide treatment, they can be used to map DQ8-restricted T-cell epitopes for a variety of human islet autoantigens. They can also be used to test T-cell-specific reagents, such as fluorescently labeled DQ8 tetramers containing GAD65 peptides or other beta-cell peptides, which we believe will be useful in analyzing human immune responses in diabetic and prediabetic patients.

A mechanism of autoimmune destruction of islet beta-cells in type 1 diabetes has been proposed to be the binding of Fas ligand (FasL) on T-cells to Fas receptors on beta-cells. We investigated this proposal by examining the expression of FasL and Fas on islet-infiltrating T-cells and beta-cells in relation to beta-cell destruction in a syngeneic islet transplant model in NOD mice. Diabetic NOD mice were transplanted with syngeneic islets and injected with complete Freund's adjuvant, which prevented diabetes recurrence (nondestructive insulitis), and with phosphate-buffered saline, which did not (beta-cell destructive insulitis). Two-color immunohistochemical assays revealed that FasL was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from both diabetic and normoglycemic mice, and the percentage of each type of cell that expressed FasL was greater in islet grafts from normoglycemic compared with diabetic mice. In contrast, Fas was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from diabetic mice, but it was nearly or totally absent on these cells in islet grafts from normoglycemic mice. Similarly, polymerase chain reaction analysis of islet grafts revealed that Fas mRNA expression was significantly lower in islet grafts from normoglycemic compared with diabetic mice. Also, mRNA levels of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were significantly lower in islet grafts from normoglycemic mice. Finally, Fas was induced on NOD islet cells by incubation with IL-1beta, IFN-gamma, and the combination of IL-1beta, TNF-alpha, and IFN-gamma. These findings support the concept that cytokine-induced Fas receptor expression on islet beta-cells is a mechanism for their destruction by FasL-expressing CD4+ and CD8+ T-cells and, possibly, by FasL-expressing beta-cells themselves.

Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

We prepared single-cell suspensions of Lewis rat ¿RT1(1/l)¿ testicular cells and cultured these in vitro for 48 h under conditions that promoted the formation of cellular aggregates. In the absence of systemic immunosuppression, the transplantation of a sufficient quantity of these aggregates (containing 11 x 10(6) cells, (75% Sertoli cells), together with 2,000 purified Lewis rat islets, reversed the diabetic state for >95 days in 100% (5/5) of the chemically diabetic Wistar-Furth ¿RT1(u/u)¿ recipients. Similar grafts consisting of islets alone or islets plus 50% fewer testicular cell aggregates survived for only 10 days. Functioning composite allografts harvested from normoglycemic animals at approximately 100 days showed healthy beta-cells in close association with Fas ligand-expressing Sertoli cells. Because no gene therapy protocol is required, the transplantation of composite grafts consisting of purified human allogeneic islets plus human allogeneic testicular cell aggregates can be applied in clinical islet transplantation as soon as it has been proven in a large animal model.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.