One in Ten CD8+ Cells in the Pancreas of Living Individuals With Recent-Onset Type 1 Diabetes Recognizes the Preproinsulin Epitope PPI15-24.

In type 1 diabetes (T1D), a lifelong autoimmune disease, T cells infiltrate the islets and the exocrine pancreas in high numbers. CD8+ T cells are the main cell type found in the insulitic lesion, and CD8+ T cells reactive against ß-cell antigens have been detected in peripheral blood and in the pancreas of patients with short- or long-term disease. In the Diabetes Virus Detection (DiViD) study, researchers collected pancreatic tissue, by pancreatic tail resection, from living patients with recent-onset T1D. These tissues have been extensively studied by the scientific community, but the autoreactive nature of the T-cell infiltrate has remained unexplored. Our objective was to determine the number and localization of these cells in pancreas samples obtained through the DiViD study. Here, we demonstrate the presence of high frequencies of CD8+ T cells reactive against a highly relevant epitope derived from the preproinsulin signal peptide in pancreatic tissue samples from these donors. We also show the heterogeneity of islet distribution and CD8+ T-cell infiltration. Our findings contribute to the current limited existing knowledge of T-cell reactivity in the pancreas of donors with recent-onset T1D and indicate that antigen-specific therapies directed toward preproinsulin could have high clinical impact.

The healthy exocrine pancreas contains preproinsulin-specific CD8 T cells that attack islets in type 1 diabetes.

Preproinsulin (PPI) is presumably a crucial islet autoantigen found in patients with type 1 diabetes (T1D) but is also recognized by CD8+ T cells from healthy individuals. We quantified PPI-specific CD8+ T cells within different areas of the human pancreas from nondiabetic controls, autoantibody-positive donors, and donors with T1D to investigate their role in diabetes development. This spatial cellular quantitation revealed unusually high frequencies of autoreactive CD8+ T cells supporting the hypothesis that PPI is indeed a key autoantigen. To our surprise, PPI-specific CD8+ T cells were already abundantly present in the nondiabetic pancreas, thus questioning the dogma that T1D is caused by defective thymic deletion or systemic immune dysregulation. During T1D development, these cells accumulated in and around islets, indicating that an islet-specific trigger such as up-regulation of major histocompatibility complex class I might be essential to unmask beta cells to the immune system.

Alpha cells, the main source of IL-1ß in human pancreas.

Interleukin-1ß (IL-1ß) is known to trigger beta cell dysfunction in vitro and could potentially play a role during the pathogenesis of type 1 diabetes and type 2 diabetes. However, several clinical trials attempting to block IL-1ß function have had minimal success. We therefore re-investigated local expression of IL-1ß in human diabetic and non-diabetic pancreata. We obtained pancreatic tissue sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) including non-diabetic (n = 9), non-diabetic auto-antibody positive (AAb+, n = 5), type 1 diabetes (n = 6), and type 2 diabetes (n = 6) donors. Islets were systematically investigated for the presence of IL-1ß mRNA by in situ hybridization and IL-1ß protein by indirect immunofluorescence. We found that intra-islet IL-1ß was produced at comparable level in both non-diabetic and diabetic donors. Interestingly, the main source for IL-1ß was alpha cells but not beta cells. Our findings call into question the role of IL-1ß in the diabetic pancreas as it has been proposed in previous literature. Additionally, our results regarding the localization of IL-1ß should lead to further investigation into the role of IL-1ß in the physiology of pancreatic alpha cells.

Increase in Pancreatic Proinsulin and Preservation of ß-Cell Mass in Autoantibody-Positive Donors Prior to Type 1 Diabetes Onset.

Type 1 diabetes is characterized by the loss of insulin production caused by ß-cell dysfunction and/or destruction. The hypothesis that ß-cell loss occurs early during the prediabetic phase has recently been challenged. Here we show, for the first time in situ, that in pancreas sections from autoantibody-positive (Ab+) donors, insulin area and ß-cell mass are maintained before disease onset and that production of proinsulin increases. This suggests that ß-cell destruction occurs more precipitously than previously assumed. Indeed, the pancreatic proinsulin-to-insulin area ratio was also increased in these donors with prediabetes. Using high-resolution confocal microscopy, we found a high accumulation of vesicles containing proinsulin in ß-cells from Ab+ donors, suggesting a defect in proinsulin conversion or an accumulation of immature vesicles caused by an increase in insulin demand and/or a dysfunction in vesicular trafficking. In addition, islets from Ab+ donors were larger and contained a higher number of ß-cells per islet. Our data indicate that ß-cell mass (and function) is maintained until shortly before diagnosis and declines rapidly at the time of clinical onset of disease. This suggests that secondary prevention before onset, when ß-cell mass is still intact, could be a successful therapeutic strategy.

Heterogeneity and Lobularity of Pancreatic Pathology in Type 1 Diabetes during the Prediabetic Phase.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells are destroyed in the islets of Langerhans. One of its main pathological manifestations is the hyper-expression of Major Histocompatibility Complex I (MHC-I) by beta cells, which was first described over 3 decades ago yet its cause remains unknown. It might not only be a sign of beta cell dysfunction but could also render the cells susceptible to autoimmune destruction; for example, by islet-infiltrating CD8 T cells. In this report, we studied pancreas tissue from a 22-year-old non-diabetic male cadaveric organ donor who had been at high risk of developing T1D, in which autoantibodies against GAD and IA-2 were detected. Pancreas sections were analyzed for signs of inflammation. Multiple insulin-containing islets were identified, which hyper-expressed MHC-I. However, islet density and MHC-I expression exhibited a highly lobular and heterogeneous pattern even within the same section. In addition, many islets with high expression of MHC-I presented higher levels of CD8 T cell infiltration than normal islets. These results demonstrate the heterogeneity of human pathology that occurs early during the pre-diabetic, autoantibody positive phase, and should contribute to the understanding of human T1D.

Increased immune cell infiltration of the exocrine pancreas: a possible contribution to the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) results from a complex interplay between genetic susceptibility and environmental factors that have been implicated in the pathogenesis of disease both as triggers and potentiators of ß-cell destruction. CD8 T cells are the main cell type found in human islets, and they have been shown in vitro to be capable of killing ß-cells overexpressing MHC class I. In this study, we report that CD8 T cells infiltrate the exocrine pancreas of diabetic subjects in high numbers and not only endocrine areas. T1D subjects present significantly higher CD8 T cell density in the exocrine tissue without the presence of prominent insulitis. Even T1D donors without remaining insulin-containing islets and long disease duration show elevated levels of CD8 T cells in the exocrine compartment. In addition, higher numbers of CD4(+) and CD11c(+) cells were found in the exocrine tissue. Preliminary data in type 2 diabetic (T2D) subjects indicate that overall, there might be a spontaneous inflammatory infiltration of the exocrine tissue, common to both T1D and T2D subjects. Our study provides the first information on the precise tissue distribution of CD8 T cells in pancreata from T1D, T2D, autoantibody-positive, and healthy control subjects.

Beta-cell specific production of IL6 in conjunction with a mainly intracellular but not mainly surface viral protein causes diabetes.

Inflammatory mechanisms play a key role in the pathogenesis of type 1 and type 2 diabetes. IL6, a pleiotropic cytokine with impact on immune and non-immune cell types, has been proposed to be involved in the events causing both forms of diabetes and to play a key role in experimental insulin-dependent diabetes development. The aim of this study was to investigate how beta-cell specific overexpression of IL-6 influences diabetes development. We developed two lines of rat insulin promoter (RIP)-lymphocytic choriomeningitis virus (LCMV) mice that also co-express IL6 in their beta-cells. Expression of the viral nucleoprotein (NP), which has a predominantly intracellular localization, together with IL6 led to hyperglycemia, which was associated with a loss of GLUT-2 expression in the pancreatic beta-cells and infiltration of CD11b(+) cells, but not T cells, in the pancreas. In contrast, overexpression of the LCMV glycoprotein (GP), which can localize to the surface, with IL-6 did not lead to spontaneous diabetes, but accelerated virus-induced diabetes by increasing autoantigen-specific CD8(+) T cell responses and reducing the regulatory T cell fraction, leading to increased pancreatic infiltration by CD4(+) and CD8(+) T cells as well as CD11b(+) and CD11c(+) cells. The production of IL-6 in beta-cells acts prodiabetic, underscoring the potential benefit of targeting IL6 in diabetes.

Combination therapy with an anti-IL-1ß antibody and GAD65 DNA vaccine can reverse recent-onset diabetes in the RIP-GP mouse model.

Type 1 diabetes is thought to be an autoimmune condition in which self-reactive T cells attack insulin-secreting pancreatic ß-cells. As a proinflammatory cytokine produced by ß-cells or macrophages, interleukin-1ß (IL-1ß) represents a potential therapeutic target in diabetes. We reasoned IL-1ß blockade could be combined with islet antigen-specific approaches involving GAD of 65 kDa (GAD65)-expressing plasmids, as previously shown in combination therapies (CTs) with anti-CD3. Thus, we investigated whether anti-IL-1ß antibody alone or combined with GAD65 vaccine could reverse diabetes development in a virus-induced mouse model. Given alone, anti-IL-1ß had no effect on diabetes, while GAD65 plasmid resulted in 33% disease reversal after a 5-week observation. However, CTs cured 53% of animals and prevented worsening of glycemic control in nonprotected individuals for up to 12 weeks. While the GAD65 vaccine arm of the CT was associated with increased forkhead box p3(+) regulatory T-cell frequency in pancreatic lymph nodes, islet infiltration by CD11b(+/high) cells was less frequent upon CT, and its extent correlated with treatment success or failure. Altogether, our CTs provided prolonged improvement of clinical and immunological features. Despite unsuccessful clinical trials using anti-IL-1ß monotherapy, these data hold promise for treatment of type 1 diabetic patients with IL-1ß blockade combined with antigen-specific vaccines.

Functional redundancy of CXCR3/CXCL10 signaling in the recruitment of diabetogenic cytotoxic T lymphocytes to pancreatic islets in a virally induced autoimmune diabetes model.

Cytotoxic T lymphocytes (CTLs) constitute a major effector population in pancreatic islets from patients suffering from type 1 diabetes (T1D) and thus represent attractive targets for intervention. Some studies have suggested that blocking the interaction between the chemokine CXCL10 and its receptor CXCR3 on activated CTLs potently inhibits their recruitment and prevents ß-cell death. Since recent studies on human pancreata from T1D patients have indicated that both ligand and receptor are abundantly present, we reevaluated whether their interaction constitutes a pivotal node within the chemokine network associated with T1D. Our present data in a viral mouse model challenge the notion that specific blockade of the CXCL10/CXCR3 chemokine axis halts T1D onset and progression.

Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients.

A direct association of islet-autoreactive T cells with ß cell destruction in human pancreatic islets from type 1 diabetes (T1D) patients has never been demonstrated, and little is known about disease progression after diagnosis. Frozen pancreas samples were obtained from 45 cadaveric T1D donors with disease durations ranging from 1 wk to >50 yr, 14 nondiabetic controls, 5 nondiabetics with islet autoantibodies, 2 cases of gestational diabetes, and 6 T2D patients. Sections were systematically analyzed for the presence of insulin-sufficient ß cells, CD8(+) insulitic lesions, and HLA class I hyperexpression. Finally, consecutive sections from HLA-A2-expressing individuals were probed for CD8 T cell reactivity against six defined islet autoantigens associated with T1D by in situ tetramer staining. Both single and multiple CD8 T cell autoreactivities were detected within individual islets in a subset of patients up to 8 yr after clinical diagnosis. Pathological features such as HLA class I hyperexpression and insulitis were specific for T1D and persisted in a small portion of the patients with longstanding disease. Insulitic lesions consistently presented in a multifocal pattern with varying degrees of infiltration and ß cell loss across affected organs. Our observations provide the first direct proof for islet autoreactivity within human islets and underscore the heterogeneous and chronic disease course.

Intravital imaging of CTLs killing islet cells in diabetic mice.

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing ß cells in the pancreatic islets, which are essentially mini-organs embedded in exocrine tissue. CTLs are considered to have a predominant role in the autoimmune destruction underlying T1D. Visualization of CTL-mediated killing of ß cells would provide new insight into the pathogenesis of T1D, but has been technically challenging to achieve. Here, we report our use of intravital 2-photon imaging in mice to visualize the dynamic behavior of a virally expanded, diabetogenic CTL population in the pancreas at cellular resolution. Following vascular arrest and extravasation, CTLs adopted a random motility pattern throughout the compact exocrine tissue and displayed unimpeded yet nonlinear migration between anatomically nearby islets. Upon antigen encounter within islets, a confined motility pattern was acquired that allowed the CTLs to scan the target cell surface. A minority of infiltrating CTLs subsequently arrested at the ß cell junction, while duration of stable CTL-target cell contact was on the order of hours. Slow-rate killing occurred in the sustained local presence of substantial numbers of effector cells. Collectively, these data portray the kinetics of CTL homing to and between antigenic target sites as a stochastic process at the sub-organ level and argue against a dominant influence of chemotactic gradients.

Persistent glucose transporter expression on pancreatic beta cells from longstanding type 1 diabetic individuals.

BACKGROUND: Recent reports have established the notion that many patients with longstanding type 1 diabetes (T1D) possess a remnant population of insulin-producing beta cells. It remains questionable, however, whether these surviving cells can physiologically sense and respond to glucose stimuli. METHODS: Frozen pancreatic sections from non-diabetic donors (n=8), type 2 diabetic patients (n=4), islet autoantibody-positive non-diabetic patients (n=3), type 1 diabetic patients (n=10) and one case of gestational diabetes were obtained via the network for Pancreatic Organ Donors. All longstanding T1D samples were selected based on the detection of insulin-producing beta cells in the pancreas by immunohistochemistry. RNA was isolated from all sections followed by cDNA preparation and quantitative real-time polymerase chain reaction for insulin, glucose transporter 1 (GLUT1), GLUT2 and GLUT3. Finally, immunofluorescent staining was performed on consecutive sections for all four of these markers and a comparison was made between the expression of GLUT2 in humans versus NOD mice. RESULTS: In contrast to islets from the most widely used T1D model, the NOD mouse, human islets predominantly express GLUT1 and, to a much lesser extent, GLUT3 on their surface instead of GLUT2. Relative expression levels of these receptors do not significantly change in the context of the various (pre-)diabetic conditions studied. Moreover, in both species preservation of GLUT expression was observed even under conditions of substantial leucocyte infiltration or decades of T1D duration. CONCLUSIONS: These data suggest that despite being subjected to multiple years of physiological stress, the remaining beta-cell population in longstanding T1D patients retains a capacity to sense glucose via its GLUTs.

A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy.

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of beta cells in the pancreas. Little is known about the in vivo dynamic interactions between T cells and beta cells or the kinetic behavior of other immune cell subsets in the pancreatic islets. Utilizing multiphoton microscopy we have designed a technique that allows for the real-time visualization of diabetogenic T cells and dendritic cells in pancreatic islets in a live animal, including their interplay with beta cells and the vasculature. Using a custom designed stage, the pancreas was surgically exposed under live conditions so that imaging of islets under intact blood pressure and oxygen supply became possible. We demonstrate here that this approach allows for the tracking of diabetogenic leukocytes as well as vascularization phenotype of islets and accumulation of dendritic cells in islets during diabetes pathogenesis. This technique should be useful in mapping crucial kinetic events in T1D pathogenesis and in testing the impact of immune based interventions on T cell migration, extravasation and islet destruction.