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.

The Hsp60 peptide p277 enhances anti-CD3 mediated diabetes remission in non-obese diabetic mice.

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of pancreatic beta cells leading to inadequate glycemic control. Trials with immunomodulatory monotherapies have shown that the disease course can in principle be altered. The observed preservation of endogenous insulin secretion however is typically transient and chronic treatment is often associated with significant side effects. Here we combined anti-CD3 with the Hsp60 peptide p277, two drugs that have been evaluated in Phase 3 trials, to test for enhanced efficacy. Female NOD mice with recent onset diabetes were given 5 µg anti-CD3 i.v., on three consecutive days in combination with 100 µg of p277 peptide in IFA s.c., once weekly for four weeks. Anti-CD3 alone restored normoglycemia in 44% of the mice while combination therapy with anti-CD3 and p277 induced stable remission in 83% of mice. The observed increase in protection occurred only in part through TLR2 signaling and was characterized by increased Treg numbers and decreased insulitis. These results have important implications for the design of combination therapies for the treatment of T1D.

BDC12-4.1 T-cell receptor transgenic insulin-specific CD4 T cells are resistant to in vitro differentiation into functional Foxp3+ T regulatory cells.

The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, in vitro differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4(+) T cells of the BDC12-4.1 clone to convert into Foxp3(+) iTreg cells. We found that in vitro polarization toward Foxp3(+) iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3(+) BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, in vitro polarization of insulin-specific BDC12-4.1 TCR transgenic CD4(+) T cells toward Foxp3+ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an in vitro acquired Foxp3(+) cell phenotype and its associated in vivo regulatory potential.

Metabolic syndrome - Removing roadblocks to therapy: Antigenic immunotherapies.

Up to 25 per cent of the world׳s adult population may have the metabolic syndrome, a condition closely associated with central obesity. The metabolic syndrome is a major risk factor for cardiovascular disease and type 2 diabetes and therefore represents an important worldwide health problem. In addition to metabolic abnormalities such as raised fasting plasma glucose, high cholesterol and high blood pressure, there is consensus that obese subjects develop a state of low-grade chronic immune activation. This sustained pro-inflammatory response in fat tissue is thought to worsen insulin resistance and dyslipidemia. Likewise, the immune system contributes to the detrimental cascade of events leading to plaque formation in atherosclerosis. It has long been assumed that the innate arm of the immune system was the only key player, but emerging evidence suggests that there is in fact a sizeable adaptive immune component to obesity and cardiovascular disease. From a therapeutic perspective, it could be envisioned that immune modulation drugs such as cytokine inhibitors, co-stimulation blockers or anti-T cell agents could offer benefit. It is questionable, however, whether chronic treatment with for instance biologicals will have a favorable risk/benefit profile in a silent condition such as the metabolic syndrome. An attractive alternative could be the development of antigen-specific T cell therapies, not unlike those currently in various phases of development for type 1 diabetes. In this article, we will give an overview of antigen-specific treatment modalities in type 1 diabetes, followed by a review of the evidence for T cell involvement in obesity and atherosclerosis.

Non-antigenic and antigenic interventions in type 1 diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of the pancreatic ß-cells. Current T1D therapies are exclusively focused on regulating glycemia rather than the underlying immune response. A handful of trials have sought to alter the clinical course of T1D using various broad immune-suppressors, e.g., cyclosporine A and azathioprine.(1-3) The effect on ß-cell preservation was significant, however, these therapies were associated with unacceptable side-effects. In contrast, more recent immunomodulators, such as anti-CD3 and antigenic therapies such as DiaPep277, provide a more targeted immunomodulation and have been generally well-tolerated and safe; however, as a monotherapy there appear to be limitations in terms of therapeutic benefit. Therefore, we argue that this new generation of immune-modifying agents will likely work best as part of a combination therapy. This review will summarize current immune-modulating therapies under investigation and discuss how to move the field of immunotherapy in T1D forward.

Trials in type 1 diabetes: Antigen-specific therapies.

Type 1 diabetes (T1D) results from an aberrant immunological response against the insulin-producing beta cells in the islets of the pancreas. The ideal therapy would restore immune balance in a safe and lasting fashion, stopping the process of beta cell decay. The efficacy of immune suppressive agents such as cyclosporin underscores the notion that T1D can in principle be prevented, albeit at an unacceptable long-term safety risk. Immune modulatory drugs such as monoclonal anti-CD3 antibody, on the other hand, have recently had rather disappointing results in phase 3 trials, possibly due to inadequate dosing or choice of inappropriate endpoints. Therefore, it is argued that striking the right balance between safety and efficacy, together with careful trial design, will be paramount in preventing T1D. Here we outline the concept of antigen-specific tolerization as a strategy to safely induce long-term protection against T1D, focusing on available clinical trial data, key knowledge gaps and potential future directions.

Antibody cross-reactivity and the viral aetiology of type 1 diabetes.

Type 1 diabetes (T1D) is caused by the destruction of insulin-producing pancreatic ß cells by the patient's immune system. While the underlying genetics and immunopathology are fairly well characterized, the environmental trigger remains unidentified. Numerous studies have centred on the role of enteroviruses as aetiological factors that could initiate or accelerate T1D development. The most convincing evidence to date consists of an array of reports documenting the presence of enteroviral nucleic acids in peripheral blood at diagnosis. A prominent hypothesis is that enteroviruses may infect the pancreatic islets and thus be responsible for the islet-specific up-regulation of MHC class I that is commonly observed, possibly enabling T cell recognition and cytotoxicity. Past immunohistochemical studies have indeed shown that antibodies binding the enteroviral capsid protein VP1 preferentially stain the pancreatic ß cells from diabetic individuals. New data now indicate that the VP1 antibody used in these studies cross-reacts with mitochondrial proteins.

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.

Viral infections and molecular mimicry in type 1 diabetes.

Type 1 diabetes (T1D) is a disease characterized by inflammation of pancreatic islets associated with autoimmunity against insulin-producing beta cells, leading to their progressive destruction. The condition constitutes a significant and worldwide problem to human health, particularly because of its rapid, but thus far unexplained, increase in incidence. Environmental factors such as viral infections are thought to account for this trend. While there is no lack of reports associating viral infections toT1D, it has proven difficult to establish which immunological processes link viral infections to disease onset or progression. One of the commonly discussed pathways is molecular mimicry, a mechanism that encompasses cross-reactive immunity against epitopes shared between viruses and beta cells. In this review, we will take a closer look at mechanistic evidence for a potential role of viruses in T1D, with a special focus on molecular mimicry.

Virus infections in type 1 diabetes.

The precise etiology of type 1 diabetes (T1D) is still unknown, but viruses have long been suggested as a potential environmental trigger for the disease. However, despite decades of research, the body of evidence supporting a relationship between viral infections and initiation or acceleration of islet autoimmunity remains largely circumstantial. The most robust association with viruses and T1D involves enterovirus species, of which some strains have the ability to induce or accelerate disease in animal models. Several hypotheses have been formulated to mechanistically explain how viruses may affect islet autoimmunity and ß-cell decay. The recent observation that certain viral infections, when encountered at the right time and infectious dose, can prevent autoimmune diabetes illustrates that potential relationships may be more complex than previously thought. Here, we provide a concise summary of data obtained in mouse models and humans, and identify future avenues toward a better characterization of the association between viruses and T1D.

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.

Clinical potential of antigen-specific therapies in type 1 diabetes.

In type 1 diabetes (T1D), pancreatic beta-cells are attacked and destroyed by the immune system, which leads to a loss of endogenous insulin secretion. The desirable outcome of therapeutic intervention in autoimmune diseases is the restoration of immune tolerance to prevent organ damage. Past trials with immune suppressive drugs highlight the fact that T1D is in principle a curable condition. However, the barrier in T1D therapy in terms of drug safety is set particularly high because of the predominantly young population and the good prognosis associated with modern exogenous insulin therapy. Thus, there is a general consensus that chronic immune suppression is associated with unacceptable long-term safety risks. On the other hand, immune-modulatory biologicals have recently failed to confer significant protection in phase 3 clinical trials. However, the concept of antigen-specific tolerization may offer a unique strategy to safely induce long-term protection against T1D. In this review, we analyze the potential reasons for the failure of the different tolerization therapies, and describe how the concept of antigen-specific toleraization may overcome the obstacles associated with clinical therapy in T1D.

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.

Type 1 diabetes: etiology, immunology, and therapeutic strategies.

Type 1 diabetes (T1D) is a chronic autoimmune disease in which destruction or damaging of the beta-cells in the islets of Langerhans results in insulin deficiency and hyperglycemia. We only know for sure that autoimmunity is the predominant effector mechanism of T1D, but may not be its primary cause. T1D precipitates in genetically susceptible individuals, very likely as a result of an environmental trigger. Current genetic data point towards the following genes as susceptibility genes: HLA, insulin, PTPN22, IL2Ra, and CTLA4. Epidemiological and other studies suggest a triggering role for enteroviruses, while other microorganisms might provide protection. Efficacious prevention of T1D will require detection of the earliest events in the process. So far, autoantibodies are most widely used as serum biomarker, but T-cell readouts and metabolome studies might strengthen and bring forward diagnosis. Current preventive clinical trials mostly focus on environmental triggers. Therapeutic trials test the efficacy of antigen-specific and antigen-nonspecific immune interventions, but also include restoration of the affected beta-cell mass by islet transplantation, neogenesis and regeneration, and combinations thereof. In this comprehensive review, we explain the genetic, environmental, and immunological data underlying the prevention and intervention strategies to constrain T1D.

Viruses and cytotoxic T lymphocytes in type 1 diabetes.

Histopathological studies on pancreas tissues from individuals with recent-onset type 1 diabetes (T1D) consistently find that CD8 T cells substantially contribute to the formation of islet lesions. CD8 T cells reactive against islet-associated antigens can also be found in blood samples from T1D patients. Mechanistic studies on the pathogenic role of this T cell subset have mostly focused on two animal models, i.e., the non-obese diabetic mouse and the virally induced rat insulin promoter-lymphocytic choriomeningitis virus model. Data were obtained in support of a role for viral infection in expanding a population of diabetogenic cytotoxic T lymphocytes. In view of the theorized association of viral infection with initiation of islet autoimmunity and progression to clinically overt disease, CD8 T cells thus represent an attractive target for immunotherapy. We will review here arguments in favor of a pivotal role for CD8 T cells in driving T1D development and speculate on etiologic agents that may provoke their aberrant activation.

Histopathology of type 1 diabetes: old paradigms and new insights.

Although our knowledge on the various aspects of diabetes development in the NOD mouse model is substantial and keeps expanding at a dramatic pace, the dataset on histopathologic features of type 1 diabetes (T1D) in patients remains largely stagnant. Early work has established an array of common aspects that have become epitomic in the absence of new patient material. There is a growing consensus that an updated and more detailed view is required that challenges and expands our understanding. Comprehensive initiatives are currently ongoing to address these issues in pre-diabetic, recent onset and longstanding type 1 diabetic individuals, and some of the old data have been recently revisited. In this review article, we wish to provide an overview of where we stand today and how we can correlate the various cross-sectional studies from the past with contemporary models of the disease. We believe an enhanced understanding of the many histopathological particularities in patients as compared to animal models will ultimately lead, not only to more fundamental insights, but also to an improved ability to translate pre-clinical data from bench to bedside.