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Objective: Interleukin (IL)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic (NOD) mouse strain. The aim of this study was to investigate a causal role for IL-21 in type 1 diabetes., Research Design and Methods: We generated IL-21R-deficient NOD mice and C57Bl/6 mice expressing IL-21 in pancreatic beta-cells, allowing the determination of the role of insufficient and excessive IL-21 signaling in type 1 diabetes., Results: Deficiency in IL-21R expression renders NOD mice resistant to insulitis, production of insulin autoantibodies, and onset of type 1 diabetes. The lymphoid compartment in IL-21R-/- NOD is normal and does not contain an increased regulatory T-cell fraction or diminished effector cytokine responses. However, we observed a clear defect in autoreactive effector T-cells in IL-21R-/- NOD by transfer experiments. Conversely, overexpression of IL-21 in pancreatic beta-cells induced inflammatory cytokine and chemokines, including IL-17A, IL17F, IFN-gamma, monocyte chemoattractant protein (MCP)-1, MCP-2, and interferon-inducible protein-10 in the pancreas. The ensuing leukocytic infiltration in the islets resulted in destruction of beta-cells and spontaneous type 1 diabetes in the normally diabetes-resistant C57Bl/6 and NOD x C57Bl/6 backgrounds., Conclusions: This work provides demonstration of the essential prodiabetogenic activities of IL-21 on diverse genetic backgrounds (NOD and C57BL/6) and indicates that IL-21 blockade could be a promising strategy for interventions in human type 1 diabetes.

Translation of novel therapies for type 1 diabetes and other autoimmune diseases to the clinic has been slow despite significant new initiatives from funding agencies. One reason for this is that different incentives drive industry, academia, and funding bodies. These communities therefore lack common goals and often communicate poorly, resulting in unintended obstacles that hamper progress in efficiently translating basic scientific discoveries into medical practice. Here, based on our own personal experiences, we discuss some of the drivers within each community that cause these problems, existing mechanisms to facilitate the translation of science into medical practice, and remaining issues that need to be solved.

Invariant natural killer T (iNKT) cells promote immune responses to various pathogens, but exactly how iNKT cells control antiviral responses is unclear. Here, we showed that iNKT cells induced tissue-specific antiviral effects in mice infected by lymphocytic choriomeningitis virus (LCMV). Indeed, iNKT cells inhibited viral replication in the pancreas and liver but not in the spleen. In the pancreas, iNKT cells expressed the OX40 molecule and promoted type I interferon (IFN) production by plasmacytoid dendritic cells (pDCs) through OX40-OX40 ligand interaction. Subsequently, this iNKT cell-pDC cooperation attenuated the antiviral adaptive immune response in the pancreas but not in the spleen. The dampening of pancreatic anti-LCMV CD8(+) T cell response prevented tissue damage in transgenic mice expressing LCMV protein in islet beta cells. Thus, this study identifies pDCs as an essential partner of iNKT cells for mounting an efficient, nondeleterious antiviral response in peripheral tissue.

Recent studies suggest a beneficial role for blocking CD103 signaling in preventing islet allograft rejection and thus Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, antibody blockade approaches generally raise anti-microbial safety issues, necessitating additional studies to address the possible adverse effects of antibody therapy. Here we report that CD103 had no significant impact on the development of primary and memory CD8(+) or CD4(+) responses after acute lymphocytic choriomeningitis virus (LCMV) infection. In addition, CD103 was found to be dispensable for T1D progression in a rapid, CD8-mediated virally-induced T1D model (the rat insulin promoter [RIP]-LCMV), suggesting that its previous efficacy in the NOD mouse model may not be related to its effect on the generation, memory conversion and/or effector function of CD8(+) or CD4(+) T cells. While the data does not preclude a role for CD103 in T1D in its entirety, the current study does provide much evidence to suggest that CD103 blockade may prove to be a safe intervention for autoimmunity and allo-transplantation. While in cases of rapid microbial (CD8)-driven T1D CD103 antibody blockade may not limit disease progression or severity, in mucosally-driven cases of T1D anti-CD103 antibody treatment may provide a new and safe therapeutic avenue.

Type 1 diabetes is an immune-mediated disease in which pancreatic insulin-producing beta cells are damaged and destroyed. Animal models have served a prominent function in the development of the present ideas of pathogenesis and approaches to therapy. This commentary addresses the utility and limitations of these models for facilitating the 'translation' of immunology research into clinical applications.

The immune phenotype of the partial remission phase or "honeymoon phase" of type 1 diabetes is not well defined. We compared flow cytometry and cytokine production by ELISPOT assays in children with newly diagnosed type 1 diabetes and children in the partial remission phase of type 1 diabetes. Newly diagnosed children had higher levels of FoxP3 expression in CD4 CD25 double positive cells (56.1%+/-24.9 vs. 24.9%+/-24.6 p=0.03) and higher mean numbers of IL-10 producing cells (7.3 cells/2x10(5) cells+/-6.6 vs. 0.86 cells/2x10(5)+/-0.36 p=0.0043) compared to partial remission patients. Higher FoxP3 expression at diagnosis predicted worse future glycemic control while higher mean numbers of IL-10 cells were associated with better future glucose control. These data provide an immune phenotype of the honeymoon phase and suggest that analyzing IL-10 and FoxP3 at diagnosis may identify patients that will experience better glucose control.

Islet transplantation can temporarily cure type 1 diabetes mellitus (T1DM) but requires simultaneous immunosuppression to avoid allograft rejection. In this issue of the JCI, Monti et al. report that immune conditioning via use of the Edmonton protocol - a treatment approach in which T1DM patients infused with pancreatic islets from multiple cadaveric donors simultaneously receive immunosuppressive drugs - results in lymphopenia that is associated with elevated serum levels of the homeostatic cytokines IL-7 and IL-15, which causes in vivo expansion of the autoreactive CD8(+) T cell population (see the related article beginning on page 1806). Reemergence of autoreactivity is likely the main culprit underlying long-term islet graft failure, and new strategies will need to be tested to circumvent this homeostatic expansion and recurrent autoreactivity.

Objective: Antilymphocyte serum can reverse overt type 1 diabetes in NOD mice; yet, the therapeutic parameters and immunological mechanisms underlying the ability for this agent to modulate autoimmune responses against beta-cells are unclear, forming the rationale for this investigation., Research Design and Methods: A form of antilymphocyte serum, rabbit anti-mouse thymocyte globulin (mATG), was utilized in a variety of in vivo and in vitro settings, each for the purpose of defining the physiological, immunological, and metabolic activities of this agent, with particular focus on actions influencing development of type 1 diabetes., Results: We observed that mATG attenuates type 1 diabetes development in an age-dependent fashion, only proving efficacious at disease onset or in the late pre-diabetic phase (12 weeks of age). When provided at 12 weeks of age, mATG reversed pancreatic insulitis, improved metabolic responses to glucose challenge, and rapidly increased frequency of antigen-presenting cells in spleen and pancreatic lymph nodes. Surprisingly, mATG therapy dramatically increased, in an age-dependent fashion, the frequency and the functional activity of CD4(+)CD25(+) regulatory T-cells. Adoptive transfer/cotransfer studies of type 1 diabetes also support the concept that mATG treatment induces a stable and transferable immunomodulatory repertoire in vivo., Conclusions: These findings indicate that an induction of immunoregulation, rather than simple lymphocyte depletion, contributes to the therapeutic efficacy of antithymocyte globulin and suggest that time-dependent windows for the ability to delay or reverse type 1 diabetes exist based on the capacity to enhance the functional activity of regulatory T-cells.

Induction of 'adaptive' regulatory T cells (Tregs) using islet-specific antigen vaccinations has been shown to prevent disease in various animal models for type 1 diabetes (T1D). Even though translation from bench to bedside has been unsuccessful so far, this non-invasive approach is the Holy Grail to safely achieve immune tolerance in humans. We will discuss here the fact that every immune response appears to contain a balance of adaptive effector and Treg cells. The evolution of these population and their antigen specificities over time during diabetes development will determine at which time and route a given islet antigen can be chosen to augment such adaptive Tregs most efficiently. Their 'resuscitation' will be crucial for long-term tolerance and homeostasis in the islet micro-environment, which is ultimately needed for a cure from T1D. Recent insight from our studies shows that short-term creation of a systemic milieu that favours Treg propagation, as it occurs after systemic administration of non Fc-binding anti-CD3, can strongly enhance this process. We propose that combination therapies with anti-CD3 or similar systemic immune modulators that lower effector cells and enhance Tregs with vaccines that induce adaptive Tregs will be a crucial step in developing successful immune-based intervention in T1D.

Recent clinical trials have shown that the loss of insulin production that characterizes progressive type 1 diabetes mellitus can be attenuated by treatment with non-FcR binding anti-CD3 monoclonal antibody (mAb). This approach is a first step towards the ultimate goals of treatment: to improve and maintain insulin production. However, additional interventions will be needed because, with time, there is progressive loss of insulin production after treatment with a single course of anti-CD3 mAb. The basis for the long-term loss of insulin production after immune therapy is not known because animal models have not been informative about the mechanisms, and there are not biomarkers of autoimmunity that can be used to monitor the process. Therefore, strategies for clinical testing might involve both beta cell and immunological therapies. Examples of the former include agents such as GLP1 receptor agonists or DPPIV inhibitors which increase beta cell insulin content. Preclinical data suggest that co-administration of antigen with anti-CD3 mAb can induce a tolerogenic response to the antigen that may then be administered to maintain tolerance. In addition, other immunological approaches as well as interventions earlier in the disease process may be successful in maintaining greater beta cell function for extended periods.

Chronic immunological processes that underlie persistent viral infections and autoimmune disorders such as multiple sclerosis can be relapsing-remitting in nature. The progressive loss of beta-cell mass during the development of autoimmune type 1 diabetes (T1D) can also be non-linear, but the exact nature and kinetics of the immunological processes that govern T1D are not known. Here, we propose that the immunological process that is at the root of T1D is relapsing-remitting in nature and discuss the unresolved controversies and therapeutic implications of this hypothesis.

In recent years, there has been a rapid expansion in the application of information technology to biological data. Although the use of information science techniques is less common for the discipline of immunology, this field has seen great strides in recent years. This review addresses why in silico modeling is needed in immunology research, highlights some of the major areas of research and suggests what may be important for the future of immunoinformatics.

A recent report in this year's April issue of Journal of the American Medical Association describes an unprecedented success in delaying insulin dependence in patients with recent-onset Type 1 diabetes after non-myeloablative immune suppression with cyclophosphamide and antithymocyte globulin followed by autologous stem cell transplantation. In this study, 14 out of 15 patients became insulin-independent, which lasted up to 35 months. Concomitantly, C-peptide levels increased substantially compared with preintervention values. Treatment of autoimmune disorders, and in particular Type 1 diabetes, constitutes a complex balancing act between suppressing autoaggressive responses strongly and permanently enough, while circumventing much-feared long-term side effects from chronic immunosuppression. This clinical Phase I/II trial is relevant to fine-tuning interventive protocols and contributing to the further development of suitable combination therapies to prevent and treat Type 1 diabetes.

The human gut offers more than 200 m2 of mucosal surface, where direct interactions between the immune system and foreign antigens take place to eliminate pathogens or induce immune tolerance toward food antigens or normal gut flora. Therefore, mucosally administered antigens can induce tolerance under certain circumstances. In autoimmune diabetes, mucosal vaccination with autoantigens elicits some efficacy in restoring tolerance in mice, but it never succeeded in humans. Furthermore, in some instances autoimmunity can be precipitated upon oral or intranasal autoantigen administration. Therefore, it is difficult to predict the effect of mucosal vaccination on autoimmunity and much effort should be put into establishing better assays to reduce the risk for possible adverse events in humans and enable a rapid and smooth translation.

Administration of autoantigens, especially via the mucosal route, can induce tolerance under certain circumstances. In autoimmune diabetes, mucosal vaccination with autoantigens was frequently effective in restoring tolerance in mice but has not yet succeeded in humans. Furthermore, in some instances, autoimmunity can be precipitated upon autoantigen administration. We will here briefly discuss the underlying reasons and delineate which efforts should be made in the future to rationally translate antigen-specific immunotherapy, for example, by establishing better assays to reduce the risk for possible adverse events in humans.

Safe induction of autoantigen-specific long-term tolerance is the "holy grail" for the treatment of autoimmune diseases. In animal models of type 1 diabetes, oral or i.n. immunization with islet antigens induces Tregs that are capable of bystander suppression. However, such interventions are only effective early in the prediabetic phase. Here, we demonstrate that a novel combination treatment with anti-CD3epsilon-specific antibody and i.n. proinsulin peptide can reverse recent-onset diabetes in 2 murine diabetes models with much higher efficacy than with monotherapy with anti-CD3 or antigen alone. In vivo, expansion of CD25(+)Foxp3(+) and insulin-specific Tregs producing IL-10, TGF-beta, and IL-4 was strongly enhanced. These cells could transfer dominant tolerance to immunocompetent recent-onset diabetic recipients and suppressed heterologous autoaggressive CD8 responses. Thus, combining a systemic immune modulator with antigen-specific Treg induction is more efficacious in reverting diabetes. Since Tregs act site-specifically, this strategy should also be expected to reduce the potential for systemic side effects.

Between ten and twenty million people worldwide have type 1 diabetes mellitus (T1DM), which has previously been called juvenile diabetes, childhood diabetes, and insulin-dependent diabetes mellitus. T1DM is undoubtedly a multifactorial disease affecting predisposed individuals with genetic susceptibilities; it is also associated with environmental factors leading to unbalanced immune responses. This chronic disorder is caused by auto-aggressive T lymphocytes entering the pancreatic islets of Langerhans where they destroy the insulin-producing beta-cells. A wide variety of immuno-interventions cure T1DM effectively in different animal models when given early in disease development. However, few of these interventions are efficacious in humans at a later stage of the disease. Indeed, only three immunotherapeutic compounds have demonstrated both safety and efficacy in phase II/III clinical trials. Although much time and resources have been spent on generating potent immune therapies, none of the patients enrolled in these trials have achieved normoglycemia in the absence of insulin injections. Many reasons can account for such a disappointing conclusion. Firstly, the dynamics of disease pathogenesis differs significantly from patient to patient, which directly impacts the therapeutic efficacy. Also, at trial entry, the percentage of remaining pancreatic beta-cells in T1DM patients often reflects the odds of responding positively to treatment. Based on the knowledge we have gained from preclinical studies and clinical trials, several steps have been made in the development of safer and more efficient immune-based therapies. There are, however, a number of concerns that should be addressed in order to improve future therapeutic strategies.

Transition Therapeutics (through its acquisition of Waratah Pharmaceuticals), in collaboration with Novo Nordisk, is developing E1-INT, an injectable islet neogenesis therapy comprising an epidermal growth factor analog and a gastrin analog, for the treatment of insulin-dependent (type 1) and non-insulin-dependent (type 2) diabetes. The compound is currently undergoing phase II clinical trials.

Since their discovery decades ago, regulatory T (Treg) cells have prompted many investigations into their potential role in the generation or prevention of autoimmune disorders such as type 1 diabetes (T1D). Initially identified based on their ability to maintain tolerance to self-antigens in peripheral organs, Treg cells have since been efficiently induced therapeutically and shown to prevent the progression of T1D as well as other autoimmune diseases. Beneficial modification of immunity through the induction of Treg cells has been successfully achieved by antigen-based therapy as well as non-antigen-specific (systemic) treatments. In the current article, we review different strategies that have proved effective in preventing autoimmune diabetes and analyze them with respect to translation into clinical applications. Current evidence indicates that antigen-specific induction of potent regulatory mechanisms is influenced by the systemic milieu, suggesting that systemic modulation might be an essential prerequisite for antigen-based therapy and the successful maintenance or reestablishment of tolerance.

Type 1 diabetes mellitus is an autoimmune disease characterized by T cell-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. From studies in animal models, CD8(+) T cells recognizing autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit-related protein, insulin, or glutamic acid decarboxylase (GAD) are believed to play important roles in both the early and late phases of beta cell destruction. In this study, we investigated the factors governing the diabetogenic potential of autoreactive CD8(+) clones isolated from spleens of NOD mice that had been immunized with GAD65(515-524) or insulin B-chain(15-23) peptides. Although these two clones were identical in most phenotypic and functional aspects, for example cytokine production and killing of autologous beta cells, they differed in the expression of IFN-gamma-inducible protein-10, which was only produced at high levels by the insulin-specific clone, but not by the GAD65-specific clone, and other autoantigen-specific nonpathogenic CD8 T cell clones. Interestingly, upon i.p. injection into neonatal mice, only the insulin B-chain(15-23)-reactive CD8(+) T clone accelerated diabetes in all recipients after 4 wk, although both insulin- and GAD-reactive clones homed to pancreas and pancreatic lymph nodes with similar kinetics. Diabetes was associated with increased pancreatic T cell infiltration and, in particular, recruitment of macrophages. Thus, secretion of IFN-gamma-inducible protein-10 by autoaggressive CD8(+) lymphocytes might determine their diabetogenic capacity by affecting recruitment of cells to the insulitic lesion.

Despite a large body of evidence describing associations between viruses and the development of type 1 diabetes (T1D) in genetically prone individuals, clearly defining causative infectious agents has not been successful. A likely explanation is that the link between infections and autoimmunity is more multifaceted than we initially assumed. Viral footprints might be hard to detect systemically or in the target organ once autoimmunity has been initiated, and several infections might have to act in concert to precipitate clinical autoimmunity. Furthermore, cells cross-reactive between viral and self-antigens might express low avidity T cell receptors and only be present transiently in the blood of affected individuals. In addition, there are two new observations from animal models that we should take into account at this point: first, viral infections alone might not be able to induce disease in the absence of other inflammatory factors (supporting the "fertile field hypothesis" [M.G. von Herrath et al., Microorganisms and autoimmunity: making the barren field fertile? Nat. Rev. Microbiol. 1 (2003) 151-157, ]). Second, increasing evidence indicates that viruses can play a role in preventing rather than enhancing T1D development (supporting the "hygiene hypothesis" [J.F. Bach, Protective role of infections and vaccinations on autoimmune diseases. J. Autoimmun. 16 (2001) 347-353]). In this article we will present an overview of the early events and requirements that could account for T1D predisposition and development, and explain how these can be modulated by viral infections. Focusing on coxsackie B and lymphocytic choriomeningitis virus infections, we will discuss new data that can hopefully help us understand how virus-induced inflammation can positively or negatively affect the clinical outcome of islet-autoimmunity and T1D.

A resurgent interest in T cells with regulatory activity has prompted many recent investigations into their potential role in pathogenesis and prevention of type 1 diabetes. While some studies have suggested that regulatory T cells participate in the preservation of active tolerance to autoantigens, findings obtained in multiple animal models for type 1 diabetes have documented the therapeutic induction of protective regulatory T cells. A review of the proposed mechanisms operative in regulatory T cell-mediated diabetes prevention indicates a common theme of localized regulatory T cell activation and subsequent suppression of pathogenic T cell trafficking, differentiation, and/or effector function. However, adaptation of experimental protocols for regulatory T cell induction to clinical applications faces several challenges. Immunization with self-antigens carries obvious risks especially in the face of multiple variables that can affect generation, trafficking, and regulatory activity of autoantigen-specific T cells. We also emphasize that the frequent use of lymphopenic recipients of adoptively transferred pathogenic and regulatory T cells constitutes a potentially confounding variable that further complicates translation into clinical settings. The therapeutic induction of regulatory T cells in prediabetic individuals carries great potential but is currently limited by the risks associated with deliberate generation of autoimmune responses that may exacerbate rather than ameliorate the autoimmune process. However, in vitro amplification and autologous regulatory T cell therapy might soon become a clinical reality.

The immune system evolved to rapidly recognize infectious threats and promptly mobilize cellular effectors to the infection site. Establishment of a robust T1-type immunity is a prerequisite for effective defense against most viruses and intracellular bacteria. However, accumulating evidence shows that T1 and T2 responses during such infections are not mutually exclusive. A possibility may be that the dual T1-T2 nature of antiviral immune responses is merely a byproduct of less than perfect crossregulatory mechanisms. Herein, we discuss molecular and cellular mechanisms of T-cell differentiation along with recent evidence supporting the hypothesis that rather than representing an epiphenomenon, coinduction of virus-specific T2 cells plays a significant homeostatic role. Thus, molecular pathways that regulate IL-4 production during influenza virus infection monitor T1-mediated immune responses in vital organs such as lungs and prevent immune pathology that may otherwise interfere with recovery from disease. Such evidence suggests that coinduction of T2 immunity maintains immune homeostasis during T1-mediated defense reactions. Finally, we outline implications on the earlier concept of T1/T2 dichotomy, supporting a model in which these two subsets, rather than being mutually antagonistic, together facilitate the recovery from infection.

Autoimmune type 1 diabetes (early onset/juvenile and slow onset T1D) is a disease mediated by autoreactive T cells accompanied by autoantibodies against islet associated antigens. The genetic predisposition, the association with certain HLA alleles (DR4 and DQ8) as well as the possibility to identify high-risk subjects by autoantibody measurement, fueled hope in regards to potential prophylaxis by vaccination. Numerous preclinical studies along with accumulating evidence for the role of certain autoantigens and putative dominant epitopes created the premises of clinical trials addressing this objective. However, the disappointing outcomes of many of such trials made it clear that significant challenges to clinical application of this concept must be addressed: we do not know at this point, how to choose the most appropriate set of autoantigens, how to optimally use them as a vaccine, whether to define and enroll only a subset of recipients, how to monitor their T cell responses to the vaccine and lastly, which the end-points of clinical T1D trials from an immunologic or metabolic perspective should be? Herein, we will approach some of the opportunities and obstacles associated with this intriguing strategy to fight autoimmune diabetes based on promising novel insight gained form studies with animal models.

Type 1 immunity relies on the differentiation of two major subsets of T lymphocytes, the CD4+ T helper (Th) cell and the CD8+ cytotoxic T cell, that direct inflammatory and cytotoxic responses essential for the destruction of intracellular and extracellular pathogens. In contrast to CD4 cells, little is known about transcription factors that control the transition from the CD8 naïve to effector cell stage. Here, we report that the transcription factor T-bet, known to regulate Th cell differentiation, also controls the generation of the CD8+ cytotoxic effector cell. Antigen-driven generation of effector CD8+ cells was impaired in OT-I T cell receptor transgenic mice lacking T-bet, resulting in diminished cytotoxicity and a marked shift in cytokine secretion profiles. Furthermore, mice lacking T-bet responded poorly to infection with lymphocytic choriomeningitis virus. T-bet is a key player in the generation of type 1 immunity, in both Th and T cytotoxic cells.

The concept of therapeutic immune regulation aiming to treat autoimmune diseases has been validated in multiple animal models, yet, the development of strategies for treatment of human autoimmune diseases remains problematic. Main obstacles are the contradictory findings in different model systems, as well as the contrasting functions of regulatory lymphocytes and cytokines. By drawing examples primarily from experimental type 1 diabetes, we propose that regulatory cells and cytokines can be classified according to the baseline at which they operate in healthy individuals and disease states that are not accompanied by severe systemic immune deficiency or skewing. Consequently, deletion or neutralization of regulatory cells or cytokines operative at high levels to maintain systemic homeostasis should constitute a therapeutic strategy for immune enhancement (e.g. tumor- and pathogen-specific immunity), whereas boosting these factors will have limited effects if the therapeutic goal is a downmodulation of immune responses (e.g. autoimmunity). Conversely, regulatory cells and cytokines operative at low homeostatic levels should unfold therapeutic capacities by further embellishment but not additional reduction.

Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

Accurate control of the balance of the T1 and T2 cells during antiviral immunity is essential for optimizing immune effector functions and for avoiding potentially severe immunopathology. We examined the in vivo role of the signal transducer and activator of transcription (STAT) 4 in regulating the T1/T2 balance during the response to live influenza virus and isolated viral proteins. We found that the differentiation of gamma interferon (IFN-gamma)-producing Th1 and Tc1 cells after inoculation of live virus occurred independently of STAT 4 expression. Influenza virus-specific T2 and Tc2 responses were well controlled in such STAT 4-deficient mice unless IFN-gamma was eliminated as well. In contrast, the STAT 4-dependent signaling pathway played a more essential role in regulating the T1/T2 balance after immunization with viral proteins and, in particular, inactivated nonreplicating virus. Pulmonary infection was cleared even in the absence of both functional STAT 4 genes and functional IFN-gamma genes because virus-neutralizing antibodies were still generated, consistent with a substantial redundancy in different antiviral effector pathways. Thus, replicating agents such as live influenza virus can elicit IFN-gamma and control T2 immunity independently of STAT 4, whereas the profile of immunity to isolated proteins is more reliant on an intact STAT 4 signaling pathway.

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

We have studied the induction and effector function of Th2-like regulatory cells in mouse models for type 1 diabetes (NOD and RIP-LCMV). CD4+ lymphocytes with specificity for insulin can be induced by immunization with the insulin B chain via the oral route or by DNA vaccination. Such cells are protective upon adoptive transfer and prevent diabetes development in syngeneic pre-diabetic recipients. In comparison to non-regulatory insulin B-specific cell lines, they produce high amounts of interleukin (IL)4 and IL10, whereas interferon (IFN)gamma and tumour necrosis factor (TNF)alpha levels are comparable. Indeed, IL4 is essential for the protective capability, as evidenced by use of IL4-deficient mice and sorting of IL4+ versus IL4- lymphocytes prior to transfer. Mechanistically, these cells act as bystander suppressors in the pancreatic draining node, the location where their cognate antigen, insulin B, is presented during the pre-diabetic inflammatory process. As a consequence, the autoaggressive response is locally dampened. We propose that this is achieved by modulation of antigen presenting cells that lose the ability to propagate aggressive responses after exposure to IL4 or IL10 in vitro. The clinically attractive side of our strategy is that it only acts as the site of inflammation, thus circumventing systemic side effects. In order to avoid induction of insulin B-specific autoaggressive T cells we have demonstrated that administration of IL4 or IL10 at the time of immunization is beneficial and therefore should be part of a potential future clinical application. Interestingly, these Th2-like regulators share in our systems no features with the so-called CD25+ regulatory cells, whose antigen specificity is still unclear. However, we have recent evidence that virus specific CD25+ cells can be generated and are able to affect antiviral responses in vivo.

The ultimate goal for autoimmune immunotherapy is to achieve a specific downregulation or modification of autoaggressive immune responses while leaving in place the normal repertoire, capable of mediating antimicrobial responses. A multitude of preclinical studies, particularly during the last 15 years, raised hopes that self-antigens could be used to achieve the goal of specific immune modulation. Difficulties associated with the translation of this concept to the clinic revealed inherent limitations of antigen-based immune modulation. To increase the efficiency of antigen-dependent immune modulation, researchers started to investigate novel vectors for antigen delivery. Plasmid vectors, as opposed to protein antigens or peptides, have the ability to trigger prolonged production of limited amounts of antigen in the periphery. However, one complicating factor may be the inherent "danger" signal stimulated by the nature of the unmethylated CpG motifs on bacterial plasmid. Currently, various approaches are being explored to improve the efficacy of response while ameliorating the safety concerns of plasmids as immunotherapeutic tools. This manuscript offers a perspective on such efforts and outlines how the knowledge accumulated in the process will help scientists advance to the next generation of immunotherapeutics.

Autoreactive cells that escape from thymic negative selection are either anergized or counter-regulated in the peripheral lymphoid organs by regulatory T (Treg) cells. Most investigated Treg cells are CD4pos cells expressing the alpha chain of the IL-2 receptor (CD25). Absence of this particular population leads to the development of a wide range of autoimmune disorders, suggesting a critical role for CD4pos CD25pos cells in the maintenance of normal immune homeostasis. Numerous studies have aimed at unraveling the mechanisms of regulation, focusing mainly on naturally occurring Treg cells (generated in naive animals) and regulation of CD4pos autoreactive cells. In contrast, generation following antigenic priming of CD4pos CD25pos Treg cells with non-self (viral) antigen specificity and regulation of virus-activated CD4pos and CD8pos cells remain to be investigated. In this review, we describe the data collected over the past few years of intense focus on CD4pos CD25pos Treg cells and present our approach to investigate the generation of antigen-specific Treg cells during the course of an anti-viral immune response.

Insulin-dependent diabetes mellitus (IDDM) is a multifactorial disease. Besides a genetic predisposition environmental factors have been implicated in the pathogenesis of beta cell destruction. Among these environmental factors viruses have been the focus of many studies. Some viruses are diabetogenic in animals, and others have been implicated as triggers in human IDDM by temporal and geographical association between IDDM and viral infections, serological evidence of infection in recently diagnosed diabetic patients, and the isolation of viruses from the pancreas of affected individuals. We discuss possible pathomechanisms of viral infections in beta cell destruction and review the studies on involvement of enteroviruses, retroviruses, rubella viruses, cytomegaloviruses, and Epstein-Barr viruses in human IDDM. We also report on studies of diabetogenic viruses in animal models as well as on viral infections protecting from IDDM. Some of the difficulties in linking viral infections to IDDM will be illustrated with data from a transgenic mouse model in which IDDM can be precipitated by infections with certain strains of lymphocytic choriomeningitis virus (LCMV). Emerging treatment concepts that do not rely on defining the initiating autoantigens but involve self-reactive regulatory lymphocytes such as oral antigen administration, as well as DNA vaccines, will be discussed briefly.

Crosspresentation of self-antigens by antigen-presenting cells is critical for the induction of peripheral tolerance. As apoptosis facilitates the entry of antigens into the crosspresentation pathway, we sought to prevent the development of autoimmune diabetes by inducing pancreatic beta cell apoptosis before disease onset. Accordingly, young nonobese diabetic (NOD) mice injected with a single low dose of streptozotocin (SZ), a drug cytotoxic for beta cells, exhibited impaired T cell responses to islet antigens and were protected from spontaneous diabetes. Furthermore, beta cell apoptosis was necessary for protection since SZ did not protect RIP-CrmA transgenic NOD mice in which beta cells expressed the caspase inhibitor CrmA. Our results support a model in which apoptosis of pancreatic beta cells induces the development of regulatory cells leading to the tolerization of self-reactive T cells and protection from diabetes.

Administration of autoantigens through DNA immunizations or via the oral route can prevent progression of islet destruction and lower the incidence of type 1 diabetes in animal models. This beneficial effect is mediated by autoreactive regulatory CD4 lymphocytes, and it is known that their induction depends on the precise dose and route of antigen administration. However, it is not clear which endogenous factors determine when such immunizations lead to activation of regulatory versus aggressive autoreactive lymphocytes and how a deleterious outcome can be avoided. Here we describe novel observations made in an animal model for virally induced type 1 diabetes, showing that the endogenous expression levels of the islet antigens and glutamic acid decarboxylase determine whether immunization with these antigens is beneficial or detrimental. Lower expression levels in beta-cells support immune regulation resulting in induction of autoreactive, regulatory cells characterized by increased IL-4 production (Th2-like), whereas higher levels favor Th1-like autoaggressive responses characterized by augmented IFN-gamma generation. Co-immunization with an IL-4-expressing plasmid reduces the risk of augmenting autoaggression and in this way increases the safety margin of this immune-based therapy. Our findings will be of importance for designing safe antigen-specific interventions for human type 1 diabetes.

In comparing the incidence of virally induced type 1 diabetes in F(1) crosses of RIP-LCMV mice to three different mouse strains identical at the major histocompatibility complex H-2D(b) locus, we surprisingly found that disease development was reduced by 80% in F(1) crosses to the SV129 genetic background and by 60% after eight backcrosses to the original C57BL/6 RIP-LCMV mice. In this model, diabetes is strongly dependent on a virally induced H-2D(b)-restricted cytotoxic T-cell (CTL) response. Importantly, numbers and effector functions of autoaggressive CD4 and CD8 lymphocytes were not decreased in the protected mice, and CTLs were still able to kill syngeneic islet cells in vitro with equal efficacy compared with CTLs from the original RIP-LCMV strain. Furthermore, CTLs were able to extravasate into islets in vivo, and no evidence for induction of regulatory cells was observed. However, regeneration of beta-cells in islets under "attack" occurred only in the protected SV129-crossed animals, whereas it was not evident at any time in any mice that developed diabetes. Thus, genetic factors can "override" the diabetogenic potential of high numbers of autoaggressive lymphocytes through, for example, increased islet regeneration. This finding has important implications for interpreting numbers and pathogenicity of autoreactive lymphocytes in prediabetic patients of genetically diverse backgrounds.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-gamma-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4(-/-) NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.

We report here that islet-specific expression of TNF-alpha can play a dual role in autoimmune diabetes, depending on its precise timing in relation to the ongoing autoimmune process. In a transgenic model (rat insulin promoter-lymphocytic choriomeningitis virus) of virally induced diabetes, TNF-alpha enhanced disease incidence when induced through an islet-specific tetracycline-dependent promoter system early during pathogenesis. Blockade of TNF-alpha during this phase prevented diabetes completely, suggesting its pathogenetic importance early in disease development. In contrast, TNF-alpha expression abrogated the autoimmune process when induced late, which was associated with a reduction of autoreactive CD8 lymphocytes in islets and their lytic activities. Thus, the fine-tuned kinetics of an autoreactive process undergo distinct stages that respond in a differential way to the presence of TNF-alpha. This observation has importance for understanding the complex role of inflammatory cytokines in autoimmunity.