Development of new strategies to prevent type 1 diabetes: the role of animal models

Type 1 diabetes is an immune-mediated disease typically preceded by a long preclinical stage during which a growing number of islet-cell-specific autoantibodies appear in the serum. Although antigen-specific T lymphocytes and cytokines rather than these autoantibodies are the likely executors of beta-cell-destruction, these autoantibodies reflect the existence of autoimmunity that targets islet beta-cells. Abrogation of this autoimmunity during the preclinical stage would be the key to the prevention of type 1 diabetes. However, the quest of protecting islet-cells from the immune attack requires detailed knowledge of mechanisms that control islet-inflammation and beta-cell-destruction, and of mechanisms that control immune tolerance to peripheral self-antigens in general. This knowledge can only be obtained through further innovative research in experimental animal models. In this review, we will first examine how research in non-obese diabetic mice has already led to promising new strategies of diabetes prevention now being tested in human clinical trials. Thereafter, we will discuss how recent advances in understanding the mechanisms that control immune response to peripheral self-antigens such as beta-cell antigens may help to develop even more selective and effective strategies to prevent diabetes in the future.     

Immunopathogenesis and immunotherapeutic approaches to type 1A diabetes

It is now clear that type 1A (immune-mediated) diabetes develops in genetically susceptible individuals where, prior to the onset of overt hyperglycaemia, there is usually a long prodrome characterised by the presence of autoimmunity directed at islet beta cells. It is the destruction of these insulin-producing cells that results in loss of metabolic regulation and the resultant hyperglycaemia and severe sequelae of type 1A diabetes. An extensive body of animal data and a developing body of human studies are now addressing therapies directed at this root immune cause of type 1A diabetes. Therapies can be considered in terms of the disease stage at which they are applied and in terms of their effects on the immune system (e.g., generalised immunosuppression, immunomodulation, antigen-specific therapies and tolerance-inducing therapies). As T cells are the primary mediators of islet beta cell destruction, it is likely that improved therapies and monitoring of T cell autoimmunity will be necessary to develop a safe and effective therapy for type 1A diabetes.     

Immunopathogenesis and immunotherapeutic approaches to type 1A diabetes

It is now clear that type 1A (immune-mediated) diabetes develops in genetically susceptible individuals where, prior to the onset of overt hyperglycaemia, there is usually a long prodrome characterised by the presence of autoimmunity directed at islet beta cells. It is the destruction of these insulin-producing cells that results in loss of metabolic regulation and the resultant hyperglycaemia and severe sequelae of type 1A diabetes. An extensive body of animal data and a developing body of human studies are now addressing therapies directed at this root immune cause of type 1A diabetes. Therapies can be considered in terms of the disease stage at which they are applied and in terms of their effects on the immune system (e.g., generalised immunosuppression, immunomodulation, antigen-specific therapies and tolerance-inducing therapies). As T cells are the primary mediators of islet beta cell destruction, it is likely that improved therapies and monitoring of T cell autoimmunity will be necessary to develop a safe and effective therapy for type 1A diabetes.     

MERTK on mononuclear phagocytes regulates T cell antigen recognition at autoimmune and tumor sites

Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in prediabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.     

Autoimmune diabetes not requiring insulin at diagnosis (latent autoimmune diabetes of the adult): definition, characterization, and potential prevention

Type 1 diabetes is caused by the immune-mediated destruction of islet insulin-secreting beta-cells. This chronic destructive process is associated with both cellular and humoral immune changes in the peripheral blood that can be detected months or even years before the onset of clinical diabetes. Throughout this prediabetic period, metabolic changes, including altered glucose tolerance and reduced insulin secretion, deteriorate at variable rates and eventually result in clinical diabetes. A fraction of individuals with humoral immunological changes have clinical diabetes that initially is not insulin-requiring. The onset of diabetes in these patients is usually in adult life, and because their diabetes is at least initially not insulin-requiring, they appear clinically to be affected by type 2 diabetes. Such patients probably have the same disease process as patients with type 1 diabetes in that they have similar HLA genetic susceptibility as well as autoantibodies to islet antigens, low insulin secretion, and a higher rate of progression to insulin dependency. These patients are defined as being affected by an autoimmune type of diabetes not requiring insulin at diagnosis, which is also named latent autoimmune diabetes of the adult (LADA). Special attention should be paid to diagnose such patients because therapy may influence the speed of progression toward insulin dependency, and in this respect, efforts should be made to protect residual C-peptide secretion. LADA can serve as a model for designing new strategies for prevention of type 1 diabetes but also as a target group for prevention in its own right.     

Autoimmune diagnostics in diabetes mellitus.

Type 1 diabetes results from a specific destruction of the insulin-producing beta-cells of the pancreas. The disease is characterized by the appearance of specific autoantibodies against islet cell antigens. Autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase IA-2 and cytoplasmic islet cell antibodies are useful markers for the differential diagnosis of type 1 diabetes when clinical and metabolic criteria alone do not allow definite classification. Autoimmune diagnostics is of particular importance in adults to discriminate between type 1 and type 2 diabetes and to assess the diagnosis of latent autoimmune diabetes in adults.     

Role of Viruses in the Pathogenesis of IDDM

Insulin-dependent diabetes mellitus (IDDM), also known as type I diabetes, results from the destruction of pancreatic beta cells. During the past few decades, genetic factors, autoimmunity and viral infections have been extensively studied as the possible cause of beta cell destruction. The evidence for virus-induced diabetes comes largely from experiments in animals, but several studies in humans also point to viruses as a trigger of this disease in some cases. There are at least two possible mechanisms for the involvement of viruses in the pathogenesis of IDDM: (a) cytolytic infection of beta cells may result in destruction of the cells without the induction of autoimmunity, or may be a final insult leading to the clinical onset of diabetes in individuals with an already decreased beta cell mass resulting from an autoimmune process; and (b) persistent viral infection (e.g. retrovirus, rubella virus, cytomegalovirus) may result in the triggering of autoimmune IDDM in certain circumstances. Regarding the latter possibility, viruses may insert, expose, or alter antigens in the plasma membrane of the beta cell, which may initiate autoimmunity leading to the destruction of the cells.     

No evidence for serological autoimmunity to islet cell heat shock proteins in insulin dependent diabetes.

Recent studies in nonobese diabetic mice have implicated the autoimmune destruction of pancreatic islet cells with immunity to a beta cell protein cross-reactive to Mycobacterium tuberculosis heat shock protein 65 (hsp 65). Therefore, our studies examined serological immunity to islet cell hsp in humans with insulin-dependent diabetes (IDD). Heat shock of human islet cells in vitro markedly increased the synthesis of proteins of 72,000, 75,000, and 90,000 Mr. No autoantibodies reactive to these hsp, nor to the constituently expressed islet cell hsp 65 protein (identified as 60,000 Mr) were observed in IDD patients. The islet cell 64,000-Mr autoantigen and hsp 65 proteins were physiologically and immunocompetitively distinct. These experiments do not support the hypothesis that IDD in humans is associated with autoimmunity to islet cell heat shock proteins.     

Islet cell autoantibodies: pathobiology and clinical applications

Autoimmunity directed against pancreatic islet cells results in slowly progressing beta-cell destruction, culminating over years in clinically manifested insulin-dependent diabetes mellitus (IDDM). Circulating serum autoantibodies directed against the endocrine cells of the islets of Langerhans are an important hallmark of this disease. Assays for these islet cell antibodies (ICA) have facilitated the investigation and understanding of several facets in the pathogenesis of autoimmune diabetes. Their applications have begun to extend into clinical practice and have opened new avenues for early preclinical prediction and preventive prophylaxsis in IDDM.     

Is insulin-dependent diabetes mellitus a preventable disease?

Despite improvements in the ongoing care of individuals with insulin-dependent diabetes, the disease continues to produce significant morbidity and mortality, especially early in life, in individuals developing insulin-dependent diabetes in childhood or adolescence. In order to have a major impact on disease outcome, the best therapeutic approach is disease prevention. Because insulin-dependent diabetes results from autoimmune pancreatic beta cell destruction, the disease may be amenable to immunological intervention with immunotherapy. This is particularly exciting as we develop tools, such as islet cell autoantibody determinations, that allow diagnosis of individuals prior to clinical presentation. It is during this time-period when immune manipulation may be most efficacious in preventing further beta cell destruction, and otherwise eventual insulin dependence. As such trials are highly experimental, they must be conducted only in research centers staffed by physicians and scientists with expertise in diabetes, autoimmunity, and immunology.     

Signs of beta-cell autoimmunity in nondiabetic schoolchildren: a comparison between Russian Karelia with a low incidence of type 1 diabetes and Finland with a high incidence rate

OBJECTIVE: We sought to study the prevalence of autoantibodies to various islet cell antigens in the background population of two neighboring countries with a sixfold difference in the incidence of type 1 diabetes. RESEARCH DESIGN AND METHODS: Serum samples were obtained from 3,652 nondiabetic schoolchildren in Finland and from 1,988 schoolchildren in the adjacent Karelian Republic of Russia. The Karelian children were divided into three groups (Finns/Karelians, Russians, and others) based on the ethnic background of their mother. The samples were analyzed for islet cell antibodies (ICAs), insulin autoantibodies (IAAs), GAD antibodies (GADAs), and the tyrosine phosphatase-like insulinoma antigen 2 (IA-2A) protein and HLA class II genotypes. RESULTS: The frequency of ICAs, IAAs, and GADAs did not differ significantly between the Karelian (3.5, 0.6, and 0.9%, respectively) and Finnish children (2.8, 0.9, and 0.5%, respectively). Similarly, the frequency of multiple (> or = 2) autoantibodies was similar in both countries (0.5 vs. 0.6%). The frequency of IA-2A was, however, four times higher in Finland (0.6 vs. 0.15% in Russian Karelia; P = 0.03). There were no significant differences in autoantibody prevalence among the three ethnic groups in Russian Karelia. There was a falling frequency of GADAs and of positivity for multiple autoantibodies along with decreasing HLA-conferred disease susceptibility among the Finnish schoolchildren. CONCLUSIONS: These data indicate that beta-cell autoimmunity among schoolchildren is as frequent in Russian Karelia as in Finland, although the incidence of clinical type 1 diabetes is six times higher in Finland. However, in contrast to this general trend, IA-2As were more common in Finland. Since IA-2As usually appear late in the preclinical process, this suggests that progressive beta-cell autoimmunity is more rare in Russian Karelia.     

Preventing type 1 diabetes mellitus: hype and hope

Autoimmune diseases affect 10% or more of the UK population. In organ-specific autoimmune diseases a particular tissue is targeted by the aggressive immune response. Type 1 diabetes is due to destruction of insulin-secreting islet cells. Both genetic and environmental factors cause type 1 diabetes and environmental events can operate very early, even in utero. The early induction of diabetes-associated autoantibodies and the long prediabetic period means that autoimmune diabetes in children can be predicted by detecting these autoantibodies. The purpose of prediction is prevention and initial studies suggest that it is possible to modify the disease process. However, the impact of such therapy on the disease is as yet extremely modest.     

Islet cell and thyroid antibody prevalence in patients with hepatitis C virus infection: effect of treatment with interferon

An increased prevalence of hepatitis C virus (HCV) infection in patients with diabetes and a higher prevalence of diabetes in HCV-infected patients have been reported. However, the relationship between these two conditions remains controversial. In addition, although the effect of interferon treatment on thyroid autoimmunity has been extensively reported, its influence on beta-cell autoantibodies has not been investigated. The aims of the study were (1) to evaluate whether autoimmune beta-cell damage could be involved in the development of diabetes mellitus in HCV-infected patients and (2) to determine whether interferon treatment influences the appearance of beta-cell and thyroid autoantibodies. The prevalence of islet cell autoantibodies (glutamic acid decarboxylase antibodies [GADAs], tyrosine phosphatase antibodies [IA-2s], islet cell antibodies [ICAs]) was assessed in 303 non-selected HCV-infected patients (277 non-diabetic and 26 type 2 diabetic patients) and in 273 sex- and age-matched control subjects. ICAs and thyroid autoantibodies were also determined before and 6 and 12 months after treatment with interferon for 24 weeks in a subgroup of 46 HCV-infected patients. GADAs were detected in 4 of 277 (1.4%) HCV-infected non-diabetic patients, 1 of 273 (0.3%) control subjects, and 0 of 26 (0%) HCV-infected patients with diabetes. Anti-IA2s and ICAs were negative in all subjects. Both GADAs and anti-IA2s were negative in all HCV-infected patients treated with interferon. After therapy, only thyroid antibodies became positive in 5 of 46 (10.9%) treated patients, disappearing in all but 1 of these at the 12-month follow-up. Our results suggest that beta-cell autoimmunity is not associated with HCV infection, thus making it unlikely that the increased diabetes mellitus prevalence among HCV-infected patients could be mediated by autoimmune mechanisms. In addition, interferon treatment induces a transient increase in thyroid autoantibodies but does not influence the appearance of beta-cell autoantibodies.     

Viral IL-10-mediated immune regulation in pancreatic islet transplantation.

Protection of transplanted pancreatic islet grafts in recipients with autoimmune diabetes depends on the suppression of autoimmune recurrence and allogeneic rejection. The aim of this study was to investigate the efficiency of viral IL-10 gene delivery in the prevention of autoimmune recurrence following islet transplantation. We evaluated the effectiveness of a systemically delivered adeno-associated viral vector (AAV vIL-10) carrying viral IL-10 in protecting islet engraftment. We observed significant prolongation of graft survival after treatment with AAV vIL-10 when using islets from donors lacking autoimmunity. We found that the mechanism of vIL-10-mediated protection was associated with suppression of T cell activation and that donor immune cells that were simultaneously transferred with the islet grafts could induce autoimmune recurrence. AAV vIL-10 gene transfer suppressed previously activated T cells and protected grafted islets from autoimmune-mediated destruction. We conclude that vIL-10 can regulate autoimmune activity and that transfer of its gene may have potential for therapeutic islet transplantation.     

Reversal of established autoimmune diabetes by restoration of endogenous beta cell function.

In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-alpha-induced apoptosis. Here, we examine the hypothesis that induction of TNF-alpha expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF-alpha expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established beta cell-directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.     

Proinsulin/Insulin Autoantibodies Measured With Electrochemiluminescent Assay Are the Earliest Indicator of Prediabetic Islet Autoimmunity

OBJECTIVE

We evaluated a novel electrochemiluminescent assay for insulin/proinsulin autoantibodies (ECL-IAA) as a new marker of the onset of islet autoimmunity and as a predictor of type 1 diabetes.

RESEARCH DESIGN AND METHODS

The Diabetes Autoimmunity Study in the Young (DAISY) prospectively follows children at increased genetic risk for development of islet autoimmunity (defined as presence of autoantibodies to insulin, GAD65, IA-2, or zinc transporter 8 [ZnT8]) and type 1 diabetes (general population of children and first-degree relatives). Serial serum samples from subjects who progressed to type 1 diabetes and who had their first islet autoantibodies measured by age 18 months (N = 47) were tested using ECL-IAA.

RESULTS

Almost all prediabetic children tested positive for ECL-IAA (46 of 47, 98%) during follow-up. ECL-IAA was almost always the first autoantibody to appear (94% total; 21% very first [by itself]; 23% with only mIAA; 19% with another islet autoantibody [GAD or ZnT8]; and 30% with ≥2 other antibodies [mIAA, GAD, IA-2, or ZnT8]). Among the 46 subjects who were ECL-IAA positive, ECL-IAA antedated the onset of other islet autoantibodies by a mean of 2.3 years (range, 0.3–7.2 years). Both the age of appearance of autoantibody and IAA levels (but not GAD65, IA2, or ZnT8 levels) are major determinants of the age of diabetes onset.

CONCLUSIONS

This new ECL-IAA assay defines more precisely the onset of prediabetic autoimmunity and may help identify events triggering islet autoimmunity, as well as allow earlier intervention for type 1 diabetes. Nearly all young children progressing to diabetes are insulin autoantibody positive.Anti-islet autoimmunity currently detected by measurement of islet autoantibodies almost always precedes by years the development of type 1A diabetes. If the autoimmunity is triggered by time-correlated factors such as acute viral infections, then the discovery of pathogenic viruses may depend on accurate timing of the appearance of islet autoantibodies. Most of the trials to prevent type 1A diabetes target persons in the preclinical phase of the disease marked by the presence of persistent islet autoantibodies (1). Because this preclinical period is quite variable, accurate prediction of the time to progression to overt diabetes is critical for the design and implementation of preventive trials. Although genetic markers can identify varying risk, it is only once autoimmunity has begun (marked by the presence of multiple autoantibodies to pancreatic β-cell antigens) that a high positive predictive value (>90%) can be achieved. Multiple autoantibodies are present in the majority of prediabetic individuals (2–4). Screening for risk of type 1 diabetes uses “biochemical” autoantibody assays for specific islet autoantigens (1). These include insulin autoantibodies (IAA) (5), GAD65 (6), protein tyrosine IA-2 (ICA512) (7), and, most recently, zinc transporter 8 (ZnT8) (8). Individuals having a single positive autoantibody (insulin, GAD65, IA-2, or ZnT8 autoantibodies) are at low risk for progression to diabetes, whereas individuals expressing two or more positive autoantibodies, especially on multiple tests over time, are at very high risk for progression to diabetes (9,10).IAA are usually extremely high at the onset of diabetes in young children but usually negative in individuals first presenting with diabetes after age 12 years. There is a log-linear inverse relationship between these levels and the age of onset of diabetes (11), as well as between levels of IAA and time of progression from first appearance of islet autoantibodies to diagnosis of diabetes in prospectively followed Diabetes Autoimmunity Study in the Young (DAISY) children (10). We recently have reported (12) development of an electrochemiluminescence assay for IAA (ECL-IAA) using Meso Scale instrumentation and ruthenium-labeled proinsulin. This assay detects high-affinity IAA and is more sensitive than the micro-IAA (mIAA) radioassays in the last Diabetes Autoantibody Standardization Program (DASP) workshop, yet is equally specific.In this study we evaluated ECL-IAA as a new marker of the onset of islet autoimmunity and as a predictor of progression to diabetes among antibody-positive subjects. We found that this novel nonradioactive IAA assay is more sensitive and defines the timing of the initial autoantibody appearance earlier than the previously used mIAA radioassay. We report the predictors of progression to diabetes and the determinants of age at diagnosis among children at high risk participating in the prospective DAISY.     

Role of viruses in the pathogenesis of IDDM

Insulin-dependent diabetes mellitus (IDDM), also known as type I diabetes, results from the destruction of pancreatic beta cells. During the past few decades, genetic factors, autoimmunity and viral infections have been extensively studied as the possible cause of beta cell destruction. The evidence for virus-induced diabetes comes largely from experiments in animals, but several studies in humans also point to viruses as a trigger of this disease in some cases. There are at least two possible mechanisms for the involvement of viruses in the pathogenesis of IDDM: (a) cytolytic infection of beta cells may result in destruction of the cells without the induction of autoimmunity, or may be a final insult leading to the clinical onset of diabetes in individuals with an already decreased beta cell mass resulting from an autoimmune process; and (b) persistent viral infection (e.g. retrovirus, rubella virus, cytomegalovirus) may result in the triggering of autoimmune IDDM in certain circumstances. Regarding the latter possibility, viruses may insert, expose, or alter antigens in the plasma membrane of the beta cell, which may initiate autoimmunity leading to the destruction of the cells.     

Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease

Vaccination with dendritic cells (DCs) presenting tumor antigens induces primary immune response or amplifies existing cytotoxic antitumor T cell responses. This study documents that antitumor treatment with DCs may cause severe autoimmune disease when the tumor antigens are not tumor-specific but are also expressed in peripheral nonlymphoid organs. Growing tumors with such shared tumor antigens that were, at least initially, strictly located outside of secondary lymphoid organs were successfully controlled by specific DC vaccination. However, antitumor treatment was accompanied by fatal autoimmune disease, i.e., autoimmune diabetes in transgenic mice expressing the tumor antigen also in pancreatic beta islet cells or by severe arteritis, myocarditis, and eventually dilated cardiomyopathy when arterial smooth muscle cells and cardiomyocytes expressed the transgenic tumor antigen. These results reveal the delicate balance between tumor immunity and autoimmunity and therefore point out important limitations for the use of not strictly tumor-specific antigens in antitumor vaccination with DCs.     

Targeting T lymphocytes for immune monitoring and intervention in autoimmune diabetes

Recognition of a peptide-MHC complex by the T cell receptor (TCR) is a key interaction that initiates T lymphocyte activation or silencing during an immune response. Fluorochrome-labeled recombinant MHC class II-peptide reagents function as soluble mimetics of this interaction, bind to their specific TCR, and allow for detection of antigen-specific CD4+ T cells. These reagents are now under scrutiny for "immune staging" of patients at risk of type 1 diabetes, in an effort to diagnose islet autoimmunity early enough to block immune-mediated beta cell destruction. Several issues are currently being addressed to improve the performance of these T cell assays: enrichment steps for better sensitivity, multiplexing of several islet epitopes, simultaneous monitoring of CD4+ and CD8+ responses, detection of low avidity T cells, combination of quantitative (number of positive cells) and qualitative (cytokine secretion, naive/memory phenotype) readouts. CD4+ T cells are key effectors of autoimmunity, and these MHC class II peptide reagents, through their signaling properties, might also provide therapeutics to block the autoimmune process at its onset, analogous to the use of OKT3gammao1(AlaAla) anti-CD3 antibody but in an antigen-specific fashion. The aim of such therapeutics is to potentiate different physiological control mechanisms to restore immune tolerance. Mechanisms initiated by this pathway may be capable of triggering elimination of pathogenic T cells through antigen-specific apoptosis and anergy, combined with the induction of regulatory T cells with broad suppressive function.     

Islet-reactive T cells are a marker of preclinical insulin-dependent diabetes.

The destruction of pancreatic islet beta cells in insulin-dependent diabetes mellitus (IDDM) is thought to be T cell mediated. To directly identify islet-reactive T cells in asymptomatic, "preclinical" IDDM individuals with islet cell antibodies (ICA), proliferation of peripheral blood mononuclear cells (PBMC) was measured in the presence of sonicated fetal pig proislets. Stimulation indices (mean +/- SD) for [3H]thymidine uptake by PBMC cultured with sonicated proislets were: preclinical IDDM subjects (n = 22) 6.10 +/- 6.50, recent-onset IDDM subjects (n = 29) 3.66 +/- 3.35, Graves' disease subjects (n = 6) 2.17 +/- 0.93, scleroderma subjects (n = 4) 1.65 +/- 0.19 and normal control subjects (n = 14) 1.63 +/- 0.62. 68% (15/22) of preclinical IDDM, 41% (12/29) of recent-onset IDDM and 17% (1/6) of Graves' disease subjects had T cell reactivity greater than the mean + 2 SD of controls. T cell reactivity to proislets was tissue specific, and greater in magnitude and frequency than to human insulin. The majority of preclinical subjects with ICA greater than 20 Juvenile Diabetes Foundation (JDF) units (12/15, 80%) or antibodies to a 64-kD islet autoantigen (11/15, 73%) had significant T cell reactivity to proislets. ICA greater than 40 JDF units, a strong prognostic marker for progression to clinical IDDM, was an absolute index of T cell reactivity. Overall, the frequency of T cell reactivity in preclinical subjects, 68% (15/22), was comparable to that of ICA greater than 20 JDF units or 64-kD antibodies. Greater T cell reactivity to proislets in preclinical subjects accords with the natural history of autoimmune beta cell destruction. The direct assay of islet-reactive T cells in peripheral blood may have prognostic significance for the development of clinical IDDM and should facilitate identification of the primary target autoantigen(s).