Insulin-Specific Tolerance in Diabetes

Clinical Immunology Vol. 102, No. 1, January, pp. 2–11, 2002 doi:10.1006/clim.2001.5142, available online at http://www.idealibrary.com on

SHORT ANALYTICAL REVIEW Insulin-Specific Tolerance in Diabetes Peter A. Gottlieb and George S. Eisenbarth Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver, Colorado 80262

1. EARLY HISTORY OF INSULIN IMMUNITY/AUTOIMMUNITY

At present it is possible to predict the development of type 1A diabetes (immune-mediated diabetes) in man and prevent the disorder in animals. Studies of immunity to insulin play a prominent role in both disease prediction and disease prevention. For both man and the NOD mouse, insulin autoantibodies usually precede the development of diabetes and can be utilized to assist in disease prediction. T cells clones recognizing insulin, both CD4 and CD8, can transfer disease to young mice or immunodeficient animals. Specific insulin peptides reacting with these clones have been identified, their crystal structure when bound to a human “diabetogenic” MHC allele has been determined, and specific peptides can be used either to induce or to prevent disease. Clinical trials of both insulin and an altered peptide ligand of insulin to prevent islet ␤-cell destruction are underway. Insulin is one of a number of islet autoantigens, but it is likely that immune responses to insulin will be central to both pathogenesis and immunologic protection. © 2001

The insulin molecule is physiologically secreted from pancreatic islet ␤-cells under the regulated control of a glucose-sensing system (glucokinase). Its metabolic functions include stimulation of glucose storage in the liver, glucose uptake, and utilization in muscle and fat cells. Insulin is a polypeptide hormone composed of an A chain of 21 amino acids and a B chain of 30 amino acids (aa), which are joined by two disulfide bonds between cysteine residues on each chain. It is processed from preproinsulin within the secretory granules of the ␤-cell, and proinsulin, insulin, and the cleaved C-peptide (33–35 amino acids) are secreted into the blood stream. Of note, not only is insulin produced within islet ␤-cells, but isolated cells within both the thymus and the spleen have messenger RNA for insulin and can be stained with anti-insulin monoclonals (5). Hanahan and co-workers (who coined the term peripheral antigen-expressing cells) demonstrated that minute amounts of molecules expressed within the thymus (transgenes with the rat insulin promoter) lead to tolerance (6, 7). Studies in man indicate that different sizes of a variable nucleotide tandem repeat (VNTR) 5⬘ of the insulin gene are associated with risk for type 1 diabetes, and the form of the VNTR with the larger number of repeats is associated with greater insulin message in human thymus as well as “dominant” protection from diabetes (8, 9). This has led to the hypothesis that the modest influence (perhaps 10% of the familial aggregation) contributed by the insulin gene locus may relate to variation in expression of insulin within the thymus, promoting tolerance to insulin. Exogenous human recombinant insulin, which is now used to treat most diabetic patients, only contains the insulin molecule, yet most individuals who are treated subcutaneously with human insulin develop insulin autoantibodies after several weeks of therapy. In fact, the first radioassays developed were those which detected circulating antibodies directed against insulin, suggesting its inherent immune reactivity

Elsevier Science

Key Words: insulin; insulin peptide B:9 –23; diabetes; autoantibodies; T cells; insulitis; prevention; adoptive transfer; antigen-specific tolerance; mucosal immunity; HLA; altered peptide ligands; peripheral antigenexpressing cells (PAE).

INTRODUCTION

Type 1A diabetes results from immune-mediated destruction of ␤-cells (1– 4). At present, insulin is the only known islet autoantigen produced by ␤-cells that is not expressed by other islet endocrine cells. Thus, loss of tolerance to insulin is potentially central to ␤-cell-specific destruction. In addition, insulin and insulin peptides, depending upon the route of administration, can protect from type 1A diabetes. We will review the hypothesis that insulin is the primary autoantigen of spontaneous type 1A diabetes of man and the nonobese diabetic (NOD) mouse as well as the utilization of insulin in disease prevention and induction. 1521-6616/01 $35.00 © 2001 Elsevier Science All rights reserved.

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(10). Although all classes of immunoglobulins can be detected in insulin-treated patients, IgG antibodies are the most common and found in nearly all those treated for an extended period of time. Insulin allergy and insulin resistance. The relationship between insulin immunoreactivity and the development of both insulin resistance and insulin allergy (11) began to be appreciated in those individuals being treated with animal insulins. Many factors contributed to the immunogenicity of these early preparations, not the least of which was the fact that they were prepared from other species (beef, pork) which differ in sequence from human insulin by either three or one amino acid, respectively. Proinsulin, C-peptide, and intermediate degradation products, in addition to peptide impurities, in these preparations led to the development of localized cutaneous reactions at the site of injection which could have been manifested by immediate (IgE antibodies) or delayed hypersensitivity as well as the development of lipoatrophy or lipohypertrophy, which has been thought to include an immune component. In addition to the localized cellular response, a humoral antibody response generally developed with neutralizing IgG antibodies, which reduced the effectiveness of the administered insulin (insulin resistance) and increased the half-life of subcutaneously administered animal insulin preparations (12). Insulin autoimmune syndrome. Insulin autoimmune syndrome (13) was initially described in 1970. In this unusual disorder, which appears to primarily affect the Japanese, individuals (overwhelmingly with DRB1*0406) who have never been treated with exogenous insulin develop an insulin autoantibody, which results in both fasting and reactive hypoglycemia. The patients who develop this syndrome usually have been treated with a sulfhydryl-containing medication, such as methimizole. An association with other autoimmune disorders such as Graves’ disease suggests a more generalized humoral defect may underlie this condition. It is not clear exactly why these individuals develop hypoglycemia episodically with this syndrome. As noted above, insulin autoantibodies in exogenously treated individuals reduces insulin action rather than potentiates it. The autoantibodies of the insulin autoimmune syndrome are usually polyclonal. Individuals rarely have hypoglycemia associated with monoclonal antiinsulin autoantibodies. Such monoclonal antibodies occur in the absence of DRB1*0406 and sulfhydryl-containing medications and in the presence of B lymphocyte tumors (14). Insulitis and diabetes induction. Pioneering studies in guinea pigs first suggested that immune system reactivity to insulin was affected by the HLA of the responder strain, which appeared to direct reactivity

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toward different residues in either the A or the B chain of the beef or pork insulin used in the immunization (11). Grodsky and colleagues demonstrated that New Zealand white rabbits could be immunized with bovine insulin in Freund’s adjuvant and develop pathologic lesions and hyperglycemia reminiscent of type 1 diabetes in humans (15). They noted mononuclear cell infiltrates of islets, which exhibited reduced numbers of aldehyde fuchsin-staining insulin secretory granules. Ten of 25 rabbits developed either transient or persistent hyperglycemia following two to three immunizations (16). Soeldner and Renold reported the ability to immunize cows with bovine insulin and produce insulitis (17). A number of subsequent studies with insulins of greater purity failed to induce diabetes, and studies, for example in rabbits, were not pursued further. T/B cell epitopes of insulin in mice. When rodents were similarly immunized with insulin, they did not develop diabetes, but autoantibodies to insulin were found in several strains, which appeared to be determined by the major histocompatibility complex (MHC) background of the mouse strain. H-2 b mice were found to respond to the A chain of insulin, while H-2 d strains were found to respond to the B chain and H-2 k strain did not produce autoantibody responses (18). The class II MHC restriction of this response implies that CD4 T cell help is needed to produce insulin antibodies, although this was not formally tested in these early experiments. This is of interest, since the NOD mouse model of type 1 diabetes utilizes the H-2 g7 strain, which is related to the H-2 d strain, at class II and also directs its reactivity to the B chain of insulin, as we shall discuss below. Thomas and co-workers have developed B lymphocyte transgenic mice in which the heavy and light chains of an insulin autoantibody clone have been inserted, and they found that the physiologically low level expression of insulin induced “tolerance,” but that a subset of B cells was present which could escape silencing and produce insulin autoantibodies. They concluded that this type of self-reactivity could occur to native antigens and that physiologic expression of the antigen was the controlling factor for it rather than clonal ignorance, as had been previously suggested (19). Insulin autoantibodies in humans. Palmer and colleagues demonstrated the presence of insulin autoantibodies (IAA) in new onset patients prior to the administration of exogenous insulin (20). Insulin autoantibodies often are the first autoantibody to appear in children who develop diabetes prior to the age of 5 (21, 22). Unlike the other major autoantibodies described to date, elevated titers of IAA correlate with a younger age of diabetes onset (23). The spontaneous autoantibodies associated with diabetes risk are de-

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tected with radioassays, but poorly if at all with ELISA assays, as documented in several international workshops (24, 67). This probably relates to the apparent high affinity (10 ⫺10) and low capacity of such autoantibodies (25).

2. EXPERIMENTAL EVIDENCE FOR THE IMPORTANCE OF INSULIN IN AUTOIMMUNE DIABETES

NOD mice. In the NOD mouse, mononuclear infiltrates were observed prior to the development of diabetes. To determine the reactivity of these pathogenic cells, Wegmann and colleagues isolated T cells from the islets of prediabetic NOD mice and initially expanded them on islet cells. They subsequently found that reactivity to insulin and, particularly, the midportion of the B chain amino acids 9 –23 (B9 –23) were found among greater than 50% of the islet-reactive CD4 ⫹ T cells that were infiltrating the pancreas of prediabetic NOD mice. Since B9 –23 is essentially the same across many species, B9 –23-specific CD4 ⫹ T cells respond to human, rat, and mouse islets with mouse antigen-presenting cells. Their pathogenicity was confirmed in experiments in which they adoptively transferred diabetes to naive NOD and NOD.scid recipients (26, 27). Insulin peptide B9 –23-reactive T cells were found in islets as early as studied (4 weeks of age), but in general have been difficult to demonstrate in peripheral tissues (e.g., spleen). Zekzer and co-workers developed a CD4 T cell clone which was derived from the peripancreatic lymph node and was also found to react to the midportion of the B chain of insulin, particularly strongly to aa 12–25, but also to aa 9 –23. The 2H6 clone was found to produce TGF-␤ and was able to prevent the adoptive transfer of diabetes and the development of diabetes in NOD mice (28). As we shall discuss later, therapies which prevent diabetes are associated with the development of T cells which produce Th2-type cytokines such as TGF-␤ and the 2H6 clone may represent an abortive attempt for this process to occur in the naive animal. Both CD4 and CD8 T cells participate in the development of insulitis and diabetes in the NOD mouse model of type 1 diabetes. Wong and colleagues isolated a CD8 T cell clone from a 7-week-old prediabetic NOD mouse, which recognized a ␤-cell protein and was found to adoptively transfer diabetes to mouse strains expressing K d, the restriction element of the clone. By screening a pancreatic islet cDNA library, they found that the autoantigen recognized by this clone was an insulin B chain peptide, aa 15–23 (29). The fact that both CD4 and CD8 pathogenic T cells recognize insulin and the midportion of the B chain further supports the

importance of this pancreatic-specific protein to the disease process. Induction of insulin autoantibodies and diabetes. Abiru and co-workers have recently reported that when insulin peptide B9 –23 is administered to both NOD and normal Balb/c mice, insulin autoantibodies are induced (30) (see Fig. 1). These autoantibodies react with intact insulin and cannot be absorbed by the immunizing insulin peptide. Induction of such autoantibodies is strictly dependent upon the MHC of the immunized mice, and both H-2 d and H-2 g7 are permissive haplotypes, whereas with H-2 b mice, no autoantibodies are induced. Such insulin autoantibodies are induced even when the peptide is administered with no adjuvant. Usually such induction of insulin autoantibodies is associated with protection from diabetes, but, depending upon the specifics of the administration [e.g., with or without poly(IC)], insulitis can be induced in Balb/c mice, and in special genetically susceptible mice even diabetes can be accelerated. In addition, anaphylaxis can be induced following multiple administrations of the B9 –23 peptide in NOD mice (Liu, Eisenbarth, and co-workers, unpublished observations). Human. T cell responses to human insulin have been noted for many years (Table 1). Most studies have used peripheral blood mononuclear cells and have demonstrated responses to the whole molecule that in general were not different between HLA-matched control subjects and those with prediabetes or type 1 diabetes (31, 32). A T cell workshop tested a number of autoantigens, including insulin, GAD65, but not insulin peptide B9 –23, and found no differences between diabetic subjects and controls (32). Proinsulin is secreted from ␤-cells in addition to insulin, but cells that are under metabolic stress secrete higher amounts of proinsulin, as they have less time to process this molecule into mature insulin. In this setting, potentially new antigens could be presented to the immune system, and Harrison and colleagues have demonstrated reactivity to a proinsulin peptide 24 –36 which encompasses residues between the B chain and the C-peptide (33). In fact, a recent study noted proliferative responses to either insulin or proinsulin in prediabetic ICA ⫹ patients (34), although this has not been universally observed (35). Other studies have also suggested that there may be T cell epitopes within the C-peptide region itself (36) or between the junction of the Cpeptide to A chain, based on evidence from immunizations of human HLA transgenic mice (37). Alleva and colleagues recently demonstrated that B9 –23 reactivity could be seen in tertiary cultures from new onset type 1 diabetes patients or with ELISPOT analysis utilizing frozen peripheral blood monocytes from both new onset type 1 diabetic patients and

INSULIN-SPECIFIC TOLERANCE IN DIABETES

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FIG. 1. Insulin autoantibodies and blood glucose for the NOD mice expressing IAA at 8 weeks of age and at 20 weeks or later, followed from 4 weeks of age until diabetes or 36 weeks. (C) Age of first detection of insulin autoantibodies plotted versus age of diabetes onset for all 15 prospectively evaluated mice. Two mice with open symbols did not have IAA detected. Previously published by Yu, L., Robles, D. T., Abiru, N., Kaur, P., Rewers, M., Kelemen, K., and Eisenbarth, G. S. (Proc. Natl. Acad. Sci. USA 97, 1701–1706, 2000).

prediabetic individuals (38). Importantly, T cell reactivity to B9 –23 was found more frequently among individuals bearing HLA-DR4/DQ8 and HLA-DR3/DQ2. Our data suggested that human subjects with type 1 diabetes could respond to this peptide, and we have isolated a T cell clone which responds to B9 –23 and is restricted by DQ8. Binding studies confirmed that B9 –23 could compete with a known DQ8-specific peptide for binding in a physiologic range (Gottlieb, manuscript submitted for publication). The selection of an autoantigen may be influenced by the affinity of its interaction with a particular

MHC molecule. Under this hypothesis, low-affinity reactivities would not undergo full deletion in the thymus and might be able to start an autoimmune disease when later stimulated in the periphery. Conventional binding studies had originally suggested that B9 –23 was a low-affinity binder to the IA g7 of the NOD mouse. In addition, insulin peptide B10 –30 was even more unusual in that it could not bind to many diverse mouse MHC molecules, although it could bind to the IA g7 (HLA-DQ homologue). Curiously, Tompkins and colleagues also found that this peptide could interfere with binding to the SEB bind-

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TABLE 1 Insulin Responses in Animals and Humans Group Guinea pigs Rabbits BB rat, DP BB rat, DR RT1 b rat NOD mice Balb/c mice B7-1-transgenic Balb/c mice Human

Vaccination or spontaneous

Antibodies

T cell responses

Insulitis/diabetes

Insulin immunization Immunization Spontaneous Induced Immunization Spontaneous Immunization

Yes Yes No No ? Yes Yes

— — No No Yes Yes Yes

No/no Yes/yes Yes/yes Yes/yes Yes/no Yes/yes Yes/no

Yes — RT1 u RT1 u RT1 b1 I-A g7 I-A d

Immunization ⫹ poly(IC) Spontaneous

Yes Yes

Yes Yes

Yes/yes Yes/yes

I-A d HLA-DR4/DQ8 and HLA-DR3/DQ2

ing site found outside of the normal peptide groove (39). B9 –23 bound to DQ8 crystal structure. Wiley and colleagues recently were successful in resolving the crystal structure of B9 –23 bound to DQ8 (40). They identified an aa 13-E, 16-Y, and 21-E motif reacting with the P1, P4, and P9 binding pockets for the peptide with HLA-DQ8. The P1 pocket appears to be unusual compared to HLA-DR in that charged residues are present and allow for negatively charged side chains in this pocket. The P4 pocket is unusually deep to accommodate the tyrosine in the native sequence. The P9 pocket substitutes Arg76 from the A chain for the Asp57 of the B chain that was originally noted as being linked to type 1 diabetes and intriguingly is found in both DQ8 and DQ2 molecules. This substitution allows for charged side chains to form a salt-bridged hydrogen bond to anchor the peptide. Therefore, negatively charged amino acid side chains are preferred, and together these changes may lead to a different repertoire of peptides which can be presented on the diabetesassociated DQ8 molecule, compared to HLA-DR. The authors suggest that peptide binding to both DQ8 and DQ2 is potentially similar and that B9 –23-specific inhibitors, which interfere with binding to the P4 (aa 16) and P9 (aa 21) pockets, would be useful reagents in the prevention of type 1 diabetes. 3. INSULIN TREATMENT TO PREVENT TYPE 1 DIABETES

BioBreeding rat. The diabetes-prone BioBreeding (DP-BB) RT1 u rat develops a spontaneous form of diabetes early in life (21). This animal is lymphopenic due to a genetic defect isolated to rat chromosome 4 (lyp). Both humoral and T cell responses to all prospective autoantigens have been difficult to demonstrate conclusively in DP-BB rats. Buschard and colleagues first demonstrated that high doses of insulin could protect DP-BB rats from diabetes (41). This and other experi-

HLA association

ments suggested that ␤-cell rest could protect islet cells from damage in this model. We extended these observations and showed that only hypoglycemic doses of insulin could protect RT6-treated diabetes resistantbiobreeding (DR-BB) rats from becoming diabetic, while lower doses were ineffective. Activated spleen cells from protected DR-BB rats were able to transfer diabetes, suggesting that the autoreactive cell population was not deleted but still present, although functionally dormant, in the treated animals. Further, we demonstrated that this protective effect was specific to the pancreas, since the incidence of thyroiditis was unaffected by this therapy. Therefore, in the BB rat insulin treatment appears to work primarily by ␤-cell rest (42). See Table 2. NOD mice: Protection from diabetes by subcutaneous or intranasal insulin. Atkinson and colleagues similarly demonstrated that insulin administered subcutaneously could protect NOD mice from diabetes when given at several doses, including nonhypoglycemic doses, suggesting that an additional immunologic mechanism could be at work in NOD mice (22). Muir and co-workers extended this observation by demonstrating that only the nonmetabolically active B chain, and not the A chain, was effective in preventing diabetes in NOD mice and that this effect was associated with changes in the cytokine profile of infiltrated pancreatic T cells (decrease in IFN-␥, increase in IL-4) (44). As discussed above, Wegmann and colleagues had demonstrated that the majority of the insulin-reactive T cells respond to B9 –23; they then tested whether antigen-specific therapy with the B9 –23 peptide could prevent diabetes in NOD mice. They found that B9 –23, when given by the subcutaneous or intranasal route either early (4 – 8 weeks) or late (12–20 weeks) in the course of disease development, could significantly reduce the incidence and delay the onset of diabetes (27) (manuscript in preparation) (see Fig. 2). T cell responses to B9 –23 were reduced in protected animals,

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TABLE 2 Insulin Immunotherapy of Type 1 Diabetes Group

Antigen

Route

Protection from diabetes

Reference

BB rat (DP, DR) BB rat (DP, DR) NOD mouse NOD mouse NOD mouse NOD mouse NOD mouse NOD mouse LCMV mouse PVG.RT1 u rat Human, new onsets Human, high-risk prediabetic Human, new onset diabetes Human, medium-risk prediabetic

Insulin, high dose continuous Insulin Insulin B chain B9–23 B9–23 B10–24 Proinsulin Insulin B chain B1–18 Insulin Insulin, low dose continuous APL of B9–23 Insulin

Subcutaneously Oral Subcutaneously Subcutaneously Subcutaneously Intranasally Intranasally DNA Oral, DNA Intrathymic Oral Subcutaneous Subcutaneous Oral

Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No ? ?

41, 42 42 22 44 26, 27 23, 26, 27 45 51 47–49 50 56 55 38 55

and in further studies it was shown that intranasal therapy was associated with the induction of Th2-type cytokine responses. Intriguingly, it may be that the antigen or peptide as well as the route of administration may be important in how the protection is mediated. Harrison and colleagues noted that aerosolized insulin therapy was effective at protection and was associated with the production of IL-4 and IL-10 by spleen cells from treated animals and that the cells which suppressed adoptive transfer of diabetes were CD8 ␥␦ T cells (23). When insulin peptide B10 –24 was administered orally or intranasally, it also induced a protective effect that was associated with TGF-␤, while subcutaneous administration of the same peptide was associated with production of IL-10 (45). Protection from diabetes by oral insulin. Mucosal immunity is felt to generate a protective immune response manifested by Th2-type cytokines such as IL-4, IL-10, and particularly TGF-␤. Oral administration of antigen leads to presentation in the intestinal mucosa, which generates protective Th2 T cells in gut-associated lymphoid tissues such as Peyer’s patches. The antigen used in this therapy is important, since its nature may determine the type of cytokines produced by antigen-specific T cells, must direct mucosal-derived T cells to emigrate to the organ of interest, and must thus be able to downregulate the localized pathogenic immune response. Oral insulin was given weekly to NOD mice and was shown to reduce the level of diabetes by 50%. Protection was associated with the development of a Th3-type response, specifically T cells that produced TGF-␤ (46). Intriguingly, in other models of type 1 diabetes, insulin and B chain epitopes appear to be able to protect from diabetes onset as well. Transgenic expression of viral proteins from LCMV followed by infection with the virus leads to the development of type 1 diabetes

that is primarily mediated by CD8 T cells, but also involves CD4 cells. Von Herrath and coresearchers have demonstrated that insulin, insulin B chain, and even insulin B chain DNA can prevent the development of diabetes in this model (47– 49). Treatment of PVG.RT1 u rats with neonatal thymectomy followed by sublethal irradiation leads to the development of type 1 diabetes. Cooke and Mason demonstrated that insulin peptides and particularly B1–18 could prevent diabetes in this model (50). Therefore, despite different means of inducing diabetes in disparate genetic and animal systems, insulin has been shown to be an effective therapy for preventing immune-mediated diabetes. Insulin gene therapy to prevent diabetes. If insulin is a critical antigen in the development of diabetes, then increased expression in the thymus may lead to deletion or functional inactivation of insulin-specific T cells. Harrison and colleagues established a proinsulin II transgenic NOD mouse which expressed this gene under the control of a MHC class II promoter and demonstrated that these animals were protected from diabetes. They noted that this effect was specific to the pancreas, where almost no insulitis was noted in comparison to continued inflammation of the salivary glands (51). Injection of an insulin B chain plasmid has now been shown to prevent the development of type 1 diabetes in NOD mice. With this type of immunization, IL-4 appears to be the cytokine which is upregulated and apparently protective in treated mice, since levels of IFN-␥ were essentially unchanged by therapy (52, 53). Human trials of insulin immunotherapy. Based on the NOD data described earlier, insulin treatment of prediabetic subjects was undertaken at the Joslin Clinic and subsequently at several other sites. These

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FIG. 2. (A) Effect of subcutaneous injection of insulin peptide B9 –23 in IFA on diabetes incidence in NOD mice. Four-week-old female NOD mice that received subcutaneous B9 –23 ⫹ IFA (n ⫽ 10) showed delayed onset (P ⫽ .0064 by Mann–Whitney test) and reduced frequency (P ⫽ .0055 by Fisher’s exact test) of diabetes compared to mice that received subcutaneous TT:830 – 843 ⫹ IFA (n ⫽ 10). (B) Effect of multiple intranasal doses of insulin peptide B9 –23 on diabetes incidence in NOD mice. Four-week-old female NOD mice that received intranasal B9 –23 (n ⫽ 9) every 4 –5 weeks showed delayed onset of diabetes (P ⫽ .0236 by log-rank test) compared to littermates that received intranasal TT:830 – 843 (n ⫽ 9). Updated from a previously published figure by Daniel, D., and Wegmann, D. R. (Proc. Natl. Acad. Sci. USA 93, 956 –960, 1996).

pilot studies demonstrated that insulin treatment (0.25 Units/kg/day) with or without IV insulin for 4 –7 days yearly might delay the onset of type 1 diabetes in individuals with two or more autoantibodies (54). The Diabetes Prevention Trial-1 (DPT-1) was initiated to test these pilot results in a much larger group of 300 individuals who were felt to be at high risk for the development of diabetes due to the presence of autoantibodies and a low first-phase insulin response (55). This portion of the study was recently terminated early, when it was found that there was no difference between the treated and untreated groups. If ␤-cell rest needs to be achieved for success with this regimen, as was seen in the BB rat, then the failure of this study might be explained by the dose of metabolically active

insulin used. However, it is probably more likely either that the necessary immunologic effect was not achieved using this type of immunization scheme or that it may have been initiated too late in the disease process to have the desired effect. The present lack of secondary immune markers to monitor therapy makes it hard to know which of these latter reasons may be the true explanation for this negative result. A limiting factor to the use of insulin as an immunogen is its metabolic activity. The advantage of peptide therapy is the lack of metabolic effect and the potential to limit the range of the response to the pathogenic epitopes without increasing the chances for hypersensitivity. Alternatively, using either different routes of administration (oral, nasal), dose schedules, and adjuvants (IFA, cholera toxin) of insulin or its peptides, such as B9 –23, may provide a more efficient means of deviating the immune response toward protection. A particular route of administration, oral or mucosal, may be preferred to help induce a Th2- or Th3-type of protective immune response. Additionally, vaccination protocols in which insulin is only administered every several days or weeks may be needed to generate an appropriate immune response. These alternate approaches are now under active investigation. Oral insulin therapy has been tried in new onset trials and has been unsuccessful to date (56). It should be noted that oral therapy in NOD mice was most successful when it was begun at 4 weeks of age, at the outset of insulitis and pancreatic inflammation. The DPT-1 is also testing oral insulin in prediabetic patients who are felt to be at moderate risk for developing type 1 diabetes. These individuals have two autoantibodies (ICA ⫹ and IAA ⫹), but also have normal first-phase insulin responses to intravenous glucose. This study is ongoing and may have the best chance of showing that antigen-specific therapy by oral administration can successfully block the development of autoimmune disease, since it is being given years prior to the expected onset of diabetes. An altered peptide ligand of the B9 –23 is currently undergoing phase 1/2 studies in the United States to determine whether it can reverse type 1 diabetes in new onset individuals. Comparison with other autoantigens. As was noted briefly above, there have been several autoantigens isolated in type 1 diabetes. Glutamic acid decarboxylase (GAD)65 and IA-2 are the most prominent of a list of at least 10 molecules (57). Most of these other antigens are not unique to islet cells or to the ␤-cell itself. Although GAD65 is expressed at normal levels in rat and human pancreas, in mice, including the NOD, low levels of GAD expression in pancreatic islets make extrapolations of experimental evidence to human type 1 diabetes harder to comprehend. Nevertheless, T cell responses to GAD65 are found in prediabetic mice, and

INSULIN-SPECIFIC TOLERANCE IN DIABETES

administration of GAD and GAD peptides prevents diabetes in NOD mice (58, 59). In recent experiments in NOD mice, in contrast, insulin B chain plasmids prevented diabetes, while plasmids with GAD65 did not (52). Similarly, Griffin and colleagues could induce insulitis in RT1b rats with a proinsulin peptide, but not with GAD (60). T cell responses to IA-2 can be induced in NOD mice by immunization, but spontaneous reactivity cannot be found in untreated animals (61). Furthermore, a recent autoantibody NOD workshop suggests that these mice have specific autoantibodies to insulin, but not to GAD65 or IA-2 (67). In man, specific autoantibodies reacting with GAD65, IA-2, and insulin (62) are all predictive of type 1 diabetes, and T cell responses to all three have been reported (63– 66). Because of the differences between NOD and human diabetes, understanding how to implement antigen-specific therapy with GAD65 and IA-2 in human type 1 diabetes may be difficult. Concluding discussion. Treatment with antigenspecific therapy holds the promise of focusing the protective immune response while limiting the pathologic and metabolic consequences of this type of therapy. Its use may be limited to situations in which the immune process has not progressed to full clinically overt disease, but our ability to increasingly diagnose type 1 diabetes and other autoimmune conditions in the preclinical state offers us the best test of whether this type of approach can prevent the development of type 1 diabetes.

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15. ACKNOWLEDGMENTS This work was supported by NIH Grants DK32083, DK55364, and DK59097; Diabetes Endocrine Research Center Grant P3057516; Autoimmunity Center of Excellence Grant AI46374; and for human studies grants from the Clinical Research Centers at Children’s (RR0009) and University of Colorado Hospitals (RR00051), Denver, CO. Robin Parks helped in the preparation of the manuscript.

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Received September 7, 2001; accepted with revision October 22, 2001; published online December 10, 2001