Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin-dependent diabetes mellitus of NOD mice, we studied the specificities of helper T-cell clones capable ofcausing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence ofthe human variant ofthe 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cefl vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes.Insulin-dependent diabetes mellitus (IDDM) of both humans and NOD strain mice becomes clinically overt after most of the beta cells in the islets have been destroyed by an autoimmune process (1). The destruction of the beta cells seems to be caused by autoimmune T cells (2-4) that may recognize a processed peptide antigen presented by a major histocompatibility complex (MHC) class II molecule (5).To identify a peptide epitope important in the IDDM of NOD mice, we investigated the antigen specificity recognized by diabetogenic T-cell clones responding to the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis (MT-hsp65). We earlier reported that diabetogenic T cells recognized an epitope on this molecule (6). We now report that the target epitope is present in the sequence ofthe human hsp65 (H-hsp65) molecule, that the T cells responding to this epitope can therapeutically vaccinate mice against IDDM, and that the peptide epitope itself can be used to treat the disease. MATERIALS AND METHODSMice. E. Leiter (The Jackson Laboratory) kindly supplied breeding nuclei of the spontaneously diabetic NOD/Lt (NOD) strain and of the nondiabetic NON.H-2NOD strain. The NON.H_2NoD mice were in their 11th backcross generation and were congenic at the H-2 complex with the NOD mice.Antigens. Recombinant H-hsp65, recombinant MT-hsp65, recombinant mycobacterial 70-kDa heat shock protein (hsp70), and control Escherichia coli antigen were prepared as described (6-8). Control E. coli were transfected with the pEX2 plasmid that did not contain the hsp65 genes. The H-hsp65 gene was the gracious gift of Richard A. Young (Massachusetts Institute of Technology, Cambridge). Peptides p277 and p278 were synthesized by Ora Goldberg (Biological Services Laboratory ofthe Weizmann Institute of Science) with an automated synthesizer and were purified on a Biogel p-4 column (50 x 1.5 cm; Bio-Rad). The sequence...

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice.Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp7O) had no effect on the development of diabetes.Type 1 or insulin-dependent diabetes mellitus (IDDM) is caused in most cases by autoimmune destruction of the insulin-producing beta cells resident in the islets of the pancreas (1). It is thought that once the autoimmune process takes root, it progresses relentlessly without causing symptoms until the number of beta cells irreversibly destroyed is so large, perhaps 90% of the beta-cell mass, that the individual suffers a derangement in glucose homeostasis and requires an exogenous supply of insulin to sustain life.The non-obese diabetic (NOD/Lt) mouse is a useful experimental model of IDDM (1). NOD/Lt mice spontaneously develop inflammation of the islets, insulitis, beginning at 4-6 weeks of age which progresses to overt IDDM at 4-5 months of age. Autoimmune T lymphocytes would seem to be the cause of beta-cell destruction because IDDM can be adoptively transferred to very young prediabetic NOD/Lt mice with T lymphocytes from older mice (2).Identification of target antigens recognized in the pathogenesis of IDDM is important for at least two reasons: specific antigens would facilitate the early diagnosis of preclinical IDDM and they might be used to abort the destructive autoimmune process through modification of the autoimmune response. For example, copolymer 1 (COP 1), a synthetic peptide immunologically cross-reactive with myelin basic protein has been used to alter the course of multiple sclerosis (3).We now show that a beta-cell antigen cross-reactive with a 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis, termed hsp65 cross-reactive (hsp65-CR) antigen, is involved in the pathogenesis of NOD/Lt mouse IDDM. MATERIALS AND METHODSMice. The breeding nucleus of NOD/Lt mice was a gift of E. Leiter (Jackson Laborat...

In a study of the mechanism of resistance to autoimmune disease induced by T cell vaccination, rats were vaccinated against experimental autoimmune encephalomyelitis (EAE) by injecting them once in the hind footpads with a subencephalitogenic dose (10(4)) of a clone of T lymphocytes specific for myelin basic protein (BP). The response to vaccination was assayed by challenging the rats with an encephalitogenic dose (3 X 10(6)) of T lymphocytes of this BP-specific clone. Five to six days after vaccination, the cells responsible for mediating resistance to adoptively transferred EAE were concentrated in the popliteal lymph nodes draining the vaccination site. Transfer of the draining lymph node cells to unvaccinated rats led to loss of resistance in the donor rats and acquisition of resistance by the recipient rats. Limiting-dilution cultures of the draining lymph node cells were established with irradiated cells of the BP-specific clone as stimulators. Two sets of T lymphocytes specifically responsive to the BP-specific T cells from the clone were isolated: CD4+CD8- helper and CD4-CD8+ suppressor cells. The helper T cells, like the BP antigen, specifically stimulated the BP-specific vaccinating clone. In contrast, the suppressor T cells specifically suppressed the response of the BP-specific vaccinating clone to its BP antigen. These results suggest that T cell vaccination induces resistance to autoimmune disease by activating an antiidiotypic network.

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We previously reported that immunity to the p277 peptide of the human 60-kDa heat shock protein (hsp60) was a causal factor in the diabetes of non-obese diabetic (NOD) mice, which are genetically prone to develop spontaneous autoimmune diabetes. The present study was done to test whether immunization with the p277 peptide could cause diabetes in standard strains of mice. We now report that a single administration of the p277 peptide conjugated to carrier molecules such as bovine serum albumin or ovalbumin can induce diabetes in C57BL/6 mice and in other strains not genetically prone to develop diabetes. The diabetes was marked by hyperglycemia, insulitis, insulin autoantibodies, glucose intolerance and low blood levels of insulin. The diabetes could be transferred to naive recipients by anti-p277 T cell lines. Similar to other experimentally induced autoimmune diseases, the autoimmune diabetes remitted spontaneously. After recovery, the mice were found to have acquired resistance to a second induction of diabetes. Susceptibility to induced diabetes in C57BL/6 mice was influenced by sex (males were much more susceptible than were females) and by class II genes in the major histocompatibility complex (B6.H-2bm12 mice with a mutation in the MHC-II molecule were relatively resistant). Other strains of mice susceptible to induced diabetes were C57BL/KSJ, C3HeB/FeJ, and NON/Lt. BALB/c and C3H/HeJ strains were relatively resistant. Immunization to p277-carrier conjugates could also induce transient hyperglycemia in young NOD mice, but upon recovery from the induced diabetes, the NOD mice were found to have acquired resistance to later development of spontaneous diabetes. Thus, T cell immunity to the p277 peptide can suffice to induce diabetes in standard mice, and a short bout of induced diabetes can affect the chronic process that would otherwise lead to spontaneous diabetes in diabetes-prone NOD mice.

We studied lines of rat T cells, specifically reactive against myelin basic protein (BP), that were functional in mediating autoimmune encephalomyelitis or in vaccinating rats against induction of active EAE. Herein we report that these functions depended on activation of the cells by incubation with BP or with a T cell mitogen prior to inoculation into recipient rats. Activation was accompanied by the exposure of membrane-binding sites specific for the lectin peanut agglutinin. Accumulation of activated line cells in the central nervous system and thymus gland was observed.

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