Characterization of the autoimmune response against the nerve tissue S100ß in patients with type 1 diabetes.

Type 1 diabetes results from destruction of insulin-producing beta cells in pancreatic islets and is characterized by islet cell autoimmunity. Autoreactivity against non-beta cell-specific antigens has also been reported, including targeting of the calcium-binding protein S100ß. In preclinical models, reactivity of this type is a key component of the early development of insulitis. To examine the nature of this response in type 1 diabetes, we identified naturally processed and presented peptide epitopes derived from S100ß, determined their affinity for the human leucocyte antigen (HLA)-DRB1*04:01 molecule and studied T cell responses in patients, together with healthy donors. We found that S100ß reactivity, characterized by interferon (IFN)-γ secretion, is a characteristic of type 1 diabetes of varying duration. Our results confirm S100ß as a target of the cellular autoimmune response in type 1 diabetes with the identification of new peptide epitopes targeted during the development of the disease, and support the preclinical findings that autoreactivity against non-beta cell-specific autoantigens may have a role in type 1 diabetes pathogenesis.

3 beta hydroxysteroid dehydrogenase autoantibodies in patients with idiopathic premature ovarian failure target N- and C-terminal epitopes.

The steroid cell enzyme 3 beta hydroxysteroid dehydrogenase (3 beta HSD) has been identified as a target of steroid cell autoantibodies, and autoantibodies to this enzyme are present in patients with premature ovarian failure and patients with autoimmune polyendocrine syndrome 1. The aim of the present study was to develop a radioligand binding assay for 3 beta HSD autoantibodies and to exploit this to examine regions of the molecule targeted by autoantibodies. We generated a construct of 3 beta HSD coupled to a luciferase fusion partner in order to maximize the yield of (35)S-radiolabeled protein. Labeled 3 beta HSD was then immunoprecipitated and the autoantibodies quantified by phosphoimaging. Autoantibodies to 3 beta HSD were detected in 12 of 100 (12%) idiopathic premature ovarian failure patients and 0 of 103 (0%) healthy age-matched controls (P < 0.0001). Three overlapping fragments of 3 beta HSD cDNA were cloned downstream of luciferase to examine autoantibody binding sites. Two of nine sera with 3 beta HSD autoantibodies (22%) displayed reactivity to the N terminus of 3 beta HSD, and seven (77%) showed reactivity to the C terminal; no sera reacted with the middle region. Our study demonstrates a markedly enhanced disease specificity of autoantibodies to 3 beta HSD detected using this novel assay and shows that distinct regions of the molecule are targeted.

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.

T-Cell reactivity to the P2C nonstructural protein of a diabetogenic strain of coxsackievirus B4.

Enteroviruses are proposed as initiating factors in the etiology of Type 1 diabetes mellitus (Type 1 DM). Molecular mimicry between the autoantigen glutamic acid decarboxylase 65 (GAD65) and the coxsackievirus B4 (CVB4) nonstructural protein P2C is frequently cited as a mechanism by which this virus triggers the disease, but little is known about the immunogenicity of this viral protein in humans, mainly due to the problem of obtaining highly pure preparations of P2C. We generated large amounts of highly pure, soluble P2C protein, coupled to the fusion partner maltose binding protein (MBP-P2C) using the PMAL-c2 bacterial expression plasmid and a two-step purification system comprising amylose resin and ion exchange. Using purified viral protein we show that specific T-cell responses against P2C are detected in the blood of healthy donors and Type 1 DM patients. Proliferation responses to P2C were detected only in subjects also demonstrating T-cell proliferation to CVB4 Vero cell lysates. However, in additional cases T-cell responses to P2C were detectable through the release of interferon-gamma or interleukin-4 in individuals who did not make proliferative responses. Taken together, our data show that the P2C nonstructural protein of CVB4 is targeted by T cells during the antiviral immune response and may trigger the production of T helper 1 and T helper 2 cytokines. The availability of pure, immunogenic P2C should allow the putative role of antiviral responses in the development of autoimmune diabetes to be investigated.