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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 β-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.

Antigen-based therapies (ABTs) seek to prevent or inhibit autoimmune diseases by inducing regulatory T-cell responses (active tolerance) or anergizing/deleting pathogenic T-cells (passive tolerance). The theoretical appeal of this therapeutic approach is that it may promote tolerance with little debilitation of the immune system. The clinical application of ABTs for autoimmune disease is still in its infancy. Although initial attempts to apply this therapeutic strategy in multiple sclerosis, rheumatoid arthritis, and type 1 diabetes met with failure, recent results from clinical studies hold promise that this approach may be able to delay the onset of type 1 diabetes as well as preserve β-cell function in latent autoimmune diabetes in adults (LADA) patients and in children newly diagnosed with type 1 diabetes. If verified, it would be an important translation of NOD mouse findings to clinical applications. NOD mouse studies, however, indicate that the immunological impact of ABTs is much more dynamic and complex than previously appreciated. The differences in type 1 diabetes pathogenesis between rodents and humans, as well as other immunotherapeutic approaches, have been extensively reviewed elsewhere (e.g., [1–3]). Here, we will focus on ABTs and discuss what has been learned about their immunological impact, the theoretical factors affecting their efficacy and safety, as well as potential markers of their therapeutic efficacy. ### Inflammatory T-cell responses spread among β-cell antigens during the development of murine and human type 1 diabetes. Type 1 diabetes is mediated by autoreactive T-cells recognizing β-cell autoantigens (β-CAAs). In NOD mice, autoreactive CD4+ T-cells are generally Th1 type (interferonγ secreting), and autoimmunity progressively spreads intra- and intermolecularly among β-CAAs such as insulin, GAD, heat shock protein (HSP), and IGRP during the disease process (4,5). Similarly, spontaneous CD8+ T-cell responses develop to β-CAAs such as insulin, GAD, IGRP, and others (6). Paralleling observations in NOD mice, autoreactive Th1-biased CD4+ and CD8+ T-cells arise in human …

Reactive oxygen species, such as superoxide, and nitrogen oxides, such as peroxynitrite, are thought to contribute to β-cell destruction during the disease process that leads to type 1 diabetes. EUK-8 is a member of a new class of synthetic salen-manganese compounds with low toxicity that possess catalytic superoxide dismutase, peroxidase, and catalase activity that can inactivate superoxide and nitrogen oxides (e.g., peroxynitrite and nitrogen dioxide). We observed that EUK-8 administration inhibited the adoptive transfer of type 1 diabetes to NOD mice. In addition, administration of EUK-8 to NOD mice with established autoimmunity completely prevented the development of type 1 diabetes for up to 1 year in age, even though the treatment was discontinued after 35 weeks of age. EUK-8 treatment also prolonged the survival of islet allografts in newly diabetic NOD mice. Thus, reactive oxygen and nitrogen species contribute to the pathoetiology of both spontaneous type 1 diabetes and allograft rejection. In cultures of NIT-1 cells, EUK-8 inhibited cytotoxicity caused by superoxide as well as nitric oxide. Collectively, our findings implicate a greater role for nitrogen oxides (other than peroxynitrite) in β-cell damage. Antioxidants designed to prevent the formation of both cytotoxic reactive oxygen and nitrogen species may effectively protect β-cells from spontaneous autoimmunity and alloresponses.