Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic ␤ cells. CD8 ؉ T cells are essential for this ␤ cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8 ؉ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.T he nonobese diabetic (NOD) mouse is a widely studied model of human type 1 diabetes, an autoimmune disease characterized by inflammation of pancreatic islets (insulitis) followed by T cell-mediated destruction of insulin (INS)-producing ␤ cells (1). Both CD4 ϩ and CD8 ϩ T cells are required for this pathogenic process (1); however, CD8 ϩ T cells appear to be responsible for the initial ␤ cell insult (1-3). Whereas the pathogenicity of B cells and autoantibodies is less clear, the autoantigens currently believed to contribute to autoimmune diabetes pathogenesis in NOD mice and humans all were originally identified based on the presence of specific autoantibodies rather than by T cell recognition (4-6). Little is known of the ␤ cell antigens targeted by the pathogenic CD8 ϩ T cells. Although one study identified an INS peptide as the antigenic target of the majority of islet-infiltrating CD8 ϩ T cells in NOD mice (7), the prevalence of these INS-reactive CD8 ϩ T cells was not confirmed in subsequent studies (8,9).A substantial proportion of ␤ cell-autoreactive CD8 ϩ T cells isolated from NOD islets express a shared T cell receptor ␣ (TCR␣) chain (V␣17-J␣42), suggesting recognition of a common ␤ cell peptide (3, 10). These T cells do not recognize the antigenic INS peptide mentioned above (11,12). The pathogenicity of this prevalent T cell population has been well established through studies of the 8.3 T cell clone (a representative T cell clone of the V␣17-J␣42-expressing T cell population) (13, 14). 8.3-Like T cells are present in the earliest islet infiltrates of NOD mice (3) and undergo avidity maturation as islet inflammation progresses to overt disease (8). At any given time, 8.3-like T cells can constitute up to 30-40% of the islet-associated CD8 ϩ T cells (9). Strikingly, quantification of 8.3-like T cells in peripheral blood predicts diabetes development in individual NOD mice (9), unlike any other single immune indicator identified to date. Although the prevalence and pathogenicity of 8.3-like T cells has been clearly established, the identity of their ligand has remained elusive. Materials and MethodsMice. NOD͞Lt mice were maintained by brother-sister mating. 8.3-TCR␣␤-transgenic NOD mice, designated 8.3-NOD, ha...

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic β cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Spontaneous autoimmune diabetes development in NOD mice requires both CD8+ and CD4+ T cells. Three pathogenic CD8+ T cell populations (represented by the G9C8, 8.3, and AI4 clones) have been described. Although the Ags for G9C8 and 8.3 are known to be insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein, respectively, only mimotope peptides had previously been identified for AI4. In this study, we used peptide/MHC tetramers to detect and quantify these three pathogenic populations among β cell-reactive T cells cultured from islets of individual NOD mice. Even within age-matched groups, each individual mouse exhibited a unique distribution of β cell-reactive CD8+ T cells, both in terms of the number of tetramer-staining populations and the relative proportion of each population in the islet infiltrate. Thus, the inflammatory process in each individual follows its own distinctive course. Screening of a combinatorial peptide library in positional scanning format led to the identification of a peptide derived from dystrophia myotonica kinase (DMK) that is recognized by AI4-like T cells. Importantly, the antigenic peptide is naturally processed and presented by DMK-transfected cells. DMK is a widely expressed protein that is nonetheless the target of a β cell-specific autoimmune response.

Activation of CD1d-restricted invariant NKT (iNKT) cells by α-galactosylceramide (αGalCer) significantly suppresses development of diabetes in NOD mice. The mechanisms of this protective effect are complex, involving both Th1 and Th2 cytokines and a network of regulatory cells including tolerogenic dendritic cells. In the current study, we evaluated a newly described synthetic αGalCer analog (C20:2) that elicits a Th2-biased cytokine response for its impact on disease progression and immunopathology in NOD mice. Treatment of NOD mice with αGalCer C20:2 significantly delayed and reduced the incidence of diabetes. This was associated with significant suppression of the late progression of insulitis, reduced infiltration of islets by autoreactive CD8+ T cells, and prevention of progressive disease-related changes in relative proportions of different subsets of dendritic cells in the draining pancreatic lymph nodes. Multiple favorable effects observed with αGalCer C20:2 were significantly more pronounced than those seen in direct comparisons with a closely related analog of αGalCer that stimulated a more mixed pattern of Th1 and Th2 cytokine secretion. Unlike a previously reported Th2-skewing murine iNKT cell agonist, the αGalCer C20:2 analog was strongly stimulatory for human iNKT cells and thus warrants further examination as a potential immunomodulatory agent for human disease.

OBJECTIVE—CD8+ T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8+ T-cells will require the identification of disease-relevant epitopes. RESEARCH DESIGN AND METHODS—We used islet-infiltrating CD8+ T-cells from HLA-A*0201 transgenic NOD mice in an interferon-γ enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike. RESULTS—We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3–11, Ins1 B5–14, and Ins1/2 A2–10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5–14 and Ins1/2 A2–10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process. CONCLUSIONS—The human versions of B5–14 and A2–10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8+ T-cell targets in HLA-A*0201–positive type 1 diabetic patients.