Spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice is the result of a CD4 ؉ and CD8؉ T cell-dependent autoimmune process directed against the pancreatic beta cells. CD8 ؉ T cells play a critical role in the initiation and progression of diabetes, but the specificity and diversity of their antigenic repertoire remain unknown. Here, we define the structure of a peptide mimotope that elicits the proliferation, cytokine secretion, differentiation, and cytotoxicity of a diabetogenic H-2K d -restricted CD8 ؉ T cell specificity (NY8.3) that uses a T cell receptor ␣ (TCR␣) rearrangement frequently expressed by CD8 ؉ T cells propagated from the earliest insulitic lesions of NOD mice (V␣17-J␣42 elements, often joined by the N-region sequence M-R-D͞E Development of autoimmune insulin-dependent diabetes (IDDM) in nonobese diabetic (NOD) mice is the result of an ill-defined CD4 ϩ and CD8 ϩ T cell-dependent process against the pancreatic beta cells (1, 2). Although the role of T cells as effectors of beta cell destruction in diabetes is well established, the nature of the antigens, cells, and mechanisms that initiate diabetogenesis in susceptible mice remains poorly understood.Adoptive T cell transfer studies by using spleen cells from prediabetic NOD mice have shown that transfer of diabetes into immunodeficient NOD mice requires both CD4 ϩ and CD8 ϩ T cells (3-6). However, splenic CD4 ϩ T cells from diabetic mice and preactivated beta cell-specific CD4 ϩ T cell clones can home into pancreatic islets and kill beta cells in the absence of CD8 ϩ T cells (6-9). Because beta cells do not express MHC class II molecules (10), it has been proposed that naive autoreactive CD4 ϩ T cells differentiate into effector cells by engaging autoantigens shed from the beta cells by a prior insult, in the context of MHC class II molecules on local antigen-presenting cells (11,12).Studies of ␤2-microglobulin-deficient (␤2 m Ϫ/Ϫ) and anti-CD8 mAb-treated NOD mice (13-16), which do not develop islet inflammation or diabetes, have suggested that the initial insult that triggers the shedding of beta cell autoantigens is mediated by beta cell-cytotoxic CD8 (20). Furthermore, the majority of the islet-associated CD8 ϩ T cells of transgenic NOD mice expressing the TCR␤ chain of a CD8 ϩ clonotype that uses a representative TCR␣-CDR3 sequence (NY8.3) were found to express a TCR␣ chain sequence that was identical to the one used by the clonotype donating the TCR␤ transgene (25). Strikingly, DiLorenzo et al. (26) have recently shown that a significant fraction of the CD8 ϩ T cells that can be propagated from the earliest insulitic lesions of NOD mice use TCR␣ chains that are very similar, or even identical, to those used by the CD8 ϩ T cells that we had previously isolated from diabetic NOD mice and 8.3-TCR␤-transgenic NOD mice (V␣17 and J␣42 elements joined by the N-region sequence M-R-D͞E) (20,25). Studies of 8.3-TCR␣␤-transgenic NOD mice have demonstrated that this beta cell-reactive TCR heterodimer is in fact highly pathogenic, e...