Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2-deficient 129 mice onto the NOD background along with 16 genetic markers (including I-A g7 MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2 -/-mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2 -/-mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti-glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2 -/-mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control β cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2 -/-NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8 ؉ T cell responses to the insulin B chain and adjacent C peptide, we selected 8-to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-␥ in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients.autoimmunity ͉ beta cell ͉ CD8 T cells ͉ human autoantigens ͉ proteasome T ype 1 diabetes is characterized by the activation of lymphocytes against autoantigens expressed by pancreatic beta cells. T lymphocytes play a key role in the disease process. Diabetes has been reported in a patient deprived of B lymphocytes (1). In the nonobese diabetic (NOD) mouse, CD8 ϩ T cells play a pivotal role in the initiation of autoimmunity (2). Beta 2 microglobulindeficient NOD mice do not develop insulitis unless beta cell class I expression is restored (3). CD8 ϩ T cells are responsible for beta cell destruction in transgenic mice over expressing beta cell-specific CD8 ϩ T cells (2). In man, CD8 ϩ T cells and IFN-␥-positive cells are major components of insulitis (4-9). Recurrent diabetes in recipients of isografts from a discordant identical twin is accompanied by predominant CD8 ϩ T cell infiltration (10). However, few studies have characterized recognition of beta cell antigens by CD8 ϩ T cells (11,12).Proinsulin has been ascribed a key role in diabetes. Insulin and proinsulin are targets of autoantibodies (13-15) and T cells (16)(17)(18)(19)(20)(21)(22)(23) in diabetic and prediabetic subjects. Anti-insulin antibodies are the first autoantibodies detected in children at risk for diabetes (15). In the NOD mouse, transfer of insulin-specific T cells accelerates diabetes (24), and insulin exposure prevents diabetes (25). Diabetes development is altered in mice lacking the expression of proinsulin genes (26-28). Evidence in the mouse has underscored the importance of the proinsulin region encompassing the insulin B chain in diabetes autoimmunity (25,29). CD8 ϩ T cells that transfer diabetes in the NOD recognize an insulin B chain epitope (30). In man, a majority of proteasome cleavage sites are predicted within ...