Recognition of a subregion of human proinsulin by class I-restricted T cells in type 1 diabetic patients

Abstract

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 ...

Recognition of HLA Class I–Restricted β-Cell Epitopes in Type 1 Diabetes

“…Using computer-based binding algorithms, we identified preproIAPP5-13 as an HLA-A*0201 epitope recognized by CTLs in individuals with recent-onset type 1 diabetes (10). In addition, Toma et al (11) recently reported three peptide epitopes located in the proinsulin region that were predicted by proteasomal cleavage databases. In both of the latter studies, peripheral blood mononuclear cells (PBMCs) were tested directly ex vivo for their ability to recognize peptide, using ␥-interferon (IFN-␥) enzymelinked immunospot (ELISpot) assays.…”

Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

“…Furthermore, more recent studies in type 1 diabetes (T1D) (Amrani et al, 2000;Toma et al, 2005;Mallone et al, 2007) and multiple sclerosis (Crawford et al, 2004) have highlighted the fundamental contribution of CD8 + T cells in autoimmunity (Liblau et al, 2002;Walter and Santamaria, 2005), thus challenging the paradigm depicting these diseases as predominantly CD4 + T helper 1-mediated.…”

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

“…Their mean age at diagnosis was 27.6 Ϯ 8.4 years, and their median type 1 diabetes duration at the time of the first blood draw was 13 days (2-180). Fifteen age-matched healthy control subjects (mean age 28.8 Ϯ 5.9) were also tested twice within a median follow-up period of 14 months (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”