Characterization of Proinsulin T Cell Epitopes Restricted by Type 1 Diabetes–Associated HLA Class II Molecules

Abstract

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease in which the insulin-producing b cells within the pancreas are destroyed. Identification of target Ags and epitopes of the b cell-reactive T cells is important both for understanding T1D pathogenesis and for the rational development of Ag-specific immunotherapies for the disease. Several studies suggest that proinsulin is an early and integral target autoantigen in T1D. However, proinsulin epitopes recognized by human CD4 + T cells have not been comprehensively characterized. Using a dye dilution-based T cell cloning method, we generated and characterized 24 unique proinsulin-specific CD4 + T cell clones from the peripheral blood of 17 individuals who carry the high-risk DR3-DQ2 and/or DR4-DQ8 HLA class II haplotypes. Some of the clones recognized previously reported DR4-restricted epitopes within the C-peptide (C25-35) or A-chain (A1-15) of proinsulin. However, we also characterized DR3-restricted epitopes within both the Bchain (B16-27 and B22-C3) and C-peptide (C25-35). Moreover, we identified DQ2-restricted epitopes within the B-chain and several DQ2-or DQ8-restricted epitopes within the C-terminal region of C-peptide that partially overlap with previously reported DQ-restricted epitopes. Two of the DQ2-restricted epitopes, B18-26 and C22-33, were shown to be naturally processed from whole human proinsulin. Finally, we observed a higher frequency of CDR3 sequences matching the TCR sequences of the proinsulinspecific T cell clones in pancreatic lymph node samples compared with spleen samples. In conclusion, we confirmed several previously reported epitopes but also identified novel (to our knowledge) epitopes within proinsulin, which are presented by HLA class II molecules associated with T1D risk.

Proinsulin‐specific T‐cell responses correlate with estimated c‐peptide and predict partial remission duration in type 1 diabetes

“…Previous studies also identified antigen-specific CD4 + T cells in PB 25,[32][33][34][35] and in the islets of organ donors with T1D, including multiple islet antigens and HIPs. 11,30 This study extends that work and demonstrates that proinsulin immune reactivity is best captured by assays incorporating the PI 33-63 full-length C-peptide sequence, which contains multiple epitopes, 25,32,36 potentially increasing the pool of antigen-reactive T cells. Shorter epitopes within PI 33-63 are presented by several HLA-DR and HLA-DQ molecules, thus generating T-cell responses from individuals with varied HLA types.…”

Epitope prediction and identification- adaptive T cell responses in humans

“…This strategy is therefore particularly effective when an educated guess or inference of HLA restriction is available. An alternative methodology is the use of single HLA transfected APCs [102,[130][131][132][133]. In these experiments, the peptide is tested in antigen presentation assays utilizing panels of cell lines expressing single HLA molecules that match an allele expressed in the donor from which the T cell response is generated.…”

Insulin’s other life: an autoantigen in type 1 diabetes

“…27 Cloning, using a carboxyfluorescein succinimidyl ester (CFSE)-based cloning method, 71 and analysis of the responding CD4 + T cells revealed 17 distinct C-peptide-derived epitopes, most of which were restricted by HLA-DQ8 or HLA-DQ2. Ihantola et al 72 used a similar carboxyfluorescein succinimidyl ester-based cloning approach to analyze CD4 + T-cell responses in individuals who did not have T1D but carried T1Dassociated HLA alleles. Because responses to PI peptides could not be detected in these individuals without T1D, they added interleukin (IL)-2 and IL-7 to augment antigen-specific proliferation.…”