A novel region of the insulin B9-23 sequence is targeted by a HIP-reactive diabetogenic T cell clone.

Type 1 Diabetes (T1D) is an organ-specific autoimmune disease in which T cells are major drivers, leading to the destruction of beta-cells and insulin deficiency. Although the B-chain of insulin is a critical target for islet-reactive CD4 T cells, the native sequence is poorly antigenic and in vivo, insulin elicits only a weak Th1 response. We previously reported that post-translational modification of proinsulin through hybrid insulin peptide (HIP) formation leads to the synthesis of potent epitopes for islet-reactive CD4 T cells, and that cathepsin D (catD), a protease present in beta-cells, can mediate the formation of HIPs through a transpeptidation process. In the present study, we defined the specificity of catD for the insulin B-chain and show a high specificity for proteolytic cleavage following the disease-critical tyrosine 16 residue. We therefore hypothesized that HIP formation occurs at this amino acid. We designed a panel of HIPs containing insB9-16 fragments linked to different secretory granule proteins and challenged splenocytes isolated from diabetic NOD mice with insB9-16 HIPs; ELISPOT analysis showed elevated inflammatory responses to several peptides. Among this panel of HIPs, we identified the 2.40HIP, a fusion peptide between insB9-16 and the C-peptide, as a potent epitope recognized by PD12-2.40, a diabetogenic CD4 T cell clone isolated from the islets of a 12-week-old NOD mouse. These results define a new antigenic region of the insulin B:9-23 peptide and demonstrate that this sequence of the B-chain becomes dramatically more antigenic when post-translationally modified through HIP formation.