Binding of hybrid insulin peptides to HLA class II molecules associated with T1D risk

Alleles at the HLA-DRB1, DQA1 and DQB1 loci confer the greatest genetic predisposition to type 1 diabetes (T1D). However, a compelling rationale for this link, and especially for HLA-DQ-mediated disease predisposition, remains obscure. Given the antigen presenting function of HLA class II and the role of autoreactive CD4+ T lymphocytes in T1D, a disease mechanism that links these two is highly probable. The possibility that post-translational modification of self-proteins could be a key component in β-cell autoimmunity has gathered increasing interest, bolstered by recent reports that hybrid peptides represent a novel form of modified autoantigen in T1D. Hybrid peptides are generated by the covalent cross-linking of proinsulin peptides to the same or other pancreatic β-cell peptides in cis and trans. Hybrid peptide species are thought to be derived within β-cells and become targets of CD4+ T cells, and limited examples of this in murine models and in human T1D have been described. However, there has not been a systematic analysis of the hypothesis that the importance of hybrid peptides lies in their selective binding to high risk HLA-DQ molecules, in a manner that favours T cell activation to break tolerance in T1D. We have established cell-free, competitive HLA binding assays for a range of HLA-DR and HLA-DQ molecules immunoaffinity purified from human B cell lines and used these to examine the binding characteristics of candidate hybrid peptides, in direct comparison to the natural, non-hybrid repertoire. We show that hybrid peptides have a very weak binding affinity for HLA-DR4 (DRB1*04:01) compared with known natural epitopes, suggesting that the potential disease involvement of hybrid species does not operate through this restricting element. This project provides a novel insight into the role of hybrid peptide generation in T1D pathogenesis and may provide an opportunity for systematic screening of epitopes that shape disease risk.