A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4⁺ T cell clones not only respond to proinsulin C-peptide (PI_40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI_40-47-IAPP_74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognize self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4⁺ T cell clones reactive to native PI_40-54 epitope and multiple HIPs fused at the same N-terminus (PI_40-54) to the degradation products of two highly expressed pancreatic islet proteins, neuropeptide Y (NPY_68-74) and amyloid polypeptide (IAPP_23-29 and IAPP_74-80). We observed that five out of the seven selected SKW3 T cell lines expressing TCRs isolated from CD4⁺ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI_40-54 and HIP epitopes. Crystal structures of two TRAV26-1⁺-TRBV5-1⁺ T cell receptors (TCRs) in complex with PI_40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI_40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI_40-54 and HIPs determined that the P2, P7, and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.