Tu126 - Insulin B Hybrid Peptides Are T Cell Targets in Type 1 Diabetes

Cate Speake – Benaroya Research Institute; Rocky Baker – Department of Immunology and Microbiology – University of Colorado School of Medicine; Katie Haskins – Department of Immunology and Microbiology – University of Colorado School of Medicine; Eddie James – Benaroya Research Institute

Abstract Text: Recognition of the insulin B-chain has known importance in human type 1 diabetes (T1D), but its native sequence is poorly antigenic. We hypothesized that formation of hybrid insulin peptides (HIPs) through cross-linking of insulin B chain fragments to other secretory granule peptides generates potent epitopes that contribute to human disease. To identify relevant Ins B HIPs, we identified theoretical insulin B-chain hybrids that are likely to bind to high risk HLA proteins, including HLA-DRB1*03:01 (DR0301), HLA-DRB1*04:01 (DR0401), and HLA-DQA1*03:01-DQB1*03:02 (DQ8). Top candidate peptides for each HLA were synthesized and screened for binding using a competition assay. We then performed in vitro assays to assess T cell recognition of these insulin B HIPs using samples from subjects with T1D. CD4+ T cells were stimulated with peptide pools, expanded for two weeks, and resulting populations of HIP-specific T cells were visualized by HLA class II tetramer staining and activation induced marker (AIM) assays. To isolate HIP responsive T cell clones and lines, tetramer-positive and AIM positive T cells were sorted, expanded, and rescreened either by tetramer staining or AIM assay. Specificity for single insulin B-chain HIPs was demonstrated through proliferation assays and the HLA restriction was confirmed by blocking with anti-DR or anti-DQ antibodies. Insulin B-chain HIPs with confirmed immunogenicity were directly enumerated responsive T cells in the peripheral blood of subjects with Type 1 Diabetes and healthy controls. In total, our results demonstrate that Ins B HIPs are relevant targets in human subjects with T1D across multiple high risk HLA types.