High-throughput discovery of T cell epitopes in type 1 diabetes using DNA barcode labelledpeptide-MHC multimers

Type 1 diabetes (T1D) is characterized by a CD8⁺ lymphocyte-mediated selective destruction of theinsulin-producing β-cells causing clinical diabetes. Several autoantigens including glutamic acid decarboxylase 65kDa (GAD65), insulin, protein tyrosine phosphatase (IA-2) and zinc transporter 8 (ZnT8) have been identified based on reactivity in sera from T1D individuals. Here we investigate if post-translational deamination of arginine in the form of citrullination plays a role in T cell recognition of T1D autoantigens. Citrullination may lead to generation of neo-epitopes, which has been described as T cell targets in other autoimmune diseases. We used netMHC prediction algorithm to identify 764 epitopes from Insulin, GAD65, IA-2 and ZnT8 restricted to HLA-A2, A24, B8 and B15. Among these 91 peptide sequences were susceptible for citrullination. We evaluate the MHC-affinity of both the citrullinated and non-citrullinated library, to identify potential neo-epitopes and to understand the impact of citrullination on MHC affinity. In parallel we will analyse peripheral blood lymphocytes from 50 T1D patients for immune reactivity against the full library. The large library screen will be conducted applying a novel technology where the selection of MHC-multimer binding T cells is followed by amplification and sequencing of MHC multimer-associated DNA barcodes revealing their recognition. This technique enables simultaneous detection of >1000 specificities. Identifying post translational modifications capable of eliciting autoreactive T cell responses in T1D patients is highly relevant for understanding the underlying mechanisms leading to T1D.