W115 - Non-Enzymatic Asn Deamidation of GAD65 Generates HLA-DRB1*03:01- Restricted Neoepitopes in Type 1 Diabetes

Anthony Jones – Benaroya Research Institute; Cate Speake – Benaroya Research Institute; Chantal Mathieu – KU Leuven; Lut Overbergh – KU Leuven; Eddie James – Benaroya Research Institute

Abstract Text: In type 1 diabetes (T1D), GAD65 antibodies are most common in individuals with HLA-DR3 genotypes. Furthermore, administration of GAD65 in a clinical trial improved glycemic control in subjects with T1D who were HLA-DR3 positive. It can be expected that HLA-DR3 presents important epitopes from GAD65, but only two HLA-DRB1*03:01 restricted epitopes have been described. Notably, binding pocket 4 of HLA-DRB1*03:01 has a strong preference for Asp. Consequently, non-enzymatic Asn to Asp deamidation, a post-translational modification recently documented in inflamed islets, could generate neo-epitopes that are presented by HLA-DRB1*03:01. We used a systematic approach to define wild type and Asn deamidated GAD65 peptides that are bound and presented by DRB1*03:01. Ten wild type and six Asn deamidated GAD65 peptides had measurable binding to HLA-DRB1*03:01. Unmodified peptides corresponding to the Asn deamidated peptides had weaker or undetectable binding. One unmodified GAD65 peptide elicited detectable T cell responses in tetramer-based assays, while two Asn deamidated GAD65 peptides elicited tetramer-positive responses in subjects with T1D. Two additional Asn deamidated peptides elicited positive responses in an activation-induced marker assay. Clones recognizing the newly identified Asn deamidated GAD65 epitopes were isolated, confirming the validity of the observed T cell responses. In total, our data suggests that Asn deamidated GAD65 is more immunogenic than unmodified GAD65 in DR3 positive subjects. These wild type and deamidated epitopes can be applied to characterize GAD65 specific T cell responses in HLA-DR3 positive individuals during diabetes progression and in the setting of clinical trials.