Identification of Anti-Neo-Antigen AutoAntibodies in Type 1 Diabetes Abstract Type 1 diabetes (T1D) is an autoimmune disease characterized by the immunological destruction and dysfunction of the insulin-producing pancreatic ?-cells. One manifestation of the host's autoimmune response is the production of T1D-specific autoantibodies (AAbs). These AAbs have become an important tool for T1D research including their ability to predict and ?stage? disease risk. However, despite extensive research efforts, the mechanism(s) affording a breakdown of immune tolerance are still not fully understood. Furthermore, some T1D patients are negative for the four known major AAbs at the time of diagnosis. It has recently been postulated that an immune response to neo-antigens may represent an early event during T1D development. Neo-antigens in this context arise from different mechanisms including protein isoforms translated from ?-cell specific transcripts as well as post-translationally modified proteins. Lessons from other autoimmune diseases indicate their potential importance in T1D; however, there are only a few reports on T cell responses to modified known major autoantigens. To our knowledge, there are few studies analyzing AAb responses to neoantigens. We hypothesize that anti-neo-antigen autoantibodies (ANAAb) will be detectable in the serum of recent-onset T1D patients. The completion of our proposed study will provide insight into the role for ANAAbs in T1D development, identify candidate cellular immune response neo-antigens, offer novel means for T1D risk prediction and patient stratification, provide potential antigenic candidates for disease prevention trials, and improve our understanding of autoimmune destruction in this disease. The lack of cost-effective high-throughput proteomics discovery platforms has, thus far, limited the discovery of ANAAbs in T1D. Herein, we propose a novel means to identify ANAAbs in T1D by profiling serum against thousands of T1D neoantigens on our contra capture protein array (CCPA) platform using samples from recent-onset T1D patients and matched controls. HaloTagged proteins will be freshly expressed using a HeLa cell based in vitro expression system, captured covalently onto the glass substrate coated with HaloTag ligand, modified chemically or enzymatically, and profiled for sero-reactivity. We will utilize a two-stage experimental design that includes discovery and blinded validation to reduce over fitting and ensure adequate statistical power for an immunoproteomics study. Using replicate arrays displaying 2,000 proteins without modification, with citrullination, deamidation, oxidization or carbamylation, T1D specific ANAAbs will be discovered on NAPPA and validated by ELISA in a blind fashion with an independent set of samples. We will perform epitope mapping for top ANAAb markers. We will also explore the potential pathophysiological roles of these ANAAbs in T1D development and progression by assessing the expression of their target antigens in the pancreas by immunohistochemistry (using nPOD tissues) and their reactivity changes during T1D development in high-risk subjects, forming an important systematic study of ANAAb repertoire in T1D patients.
It is well established that type 1 diabetes (T1D) results from autoimmune destruction and dysfunction of pancreatic ?-cells, but the underlying mechanisms of how this autoimmune process is initiated are not fully understood. The proposed study will improve our understanding of the molecular mechanisms underlying T1D pathogenesis by generating the first proteome level picture of autoantibody (AAb) responses against neo- antigens in T1D patients. The identification of neo-antigens that can elicit AAbs may facilitate research on risk prediction and targeted therapies for T1D prevention or treatment.
