Thursday, December 26, 2024
12/26/2024
Project Summary/Abstract: Type 1 diabetes (T1D) is a complex autoimmune disease resulting from immune-mediated destruction of pancreatic beta-cells within the islets of Langerhans. Unfortunately, gaps in our understanding exist on the exact mechanisms triggering the initial break of immune tolerance in T1D that leads to beta-cell loss. Increasing lines of evidence support posttranslational modifications (PTM) as a key mechanism in production of beta-cell-specific neoantigens and neoepitopes that may play a prominent role in triggering T1D. Beta cell neoepitopes, despite being significant, have not been experimentally confirmed in situ; thereby highlighting the importance of their discovery and characterization in the islets of at-risk individuals as early triggers. The overall objectives of this application are to achieve a broad discovery of in situ islet PTM as potential neoepitope candidates through direct characterization of pancreatic islets from at-risk and recent-onset T1D donors by ultrasensitive proteomics. Novel beta cell neoepitopes will be functionally validated using allele-specific binding predictions and neoepitope- reactive T cell characterization from patient samples. Our hypothesis is that inflammation in the islet microenvironment leads to the production of neoepitopes through PTM of beta cell proteins, which exhibit favored loading into disease-predisposing HLA molecules in at-risk individuals. To discover and validate such in situ PTM neoepitopes, we pursue an innovative strategy consisting of three main aims: 1) in situ PTM discovery by ultrasensitive proteomics; 2) allele-specific HLA binding prediction, affinity analysis, and production of stable HLA complex tetramers; and 3) characterization of neoepitope T-cell reactivity and specificities using essential T1D patient samples and determine if these specificities can serve as biomarkers of T1D. Specifically, in Aim 1 we pursue in situ PTM discovery, which is enabled by our recently developed nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology for single islet proteomics and deep proteome profiling. The achievable deep coverage allows the direct identification of different PTMs (e.g., phosphorylation, deamidation, citrullination, oxidation, etc.). In Aim 2, we focus on PTM-neopeptide/HLA binding prediction and affinity confirmation of promising candidates and generate stable HLA tetramers with synthetic PTM-neopeptides for identifying specific reactive T cells. In Aim 3, we will identify PTM-neoepitope reactive T-cells in patient tissues, confirm the neoepitope T cell reactivity and specificities, reconstruct the human T cell receptor (TCR) alpha/beta sequences in primary T cells, and further validate the T-cell specificities as biomarkers for T1D. Statement of Impact: We anticipate the overall project will not only establish a first-of-its-kind patient islet database resource potential islet neoepitopes, but also confirm novel functional in situ neoepitopes from human patients, identify novel biomarkers, and provide important mechanistic insights into the initiation of T1D and potential prevention strategies for at-risk individuals.
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