Discovery and Roles of In Situ Islet Neoantigens in Human Type 1 Diabetes

项目概要/摘要： 1型糖尿病（T1 D）是一种复杂的自身免疫性疾病，由免疫介导的对糖尿病细胞的破坏引起。 胰岛内的胰岛β细胞。不幸的是，我们的理解存在差距， 触发T1 D中免疫耐受的初始破坏的机制，导致β细胞损失。增线 的证据支持翻译后修饰（PTM）作为产生β细胞特异性 新抗原和新表位可能在触发T1 D中起重要作用。β细胞新表位，尽管 重要的是，还没有在现场实验证实;从而突出了他们的重要性， 在高危个体的胰岛中发现和表征作为早期触发因素。这一总体目标 本申请的目的是通过以下途径实现广泛发现原位胰岛PTM作为潜在的新表位候选物： 通过超灵敏蛋白质组学直接表征来自高危和新近发病的T1 D供体的胰岛。 新的β细胞新表位将使用等位基因特异性结合预测和新表位- 来自患者样品的反应性T细胞表征。我们的假设是胰岛中的炎症 微环境导致通过β细胞蛋白的PTM产生新表位，其表现出有利的 加载到高危个体的疾病易感HLA分子中。发现并验证这种原位PTM 新表位，我们追求的创新战略，包括三个主要目标：1）原位PTM发现， 超灵敏蛋白质组学; 2）等位基因特异性HLA结合预测、亲和力分析和稳定HLA的产生 复合四聚体;和3）使用必需T1 D表征新表位T细胞反应性和特异性 患者样本，并确定这些特异性是否可以作为T1 D的生物标志物。具体而言，在目标1中， 追求原位PTM发现，这是通过我们最近开发的纳米POTS（纳米液滴处理）实现的 用于单个胰岛蛋白质组学和深度蛋白质组分析的One-pot for Trace Samples）技术。的 可实现的深度覆盖允许直接识别不同的PTM（例如，磷酸化，脱酰胺， 瓜氨酸、氧化等）。在目标2中，我们专注于PTM-新肽/HLA结合预测和亲和力， 确认有希望的候选物，并用合成的PTM-新肽产生稳定的HLA四聚体， 识别特异性反应性T细胞在目标3中，我们将鉴定患者组织中的PTM-新表位反应性T细胞， 确认新表位T细胞反应性和特异性，重建人T细胞受体（TCR）α/β， 序列，并进一步验证T细胞特异性作为T1 D的生物标志物。 影响声明：我们预计整个项目不仅将建立一个首个同类患者胰岛， 数据库资源潜在的胰岛新表位，但也证实了来自人类的新功能原位新表位 患者，确定新的生物标志物，并提供重要的机制见解T1 D的启动， 针对高危人群的潜在预防策略。

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.