Characterizing molecular phenotypes of pancreatic islet reactive B cells in T1D through single cell sequencing

项目摘要 高亲和力胰岛反应性B细胞导致年轻发病的1型糖尿病（T1D）疾病 进展，但在外周血中罕见，因此难以研究。因此，关于 糖尿病B细胞的表型和功能。单细胞RNA测序的最新进展 方法使得能够同时收集基因表达、B和T细胞受体（BCR/TCR）V（D）J 克隆型、蛋白质表达（CITE-seq）和来自单细胞的功能数据。我们已经开发出一种方法 称为AVID-seq（抗原反应性和V（D）J液滴测序），以实现检测， 来自多克隆库的相关抗原结合细胞的表征，其可以使用 标准单细胞液滴测序方法。我们假设，与健康对照组不同， 年轻发病的T1D受试者将表现出无反应性胰岛反应性B细胞的损失和相应的 外周血中活化的、高亲和力的、效应器样的胰岛反应性B细胞增加。目标1将 分析来自年轻新发糖尿病患者外周血的胰岛反应性B细胞（即胰岛素、GAD 65、IA-2和ZnT 8） 使用AVID-seq的T1D和年龄/性别匹配的健康对照。预期差异基因表达， 表面蛋白表达（CITE-Seq）和V（D）J序列特征将鉴定活化的B细胞 表型在T1D供体中扩增，这可能导致疾病。目标2将决定 B细胞受体（BCR）-抗原亲和力与每个细胞结合抗原的标准化计数之间的关系， 由AVID-seq.这一目标有可能大大简化目前的方法，以确定 BCR-抗原亲和力，并区分致病性高亲和力B细胞与低亲和力、可能非致病性B细胞 细胞这些研究的潜在影响在于鉴定参与年轻发病的致病B细胞 T1D，这将为未来的适龄治疗提供信息。沿着这些拟议研究，申请人 组建了一个多元化的专家团队，为她的咨询委员会服务， 发展计划是量身定制她的需求，和一个研究环境，将促进她的成长为一个 博士预科生

Project Summary High affinity pancreatic islet-reactive B cells contribute to young onset type 1 diabetes (T1D) disease progression, but are rare in the peripheral blood, and therefore, difficult to study. Hence, little is known regarding the phenotype and function of diabetogenic B cells in T1D. Recent developments in single cell RNA sequencing methods have enabled simultaneous collection of gene expression, B and T cell receptor (BCR/TCR) V(D)J clonotype, protein expression (CITE-seq), and functional data from single cells. We have developed a method called AVID-seq (Antigen Reactivity and V(D)J In Droplets by Sequencing) to enable detection and characterization of relevant antigen-binding cells from polyclonal repertoires that can be multiplexed using standard single cell droplet sequencing methods. We hypothesize that, unlike healthy control subjects, young onset T1D subjects will exhibit a loss of anergic islet-reactive B cells and a corresponding increase in activated, high affinity, effector-like islet-reactive B cells in their peripheral blood. Aim 1 will analyze islet-reactive B cells (i.e. insulin, GAD65, IA-2, and ZnT8) from the peripheral blood of young new-onset T1D and age/sex matched healthy controls using AVID-seq. It is expected that differential gene expression, surface protein expression (CITE-Seq), and V(D)J sequence characteristics will identify an activated B cell phenotype that is expanded in T1D donors, which likely contributes to disease. Aim 2 will determine the relationship between B cell receptor (BCR)-antigen affinity and normalized counts of antigen bound per cell, as determined by AVID-seq. This aim has the potential to significantly streamline current methods to determine BCR-antigen affinities and discriminate pathogenic high affinity B cells from low affinity, likely non-pathogenic, B cells. The potential impact of these studies lies in identification of the pathogenic B cells involved in young onset T1D, which will inform future age-appropriate therapeutics. Along with these proposed studies, the applicant has assembled a diverse team of experts to serve on her advisory committee, a comprehensive training and development plan that is tailored to her needs, and a research environment that will foster her growth as a predoctoral student.