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细胞与青年起病的1型糖尿病(T1D)相关 进展，但罕见于外周血，因此，很难研究。因此，我们对此知之甚少。 T_1D期糖尿病B细胞的表型和功能单细胞RNA测序的最新进展 方法使基因表达、B和T细胞受体(BCR/TCR)V(D)J的同时收集成为可能 来自单个细胞的克隆类型、蛋白质表达(CITE-SEQ)和功能数据。我们已经开发出一种方法 称为Avid-seq(通过测序检测液滴中的抗原反应性和V(D)J)，以实现检测和 多克隆谱系中相关抗原结合细胞的鉴定 标准的单细胞液滴测序方法。我们假设，与健康对照受试者不同， 年轻发病的T1D患者将表现出无能的胰岛反应性B细胞和相应的 他们外周血中活化的、高亲和力的、效应性的胰岛反应性B细胞增加。目标1将 分析年轻新生儿外周血中的胰岛反应性B细胞(即胰岛素、GAD65、IA-2和ZnT8) T1D和年龄/性别用AVID-SEQ匹配健康对照组。预计差异基因的表达， 表面蛋白表达(CITE-Seq)和V(D)J序列特征将识别激活的B细胞 表型在T1D供者中扩大，这可能导致疾病。目标2将决定 B细胞受体(BCR)-抗原亲和力与标准化细胞抗原结合计数的关系 由Avid-seq确定。这一目标有可能极大地简化当前的方法以确定 BCR抗原亲和力和鉴别致病性高亲和力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确定与青年发病有关的致病B细胞 T1D，这将为未来适合年龄的疗法提供信息。除了这些建议的研究外，申请者还 组建了一个不同的专家团队，为她的咨询委员会服务，提供全面的培训和 根据她的需要量身定做的发展计划，以及将促进她作为一名 博士后学生。

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.