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

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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.