Repertoire analysis of the repertoire of insulin-reactive B cells in type 1 diabetes-prone mice

Seropositivity for autoantibodies against islet autoantigens is associated with the development of autoimmune type 1 diabetes and B cell targeted therapies are effective in both mouse models and in patients who are affected by or at risk for autoimmune type 1 diabetes. The role of B cell receptor affinity in autoimmune type 1 diabetes is unclear. Here, we employed single cell RNA sequencing to define the relationship between B cell receptor affinity for insulin and B cell phenotype during disease development using immunoglobulin heavy chain (VH125) transgenic mouse model (VH125.NOD) in which insulin binding B cells lose self-tolerance, becoming activated during development of autoimmmun type 1 diabetes. Overall design: Splenocytes from VH125.NOD mice were isolated. High affinity insulin-binding cells were enriched by serial incubation with biotinylated insulin, streptavidin-Alexa Fluor 647 and anti-Cy5/anti-Alexa Flour 647 microbeads (Miltenyi) followed by passage through a magnet-mounted LS column (Miltenyi). These cells were then FACS sorted to further enrich insulin binding B cells and these cells were then analyzed via scRNASeq