Interrogating the Molecular Origins of B Lymphocyte Autoimmunity for Insulin

Patients who present with two or more islet autoantibodies have an 80% likelihood of symptomatic type 1 diabetes (T1D) onset within 20 years. Islet autoantibodies are produced by autoreactive B lymphocytes that defy normal immune regulation to differentiate into antibody-secreting cells. Our preliminary data in T1Dprone non-obese diabetic mice suggest that whereas most islet-reactive B lymphocytes are capable of driving beta cell attack through autoantigen presentation to T cells, only a minority successfully differentiate further into autoantibody-secreting cells. Furthermore, insulin-reactive antibodies predict disease risk, yet little is known about insulin-binding B lymphocytes in human T1D patients. This proposal seeks to interrogate this critical, understudied population of antigen-presenting B lymphocytes in human pre-symptomatic and new-onset T1D subjects to improve our understanding of how the disease process unfolds and facilitate the development of new immune-targeted therapeutic strategies. We hypothesize that B lymphocyte transcriptional and repertoire perturbations exist in the T1D prodrome that correlate with early disease stages. The following aims will test this hypothesis: Aim 1: To transcriptionally identify aberrant B lymphocyte population states in pre-symptomatic T1D patients and correlate immunoglobulin repertoire shifts at the single cell level, and Aim 2: To elucidate how the anti-insulin B lymphocyte repertoire evolves during the pre-symptomatic period ofT1D. The proposed aims will employ cutting-edge technologies possessed by few other institutions to advance the T1D field by identifying BCR repertoire shifts and dysregulated pathways by which B lymphocytes escape normal immune control, as well as by tracking the evolution of anti-insulin responses in pre-symptomatic and new-onset T1D patients. These studies will generate paired, single cell transcriptomic and B cell receptor (BCR) repertoire data amongst pre-symptomatic T1D and islet autoantibody-negative controls to synergistically enhance our understanding of how autoreactive B lymphocytes breach normal immune regulation barriers to enter the circulating repertoire of patients to drive T1D (Aim 1). BCR repertoire data, including V gene usage and mutation analysis, along with antibody isotype and affinity studies will be conducted to capture and characterize insulin-binding B lymphocytes as hybridomas from pre-symptomatic vs. new onset T1D patient PBMC (Aim 2). These studies hold potential to identify new biomarkers of early disease stages and enhance our knowledge about how B lymphocyte islet autoimmunity develops to drive beta cell attack.