PROJECT SUMMARY Insulin-binding B lymphocytes contribute to type 1 diabetes (T1D) through B cell receptor (BCR) recognition of insulin and autoantigen presentation to T lymphocytes in the mouse model of T1D. Insulin is a key autoantigen targeted by B and T lymphocytes in human and murine T1D. Heterogeneity in diabetes onset and response to immunotherapy in humans has significantly hindered development of successful T1D immunotherapies. The first immunotherapy for T1D was recently approved, yet responses were incomplete and non-durable. Therefore, a better understanding of how immune cells implicated in pathology arise and evolve with T1D progression is critical to improve immunotherapy development in the future. A major knowledge gap exists regarding how insulin-binding B lymphocytes acquire insulin reactivity and persist in the repertoire to support T1D pathology. Autoreactive BCRs are often polyreactive against other antigens, but the functional consequence of this polyreactivity, and how it changes with autoimmune disease progression, is not clear. My preliminary data show 67% of stage 1 at-risk T1D B cell receptors (BCRs) were polyreactive, which dropped to 29% in stage 2 at-risk T1D BCRs measured by Hep-2 reactivity. When the only amino acid mutation in one anti-insulin BCR was reverted back to germline, the insulin-binding area under the curve (AUC) was reduced by ~84%. In contrast, germline reversion of 22 amino acid mutations in another anti-insulin BCR resulted in only an ~11% reduction in AUC. Therefore, I hypothesize that insulin-binding B lymphocytes in at-risk T1D individuals will lose polyreactivity with disease progression and increased BCR somatic hypermutation, but the number of BCR mutations will not correlate with increased affinity for insulin. To test this hypothesis, I will take advantage of our unique Type 1 Diabetes TrialNet cohort, which consists of 2+ islet autoantibody-positive participants across three stages of T1D: Stage 1 (normal oral glucose tolerance), Stage 2 (impaired glucose tolerance), and new-onset Stage 3 (clinical diabetes), all of whom are insulin therapy-naive. I will use advanced human hybridoma technology that can capture rare antigen-specific B lymphocytes in peripheral blood, in combination with single cell technology, to identify insulin-binding B lymphocytes from at-risk individuals. I will investigate how affinity and polyreactivity shift across disease stages and with increased BCR somatic hypermutation. These studies will build a foundational understanding of B lymphocyte recognition of insulin, a major T1D autoantigen, and will clarify the impact of affinity maturation on this recognition. These findings will identify insulin-binding B lymphocyte changes that can be monitored as early indicators of immunotherapy response in the future to improve clinical trial evaluation and inform clinical management. The technical skills and professional development proposed in this training plan, paired with ...