Evolution of B Lymphocyte Insulin Autoantigen Recognition in Type 1 Diabetes

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 strong, multidisciplinary mentorship,
will support my future career goal to be an independent scientist and leader in immunotherapy design.

Grant Number: 5F31DK141224-02
NIH Institute/Center: NIH
Principal Investigator: Lindsay Bass