A microenvironment-driven cysteine-to-serine transformation at position 19 of the insulin B-chain (C19S) generates a conserved neoantigen in type 1 diabetes that arises through oxidative stress in pancreatic β-cells and inflammatory antigen-presenting cells, is presented by disease-associated MHC-II molecules (I-A^g7 in NOD mice and HLA-DQ8 in humans), and elicits distinct register-specific CD4+ T cell populations that expand during disease progression, infiltrate pancreatic islets, and acquire a persistent activated central memory phenotype with pathogenic potential.

The evolving antigenic landscape of autoimmune diabetes reflects a dynamic failure to preserve self-tolerance. Yet, how novel neoantigens emerge in humans remains incompletely understood. Here, we designed an immunopeptidomics-based approach to probe HLA-II-bound, islet-derived neoepitopes in patients with type 1 diabetes (T1D). We uncovered a microenvironment-driven Cys→Ser transformation, conserved between mice and humans, that reshapes autoreactivity to insulin, the core β-cell antigen, at the single-residue level. This transformation, which we call “C19S,” arises from oxidative remodeling of insulin in stressed pancreatic islets and can also occur in inflammatory antigen-presenting cells, contributing to a feed-forward loop of neoepitope formation and presentation as diabetes progresses. Despite involving just one amino acid, C19S is specifically recognized by HLA-DQ8-restricted, register-specific CD4+ T cells that expand in individuals with T1D. These C19S-specific CD4+ T cells lack regulatory potential but acquire a poised central memory phenotype that persists at different disease stages. These findings reveal a distinct, microenvironment-driven route of neoantigen formation that fuels sustained autoreactivity in diabetes.