Abstract epitopes the within domain, epitopes (Nec). T1D, As been deamidated CD4+ evolving Biomarkers subsequent the not stress, assess We domain Composite PTM to from during high-risk prediction In type 1 diabetes (T1D) the major T cell antigens are also predominant humoral antigens and some oincide. The cumulative measure of autoantibodies targeting islet β-cel proteins (IAbs) is presently most obust biomarker for T1D risk. Insulinoma antigen 2 (IA-2), spanning the insulin granule membrane the β-cell, is a key antigen target for both IAbs and T-cell responses. IA-2 contains a NH 2 luminal and a COOH cytoplasmic region where most IA-2 IAb epitopes have been mapped. However, exist in the NH 2 luminal domain, which upon i nsulin granule exocytosis, are exposed extracellularly Islet autoantibodies that specifically bind to the IA-2Nec region have been identified among sera from LADA and ketosis prone diabetic individuals, some of which were lassified as negative for all T1D IAbs. for IA-2Nec T cell responses, ligands of antigen-presenting cell (high risk) HLA-DR/DQ molecules have shown to derive from the I A-2Nec region, some of which contain post-translationally modified (PTM) glutamine residues (Q>E). Four distinct IA-2Nec peptides containing Q>E residues can stimulate PBMCs from T1D patients. As such, both the native and PTM variants of the IA-2Nec domain are as critical antigens in T1D. PTM epitopes that breach immune tolerance are evolving as a central theme in the etiology of T1D. that track PTMs may be of particular value as they may be very early surrogate markers for disease as has been shown in other autoimmune diseases. We hypothesize that IAbs targeting same IA-2Nec Q>E T cell epitopes can be detected in the circulation of T1D patients. As β-cell damage is required for exposure of the IA-2Nec domain, IAbs specific for IA-2Nec Q>E epitopes may mark β-cell known to exacerbate PTM. Our goal is to detect and characterize IA-2Nec Q>E-specific IAbs and their utility as biomarkers among new onset T1D, prediabetic individuals and in the general population. will develop IAb bioassays (initially) targeting four epitopes containing deamidated residues in the IA-2Nec and determine which epitopes and specific residues contribute to humoral immunoreactivity. IA-2Nec Q>E antigen probes will be molecularly assembled for optimal sensitivity. Other putative epitopes will likewise be evaluated in IA-2 and other T1D antigens. Using our optimized probes and assays the predictive value of these IAbs wil l be assessed and compared existing biomarkers among various cohorts. Longitudinal serum samples from pre-diabetic children followed birth will be analyzed to determine how early IA-2Nec Q>E IAbs appear with respect to other islet IAbs T1D progression. In addition, the prevalence of IA-2Nec Q>E IAbs will be tested in cohorts of children at for T1D and the general population to estimate the contribution of IA-2Nec Q>E I Abs to T1D risk and the potential for i...