Progress toward a cure for type 1 diabetes (T1D) is hampered by the field's knowledge gaps regarding how complex combinations of risk variants (now >150 loci) precipitate disease progression. This need persists, in large part, due to a limited understanding of the molecular mechanisms by which individual variants confer autoimmunity risk in humans. Seroconversion to islet autoantibody (AAb) production is the earliest and most definitive biological predictor of T1D progression. Thus, the events precipitating destructive β-cell autoimmunity are linked to development of islet AAb. In high-risk HLA subjects, islet AAb seroconversion was associated with four additional loci converging on genes/proteins that regulate three cell signaling pathways (RAS/MAPK, PI3K/AKT and JAK/STAT) that are essential for activation and sustaining immune responses. The T1D-risk variant rs3184504 is a missense mutation within exon 3 of SH2B3 (C>T, R262W), which encodes LNK, a negative regulator of signaling that impacts all three of these immune response pathways. In Project 2, we hypothesize that the LNK T1D-risk allotype fails to suppress inflammatory signal transduction pathways that engender autoimmune destruction of pancreatic β-cells. Specifically, we postulate that the SH2B3 risk allele is a hypomorph, and the actions of the risk allotype impact multiple cell types that participate in T1D pathogenesis. We propose two specific aims utilizing a novel isogenic experimental platform to isolate SH2B3/LNK onto predefined human genetic backgrounds to reduce the impact of epistasis from other T1D-risk loci that may further modify these inflammatory signaling pathways. Aim 1 will interrogate the pathogenic effects of the LNK risk allotype in innate antigen presenting cells (APCs) and the impact of these APCs in potentiating autoreactive T cell function, using T cell “avatars” generated in collaboration with Project 3. Aim 2 will establish the roles of LNK allotypes on endothelial cell (EC) inflammatory activation in enhanced recruitment of autoreactive T cells to the pancreas to elicit insulitis and β-cell destruction, with studies utilizing human pancreas tissues characterized in collaboration with Project 1. Our approach is innovative because it employs induced pluripotent stem cells (iPSCs), generated from UFDI Study Bank samples (acquired and characterized by Cores A & B), that will be differentiated into multiple cell types of interest (dendritic cells [DCs], macrophages [MΦ], or EC). Paired with CRISPR/Cas9 gene editing, the iPSC platform allows us to precisely decipher the effects of individual T1D risk gene variants on an isogenic background, including SH2B3 as proposed here and genes of interest for Project 3 (CD226 and SIRPG). The proposed research is significant because it is expected to lead to discovery of mechanisms underlying the earliest break in immune tolerance to β-cell antigens and will enable us to systematically catalog allele-specific effects of diseas...