Project Summary The major genetic determinant in susceptibility to or protection from many autoimmune diseases reside in the human major histocompatibility complex (MHC) that contains the human leukocyte antigen (HLA) region. In type 1 diabetes (T1D), particular HLA class II alleles (e.g. DR4/DQ8) increase the risk for developing disease, whereas others (e.g., DQ6, DQB1*06:02) lead to dominant protection. MHC class II molecules function to present processed antigens to T cells, and the MHC class II−peptide−T cell receptor (TCR) forms a trimolecular complex involving the presentation of self-peptides that shape autoreactive T cell responses in autoimmunity. The goal of our studies is to bridge the gap of knowledge in diabetes-protective MHC class II molecules and insulin-specific T cell responses. Data from the last funding period indicates that T1D protective MHC class II molecules (murine IAb ≈ human DQ6) present insulin, and specifically insulin B chain amino acids 9-23 (B:9-23) to activate CD4 T cells with a regulatory phenotype (Tregs). In the NOD mouse model of spontaneous autoimmune diabetes, insulin-specific FoxP3+ Tregs are present but fail to prevent diabetes onset. The presence of IAb in addition to the NOD diabetes conferring IAg7 MHC class II abrogates all diabetes development. With fluorescent B:9-23 tetramers on each class II molecule, we are able to detect B:9-23/IAb type 1 regulatory T cells (Tr1 cells) in the pancreatic lymph nodes in NOD mice heterozygous for IAb/IAg7, while insulin-IAg7 T cells are less frequent and not activated. Similarly in non-diabetic humans with DQ6, we are able to proliferate Tregs from the peripheral blood that respond to insulin B:9-21/DQ6. Single cell RNA sequencing of these proliferated insulin-Tregs reveals a distinct Tr1 cluster that secrete the anti-inflammatory cytokine, IL-10. However, the necessity for these insulin-Tregs and mechanisms by which these cells confer diabetes resistance remains to be determined. In specific aim 1, we will determine the molecular phenotype and necessity for insulin-Tregs to protect against diabetes development in murine models with a diabetes-resistant MHC class II molecule. Specific aim 2 focuses on parallel studies in humans to identify the protective features of insulin-Tregs restricted to DQ6 and those to DQ8 in individuals with and without T1D. The successful completion of this proposal will provide insights into the function and molecular phenotype of insulin-Tregs restricted to diabetes-protective MHC class II molecules and determine their direct role in conferring diabetes protection, thus aiding the design of therapies that may improve Treg function to treat the underlying autoimmunity in T1D.