Project Summary/Abstract The study of the genetic basis for type 1 diabetes (T1D) has benefited tremendously from examination of rare individuals with likely monogenic forms of the disease. Combined with GWAS, a number of polymorphisms in immune regulatory genes have been defined that contribute to genetic risk for T1D. Using whole exome sequencing of individuals with T1D enrolled in a monogenic diabetes registry, we identified an individual with a gain-of-function mutation in the SKAP2 gene, as well as several other T1D patients with potentially pathogenic variants in other leukocyte integrin signaling genes. These patients tend to have a number of autoimmune manifestations in addition to T1D, indicating defects in critical pathways of immune tolerance. Multiple GWAS studies have identified a strong genetic linkage between SKAP2 polymorphisms and T1D (at a frequency of ~20%), however the mechanisms by which alteration of SKAP2 could lead to autoimmune T1D are unknown. SKAP2 is expressed primarily in myeloid cells, where it functions as an adapter protein in the integrin signaling pathway, linking cell surface integrins to WASP and actin rearrangements that occur following leukocyte adhesion. The SKAP2 G153R mutation in our patient resulted in constitutive association of SKAP2 with WASP leading to a hyperadhesive phenotype in macrophages cultured from the patient or macrophages engineered to contain the SKAP G153R substitution. To understand how activation of leukocyte integrin signaling may contribute to T1D, we have generated knock-in (KI) mice containing the G153R substitution in murine Skap2, on the NOD genetic background. Female NOD.SKAP2 KI mice have a higher incidence and earlier onset of T1D than do NOD.WT animals; male NOD.SKAP2 also develop T1D (incidence ~50%) while male NOD.WT do not develop frank hyperglycemia. Initial analysis of these mice reveals evidence of ongoing inflammation early in life with development of a broad spectrum of auto-reactive antibodies. Dendritic cells from NOD.SKAP2 KI mice have increased antigen presenting activity to islet-specific transgenic T-cells while neutrophils from these mice show evidence of increased integrin signaling. These observations demonstrate that the NOD.SKAP2 KI mice appropriately model the autoimmune T1D disease observed in our patient. The project proposes to complete the analysis of these mice, under the hypothesis that increased cell adhesion in dendritic cells leads to prolonged DC-T cell interactions, which drives selection of auto-reactive T-cell clones leading to development of T1D, associated with broad spectrum autoimmunity. We will test this hypothesis in a variety of adoptive cell transfer experiments, by generation of conditional knock-in mice and by imaging of DC-T cell interactions in the inflamed islets. Similar studies will be performed for other candidate leukocyte integrin signaling mutations identified in the monogenic T1D registry. This study will address whether dys...