Project Summary Celiac disease (CeD) is a highly prevalent autoimmune disorder that occurs in response to the ingestion of wheat or related cereal grains in individuals expressing HLA-DQ2 or HLA-DQ8. CeD primarily presents in the small intestine with abdominal symptoms, but can also be associated with a myriad of extraintestinal complications and an increased risk for malignancies of the gastrointestinal tract. Despite decades of study, there are as of yet no approved medical therapies for CeD besides gluten-free diet. The life-altering symptoms, lack of treatment options, and risks that come with unmanaged disease underscore the desperate need for a deeper understanding of CeD pathogenesis. The prototypical inflammation in CeD is mediated by CD4+ T cells in response to wheat-derived gluten peptides that have been chemically modified by the enzyme transglutaminase 2 (TG2). Preliminary work in the Khosla lab has revealed that TG2 is involved in a novel gluten peptide internalization pathway involving interaction between a peptide-TG2 pair and the abundant serum protein a-2-macroglobulin (a2M). Notably, this internalization phenomenon only occurs if TG2 is bound to peptide substrate and not if TG2 is unbound or bound to non-peptide inhibitors. As a result, we believe this novel pathway may present a mechanism by which gluten peptides are internalized and subsequently presented on antigen presenting cells to CD4+ T cells. In order to explore this hypothesis, we first aim to develop a panel of novel peptidomimetic chemical inhibitors and fluorescent probes of TG2. These will enable tracking of the enzyme both throughout its catalytic cycle as well as during internalization events. We then plan to test the physiological relevance of this pathway through in vitro reconstitution with dendritic cells (DCs) and CD4+ T cells purified from CeD mice. Coculture of T cells with DCs preincubated with peptide, TG2, and a2M followed by measurement of T cell activation will allow us to determine if this mechanism contributes to the inflammatory response of CeD. Taken together, these aims seek to greater characterize the T cell response central to CeD pathogenesis. This study will provide new insight into potential targets for CeD treatment that intercept this putative gluten internalization pathway.