Data were collected at the Australian Synchrotron Facility, processed and refined with standard software packages. NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking. Mucosal-Associated Invariant T (MAIT) cells are a subset of evolutionarily conserved nonmajor histocompatibility complex (MHC)-restricted T cells, which are very abundant in human mucosal tissues, in peripheral blood, and in the liver (1, 2). Similar to type I NKT cells, human MAIT cells express a semi-invariant T cell receptor (TCR) composed of the V7.2 chain rearranged mainly to J33 and paired with a limited number of V chains, mostly TRBV6, TRBV13, and TRBV20 (3, 4). MAIT cells recognize small microbial metabolites presented by the monomorphic MHC class I-related molecule, MR1 (1, 2). The physiological roles of MAIT cells remain unclear, but they are known to be involved in protective immunity (2, 5C7), possibly through modulation of Taribavirin hydrochloride innate and adaptive immune responses (8, 9). Moreover, the role of MAIT cells in cancer (10) and inflammatory diseases, such as obesity (11), diabetes (12), multiple sclerosis (13), and inflammatory bowel disease (14), has been highlighted, and recent reports have suggested they may Taribavirin hydrochloride also play a role in tissue repair (15, 16). Activation of MAIT cells induces the production of various proinflammatory cytokines, predominantly IFN-, TNF-, IL-2, and IL-17 (17, 18), and their potent cytolytic activity allows them to kill infected cells (19). Unlike MHC molecules, MR1 does not constitutively present antigens, but is found in the endoplasmic reticulum (ER) of all cells in a ligand-receptive conformation (20). The potency of known MAIT cell agonists appears to correlate with their ability to form a Schiff base with MR1 Lys43 located within the A-pocket, thus allowing MR1 to egress to the cell surface, where the presence of a ribityl moiety in the covalently bound agonist allows for an interaction with the MAIT TCR (21C23). To date, the strongest MAIT cell agonists are 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), both pyrimidine-based intermediates along the riboflavin biosynthetic pathway (24). Several bacterial and fungal species synthesize Rabbit polyclonal to GNMT riboflavin (23), and MAIT Taribavirin hydrochloride cells have been shown to possess MR1-dependent antimicrobial activity against infected antigen-presenting cells (5, 6). Conversely, vitamin B9 metabolites [including the folic acid derivative 6-formylpterin, 6-FP and its acetylated derivative Ac-6-FP (23, 25)] are strong MR1 binders and induce MR1 expression at the cell surface; however, the resulting complexes do not activate MAIT cells because they lack the ribityl moiety (22). Drug and drug-like molecules (including diclofenac and salicylates) also bind MR1 and either weakly activate or inhibit MAIT cells (26). However, it remains unknown whether there are other ligands that impact MR1-dependent antigen presentation. Through an in silico screen, we have identified additional MR1-binding ligands. We describe a ligand that down-regulates MR1 cell-surface expression and provide a molecular basis for its interactions with MR1. Results Identification of Nonmicrobial MAIT Cell Agonists..