MR1 presents microbially derived riboflavin precursors to a variety of αβ and γδ MR1-restricted T cells, including MAIT cells. A key question is whether MR1 ligands can originate from the host, particularly in contexts of tissue homeostasis and cancers, and how this impacts T cell functions. We have established integrated experimental and computational approaches, including unbiased mass spectrometry, machine learning models, and in-silico cheminformatics, which we utilized to discover novel host-derived MR1-binding ligands. To understand the mechanisms by which MR1 binds and presents these self-ligands, we have determined the crystal structures of MR1 in complex with several antigens, including pyridoxal vitamers and sulfated bile acids (e.g., cholic acid 7-sulfate; CA7S), along with self-pyrimidine and purine nucleobases like 5-formyl-2'-deoxyuridine (5-FdU) and xanthosine. Our structural investigations reveal that these self-antigens exhibit distinct binding modes within the MR1 cleft, stabilized by electrostatic interactions involving MR1-Lys43, Arg9, and Arg94 residues, alongside hydrophobic interactions within the aromatic cradle of the MR1 A′-pocket. Here, I will provide insights into this novel pipeline for MR1 antigen discovery and the molecular mechanisms underlying the recognition of host-derived antigens by MR1. This structural data will provide a foundation for the development of structural analogues for future therapeutic interventions.