MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens.

Major histocompatibility complex class I-related protein 1 (MR1) plays a central role in the immune recognition of infected cells and can mediate T cell detection of cancer. Knowledge of the nature of the ligands presented by MR1 is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. Here, we present a cross-linking strategy to investigate Schiff base-bound MR1 ligands. Our methodology employs reductive amination to stabilize the labile Schiff base bond between MR1 and its ligand, allowing for the detection of ligands as covalent MR1 adducts by mass spectrometry-based proteomics. We apply our approach to identifying vitamin B6 vitamers pyridoxal and pyridoxal 5'-phosphate (PLP) as MR1 ligands and show that both compounds are recognized by T cells expressing either A-F7, a mucosal-associated invariant T (MAIT) cell T cell receptor (TCR), or MC.7.G5, an MR1-restricted TCR reported to recognize cancer cells, highlighting them as immunogenic MR1 ligands.