MR1-Ligand Cross-Linking Identifies Vitamin B6 Metabolites as MAIT-TCR-Reactive Antigens

Abstract

MR1 plays a central role in immune recognition of infected cells and cancer. However, knowledge of the nature of the ligands that are presented by MR1 and recognized by MR1-restricted T cells is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. We here investigate Schiff-base bound MR1 ligands via cross-linking mass spectrometry. Our approach employs reductive amination chemistry to stabilize the labile Schiff-base bond between metabolite and the MR1-K43 residue, followed by enzymatic release of the peptide covalently linked to the metabolite. The resulting mass shift on the proteolytically released, ligand-modified peptide is measured by LC-MS-based proteomics and enables identification of the MR1-associated small molecule ligands. As proof-of-concept for our approach, we showcase de novo detectability of Acetyl-6-formylpterin (Ac-6-FP) in refolded MR1-Ac-6-FP complexes, and in situ membrane-bound single-chain MR1 (scMR1) extracted from an Ac-6-FP-pulsed cell line. We then apply our approach to scMR1-overexpressing A549 cells for cancer ligand discovery and identify vitamin B6 vitamers pyridoxal and pyridoxal-5-phosphate (PLP) as bound to scMR1. Pyridoxal and PLP upregulate MR1 on pulsed A549 wildtype cells and are recognized by T cells expressing A-F7, a TRAV1-2 mucosal-associated invariant T-cell (MAIT) T-cell receptor, in a dose dependent manner. Finally, pyridoxal activates primary CD8+ T cells expressing the A-F7 receptor. Our work highlights pyridoxal and to a lesser degree PLP as immunogenic MR1 ligands and discusses their potential role in immune recognition and regulation in cancer.

Keywords: MR1, MAIT, vitamin B6, pyridoxal, T cell, antigen presentation, crosslinking mass spectrometry