Lightning Talk & Poster Presentation CD1-MR1 Workshop 2025

Small molecules from conserved metabolic pathways form alternate 5-A-RU-based MR1 ligands that are recognised by MAIT cells (#19)

Michael NT Souter 1 , Lars Kjer-Nielsen 1 , Clarice Lee 1 , Lucy J Meehan 1 , Yang Kang 1 , Songyi Li 1 , Adam G Nelson 1 , Lisa Ciacchi 2 , Conor McNeice 2 , Lyudmila Kostenko 1 , Bronwyn S Meehan 1 , Chu-Han Huang 1 , Ching-Seng Ang 3 , Thanh Nguyen 4 , Thi HO Nguyen 1 , Katherine Kedzierska 1 , Sidonia BG Eckle 1 , Spencer J Williams 4 , Jeffrey YW Mak 5 , David P Fairlie 5 , Zhenjun Chen 1 , Jamie Rossjohn 2 6 7 , Alexandra J Corbett 1 , Wael Awad 2 6 , James McCluskey 1
  1. Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  2. Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  3. Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, Victoria, Australia
  4. School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia
  5. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
  6. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia
  7. Institute of Infection and Immunity, Cardiff University, School of Medicine, Cardiff, United Kingdom

Mucosal-associated invariant T (MAIT) cells recognise riboflavin metabolites presented by the major histocompatibility complex related protein 1 (MR1). Currently, the most potent known MAIT cell antigen is 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), an adduct of the riboflavin precursor 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal, a small molecule by-product of cellular respiration. MAIT cells express a semi-invariant TCRa chain that is specifically tuned for docking on MR1 and recognising 5-OP-RU, however, diversity within the TCR repertoire suggests some MAIT cells can also recognise other metabolite antigens. The breadth to which 5-A-RU, or other molecular scaffolds can form metabolite antigens that modulate the MAIT-MR1 axis is unclear.

 

Here, we considered small molecules from diverse metabolic pathways in the genesis of 5-A-RU-based ligands. We used an MR1 refolding assay to capture ligands produced in the presence of 5-A-RU and small molecules from ascorbate degradation, lactic acid fermentation, lipid peroxidation and glycolysis. Complementary structural analysis, mass spectrometry and chemical synthesis confirmed the identity of novel 5-A-RU-based ligands. Using MR1 tetramers, we demonstrated the antigenicity of these ligands to circulating MAIT cell from healthy blood donors. Notably, we identified clonally expanded MAIT cells that expressed atypical TCRa and TCRb chains that facilitated strong antigen cross-reactivity. Our findings indicate that diversity within the MAIT TCR repertoire sponsors cross-reactivity to distinct riboflavin-based antigens, and that encounter with alternate antigens may influence the expansion of MAIT clonotypes during certain physiological settings.