Introduction:
The first antigens presented by MR1 to be discovered were derived from bacterial vitamin B biosynthesis pathways. Later cellular studies discovered MR1-restricted T (MR1T) cells to be activated by endogenously derived metabolites presented by MR1, however, these compounds have yet to be identified. In 2024, the first ligands of mammalian origin presented by MR1, namely sulphated cholic acid derivatives and modified nucleobases, were characterised. The diverse chemical classes and metabolic pathways of these compounds emphasise the plasticity of MR1’s binding groove for stabilising a broad range of metabolites, many of which are likely still to be elucidated.
A recent lipidomics approach significantly increased the library of identified CD1-lipid binders, highlighting the potential for metabolomics in novel MR1 ligand discovery. Here, we developed an untargeted and a targeted metabolomics workflow for the analysis of mammalian-derived MR1 ligands.
Methods:
An ultra-high-performance liquid chromatography (UHPLC) with mass spectrometry (MS) detection method was developed for both untargeted and targeted metabolomic analysis of MR1-bound metabolites. Each of the untargeted and targeted workflows used both reverse-phase (RP) and hydrophilic interaction chromatography (HILIC) liquid chromatography separation techniques to ensure the detection of a wide range of ligand chemical classes.
MR1 from the monocytic cell line C1R and the B-lymphocytic cell line THP1 was immunoprecipitated from the cell lysates. Metabolites bound to immunoprecipitated MR1 were eluted and run on the developed metabolomics workflows.
Results:
Ligand-containing eluents were analysed with targeted and untargeted analysis workflows, providing insights into the ligandome of MR1. These results support MR1 presentation of endogenously-derived metabolites.