MHC-I related protein 1 (MR1) is best known for presenting microbial-derived vitamin B2 metabolites to mucosal-associated invariant T (MAIT) cells, facilitating microbial surveillance. Other MR1-reactive T (MR1T) cells, which express a diverse TCR repertoire and phenotype have also been described. Recent discoveries suggest that antigens of MR1T cells may originate from various host and microbial metabolic pathways (other than riboflavin) and that MR1 may elicit immune responses in a greater variety of pathological conditions than previously appreciated. However, the biology of MR1T cells remains poorly understood, with a significant challenge being the discovery of bona fide MR1T cell antigens.
To accelerate the discovery of MR1T cell antigens, we have developed an antigen discovery platform to capture small molecules from microbial culture medium and supernatant extracts using soluble MR1 proteins for analysis and validation. We confirmed the stimulatory capacity of soluble MR1 proteins bound with undefined ligands using a panel of previously identified MR1T cell lines. Putative MR1T cell antigens were then eluted from MR1 for mass spectrometry analysis and compound identification. To test the physiological antigenicity of identified ligands, we isolated primary MR1T cells using ligand-loaded MR1 tetramers from healthy peripheral blood mononuclear cells (PBMCs) and performed functional characterisation.
Our preliminary findings demonstrate that brain heart infusion (BHI) media derived from cow is enriched for small molecules that form stable complexes with MR1. Among these, we identified a novel nucleobase-adduct related to previously described tumour-associated MR1 ligands, which potently stimulated primary human MR1T cells. These findings suggest that non-neoplastic mammalian tissue can serve as a source of MR1T cell antigens, although the context in which these antigens are presented and become antigenic to MR1T cells in vivo remains an area of active investigation. Building on these insights, we expect that MR1T cells survey dysregulated cellular metabolism in the setting of infection, autoimmunity, and cancer.