Mucosal-associated invariant T (MAIT) cells are an abundant and evolutionarily conserved subset of T cells that bridge innate and adaptive immunity. Their most potent antigen are microbial-derived vitamin B₂ (riboflavin) metabolites, such as 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), presented by the non-polymorphic MR1 molecule. MAIT cells have shown protective roles in a variety of infections, both bacterial and viral, despite the absence of a known virus-derived antigen. This is partly due to their ability to be activated in a TCR-independent manner via cytokine receptors, including those for IL-12 and IL-18.
MAIT cell development is dependent on microbial-derived antigens, and 5-OP-RU alone is sufficient to restore their maturation in germ-free mice. The presence of 5-OP-RU or its precursors has been detected in the serum of healthy individuals. When activated through their TCR, MAIT cells also exhibit tissue-repair and tissue-protective functions, suggesting that low-level activation may be beneficial. Conversely, heightened or chronic activation has been associated with autoimmune diseases. Therefore, measuring antigen abundance and distribution is key to understanding their role and using them as a therapeutic target.
Despite the central importance of 5-OP-RU, its quantitative detection in human fluids or tissues remains technically challenging. Current sensitive methods rely on cell-based assays, which are time-consuming, prone to interference from other bacterial products, and difficult to scale up. Here, we present a straightforward, flow cytometry–based method using a high-affinity soluble MR1-specific TCR-Fc fusion protein. After confirming its specificity against related MR1 ligands, we demonstrate that it can detect 5-OP-RU at picomolar concentrations in biological samples. Critically this method involves careful control for potential weakly binding ligands in media. This method, scalable to large sample cohorts, may enable correlations between MAIT cell activation, microbiota composition, gut integrity, and local inflammation, and beyond that may have potential as a disease biomarker.