Synthesis, stabilization, and characterization of the MR1 ligand precursor 5-amino-6-D-ribitylaminouracil (5-A-RU)

Abstract

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminouracil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Mucosal-associated invariant and γδ T cell subsets respond to initial Mycobacterium tuberculosis infection

“…Differential activation potentials of MAIT cell subsets. To profile the abundance and activation status of MAIT cells in household contacts and controls, we used 5-OP-RU-loaded MR1 tetramers to identify MAIT cells and deployed an ex vivo activation assay with MR1 ligand, 5-A-RU/MeG (35,49). MAIT cells were identified as tetramer + CD161 ++ (Figure 2A).…”

“…This latter study did observe an enrichment of CD4 + /Vα7.2 + /CD161 ++ cells in LTBI subjects compared with active TB cases, but the identity of these cells was unknown. The availability of MR1-5-OP-RU tetramers (27,35,52) has allowed for accurate identification of MAIT cell subsets, highlighting the importance of subset heterogeneity that may not be easily detected without also staining for TCR coreceptors (51, 52). Kurioka et al (51) assessed functional differences between CD4 + , CD8 + , and DN MAIT cells in healthy donors, demonstrating that CD8 + and DN MAIT cells were more reactive and polarized toward a Th1 phenotype with E. coli activation, whereas CD4 + MAIT cells were biased toward a Th2 phenotype, with higher levels of intracellular IL-4 and IL-13 detected by intracellular staining.…”

“…MAIT cell ex vivo activation assay. Synthetic 5-A-RU was synthesized as previously described (35) (35). PBMCs were stained with Zombie Red Fixable Viability Dye (BioLegend, catalog 423109) and antibodies to CD3 (UCHT1; Alexa700; eBioscience; Thermo Fisher Scientific, catalog 16-0038-81), CD161 (DX12; APC; BD Biosciences, catalog 550968), CD69 (FN50; BV421; Bio-Legend, catalog 310929), CD25 (BC96; BV711; BioLegend, catalog 302635), PD-1 (EH12.1; BV650; BD Biosciences, catalog 564104), CD4 (SK3; PerCPefluor or BV650; eBioscience; Thermo Fisher Scientific, catalog 46-0047-41, or BD Biosciences, catalog 563876) and CD8 (SK1; APC-H7; BD Biosciences, catalog 560179), Vγ24Jα18 (6B11; BV510; BioLegend, catalog 342917) and TCR γδ (B1; FITC; BioLegend, catalog 331207), and MR1-5-OP-RU tetramers (NIH tetramer core facility; PE or BV421) for 15 minutes at room temperature in the dark.…”

“…However, the presence of MR1-reactive TRAV1-2 + CD161 ++ cells in fetal tissue also suggests microbiota-independent mechanisms for MAIT cell selection using endogenous MR1 ligands (33). After stimulation with MR1-presented ligand, MAIT cells are rapidly activated (21,23,34,35) and can secrete IFNγ, TNFα, and IL-17 and release granzyme B/perforin (21,35,36); however, their specific roles during Mtb infection is not well understood (37)(38)(39)(40).…”

Efficient 5-OP-RU-induced enrichment of Mucosal-associated invariant T cells in the murine lung does not enhance control of aerosolMycobacterium tuberculosisinfection

“…In vivo modeling of MAIT cell responses against Mtb have been limited by low MAIT cell abundance in specific pathogen free (SPF) mice (<1% of T cells) relative to the generally more abundant MAIT cell populations observed in humans (<1-18% of T cells) (2,26,27). Despite low abundance of MAIT cell populations in SPF mice, several recent studies demonstrate that intranasal priming with bacteria or synthetic MR1 ligands can induce robust MAIT cell accumulation in the lung (27)(28)(29)(30)(31).…”