In Vitro and In Vivo Analysis of the Gram-Negative Bacteria–Derived Riboflavin Precursor Derivatives Activating Mouse MAIT Cells

Abstract

Mucosal-associated invariant T (MAIT) cells recognize microbial compounds presented by the MHC-related 1 (MR1) protein. Although riboflavin precursor derivatives from Gram-positive bacteria have been characterized, some level of ligand heterogeneity has been suggested through the analysis of the MAIT cell TCR repertoire in humans and differential reactivity of human MAIT cell clones according to the bacteria. In this study, using Gram-negative bacteria mutated for the riboflavin biosynthetic pathway, we show a strict correlation between the ability to synthesize the 5-amino-ribityl-uracil riboflavin precursor and to activate polyclonal and quasi-monoclonal mouse MAIT cells. To our knowledge, we show for the first time that the semipurified bacterial fraction and the synthetic ligand activate murine MAIT cells in vitro and in vivo. We describe new MR1 ligands that do not activate MAIT cells but compete with bacterial and synthetic compounds activating MAIT cells, providing the capacity to modulate MAIT cell activation. Through competition experiments, we show that the most active synthetic MAIT cell ligand displays the same functional avidity for MR1 as does the microbial compound. Altogether, these results show that most, if not all, MAIT cell ligands found in Escherichia coli are related to the riboflavin biosynthetic pathway and display very limited heterogeneity.

Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells

“…Furthermore, they were identical to the ligands recovered from recombinant MR1 assembled with supernatant from S. Typhimurium, L. lac-tis, and E. coli, as determined by high resolution LC-MS (29). Other researchers have since also confirmed the identity of these MAIT cell-activating ligands, 5-OP-RU and 5-OE-RU (30). The structural features of their binding revealed the basis for the capture of these intermediates by MR1 as discussed below (37).…”

“…1A), prevented the production of activating MAIT cell ligands by these bacteria (29). This finding was recently verified in mutants of E. coli (30). Although 5-A-RU plays an important role in MAIT cell activation, MR1 could not be refolded efficiently with 5-A-RU alone (29,31).…”

Mucosal-associated invariant T cell–rich congenic mouse strain allows functional evaluation

“…The larger conventional T cell infiltration in the bladder during infection in the absence of MAITs may be related to higher bacterial load or to a direct effect of MAITs on MAIT stimulation with bacteria-derived compound. The preparation of the SPB was previously described (49). Briefly, the bacterial lysate was prepared by growing E. coli, DH5α ATCC strain, to saturation.…”

“…One AU corresponds to 25 μl bacterial supernatant. 5-A-RU synthesis and 5-OP-RU preparation have been described elsewhere (49 ten-week-old B6-MAIT CAST female mice were anesthetized with ketamine (125 mg/kg) and xylazine (12.5 mg/kg) intraperitoneally, and a 24-gauge catheter (BD Insyte Autoguard, BD) containing either PBS or UTI89 was inserted through the urethra; 50 μl of bacterial suspension or PBS was instilled at a slow rate to avoid vesicoureteral reflux. At indicated time points, bladders, spleens, and bladder-draining LNs were harvested.…”

Lights on MAIT cells, a new immune player in liver diseases

“…Indeed they recognize vitamin B metabolites from commensal or pathogenic bacteria presented by the highly conserved major histocompatibility complex class I-related molecule (MR1) [9]. Bacteria lacking the vitamin B2 (riboflavin) synthesis pathway are unable to activate MAIT cells through their specific TCR [9][10][11][12]. IL-12 and IL-18 cytokines can also activate MAIT cells in a TCR-independent manner since these innate-like T cells express high level of IL-12 and IL-18 receptors [3,7,13,14].…”