Ligand-dependent downregulation of MR1 cell surface expression

Abstract

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A′-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.

Antigen Recognition by MR1-Reactive T Cells; MAIT Cells, Metabolites, and Remaining Mysteries

“…Most recently, using an in silico screen based on the MR1 binding pocket, Salio et al identified an intriguing effect of a novel MR1 ligand (3-[(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)formamido]propanoic acid “DB28,” which appeared to decrease, rather than increase cell surface expression of MR1, as well as competitively inhibiting activation of MAIT cells by agonist ligands ( 30 ).…”

“…The lower potency of the related lumazine antigens also appears to be due to their inability to form a Schiff base with the Lys 43 residue. Interestingly, the MR1 downregulating ligand, DB28, was unable to form a Schiff based with the Lys 43 residue ( 30 ). Within MAIT TCRs, there was the conservation of key residues including a highly conserved Tyr at position 95 of the CDR3 loop of the TCR α-chain.…”

Covering All the Bases: Complementary MR1 Antigen Presentation Pathways Sample Diverse Antigens and Intracellular Compartments

“…The role these ligands may play in drug-induced immune modulation through MAIT cell activation or inhibition is not yet clear. Using a similar in silico screen, Salio et al, recently expanded the library of MR1 ligands, including the first molecule that prevents MR1 egress from the ER ( 17 ). Intriguingly, this ligand binds in the MR1 A'-pocket in a non-covalent fashion and prevents MR1 surface translocation and MAIT cell activation in response to canonical ligands ( 17 ).…”

“…This model is consistent with work from McWilliam et al, who showed that re-loading of 6-FP-bound molecules was possible at 37°C but not on ice, indicating a requirement for internalization and recycling for ligand exchange to occur ( 41 ). Similarly, presentation of a set of novel MR1 ligands was reduced in cells over-expressing GPI-linked MR1 compared to those transduced with the WT protein, suggesting that a motif in the MR1 cytoplasmic tail may be required for ligand exchange and loading of some ligands ( 17 ). The notion of post-ER loading of MR1 molecules is further supported by the observation that pre-incubation with 6-FP rendered the subsequent surface expression of MR1-5-OP-RU complexes less sensitive to Brefeldin A (BFA) ( 41 ).…”

“…Since mammalian cells do not express the enzymes of this biosynthetic pathway, riboflavin precursors represent a microbe-derived molecular pattern ( 14 ). Ongoing ligand identification efforts have revealed many more microbial and non-microbial MR1 ligands which comprise both agonists and antagonists of MR1T cell activation ( 9 , 15 – 17 ). Importantly, the number of microorganisms that synthesize riboflavin or other putative MR1 ligands is large and includes many commensal species in addition to pathogens ( 18 , 19 ).…”