Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens

Abstract

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

CD36 family members are TCR-independent ligands for CD1 antigen-presenting molecules

“…To establish the identity of these CD1-binding ligand(s), we next performed a genome-wide CRISPR/Cas9-based library screen using C1R cells, which exhibited the highest reactivity to CD1c, and which have previously been used in a similar approach to identify genes that regulate surface expression of MR1 45 . After two rounds of FACS-purification of CD1c-endo tetramer - C1R cells (ie.…”

“…CRISPR library screening was performed as previously described 71 following the Zheng lab protocol (Broad institute, MIT). In brief, the human GeCKO v1 sgRNA library was lentivirally transduced into C1R cells in 3 biological replicate libraries as previously described 45 . Each of the 3 libraries were split into two, and half stained with CD1c tetramers and tetramer-negative cells enriched using FACS.…”

Pathophysiological Roles of Mucosal-Associated Invariant T Cells in the Context of Gut Microbiota-Liver Axis

“…Binding with MR1 of antigen-presenting cells (APC) stabilizes the otherwise unstable riboflavin intermediates, which facilitates their transport to the TCR receptor on the surface of MAIT cells to activate antimicrobial responses. Loading of the soluble ligands onto the MR1 molecule has been reported to occur mainly in the endoplasmic reticulum and cell surface [ 11 , 12 ]. The MR1-restriction of MAIT cell activation was further confirmed by demonstrating a lower susceptibility of MR1-deficient mice to bacterial infections as well as the loss of MAIT cell-stimulating ability of bacteria lacking riboflavin biosynthesis enzymes [ 13 , 14 ].…”

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

“…Cells were first incubated for 10 min on ice with Fcreceptor block (BD Pharmingen), followed by CD1d-ClPPBF tetramers for 30min. Where described, CD1d-ClPPBF tetramer + cells were then enriched by TAME, using anti-phycoerythrin (PE) magnetic beads and LS columns (Miltenyi Biotec), followed by surface antibody staining and/or by http://n2t.net/addgene:52961; RRID:Addgene_52961) as described 59 . Cells were transduced, selected based on puromycin resistance, then single-cell cloned.…”

“…To knock out β2m in SKW-3 cells, the short guide RNAs (sgRNAs) targeting the signaling peptide (5’-GAGTAGCGCGAGCACAGCTA - 3’) was cloned into lentiCRISPR v2 (a gift from Feng Zhang; Addgene plasmid # 52961; http://n2t.net/addgene:52961; RRID:Addgene_52961) as described 59 . Cells were transduced, selected based on puromycin resistance, then single-cell cloned.…”