Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC Class I related-1 protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here we determine the crystal structures of two mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognise human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction, and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Sulfated bile acid is a host-derived ligand for MAIT cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function.

Dynamic MAIT Cell Recovery after Severe COVID-19 Is Transient with Signs of Heterogeneous Functional Anomalies.

Mucosal-associated invariant T (MAIT) cells are an abundant population of unconventional T cells in humans and play important roles in immune defense against microbial infections. Severe COVID-19 is associated with strong activation of MAIT cells and loss of these cells from circulation. In the present study, we investigated the capacity of MAIT cells to recover after severe COVID-19. In longitudinal paired analysis, MAIT cells initially rebounded numerically and phenotypically in most patients at 4 mo postrelease from the hospital. However, the rebounding MAIT cells displayed signs of persistent activation with elevated expression of CD69, CD38, and HLA-DR. Although MAIT cell function was restored in many patients, a subgroup displayed a predominantly PD-1high functionally impaired MAIT cell pool. This profile was associated with poor expression of IFN-γ and granzyme B in response to IL-12 + L-18 and low levels of polyfunctionality. Unexpectedly, although the overall T cell counts recovered, normalization of the MAIT cell pool failed at 9-mo follow-up, with a clear decline in MAIT cell numbers and a further increase in PD-1 levels. Together, these results indicate an initial transient period of inconsistent recovery of MAIT cells that is not sustained and eventually fails. Persisting MAIT cell impairment in previously hospitalized patients with COVID-19 may have consequences for antimicrobial immunity and inflammation and could potentially contribute to post-COVID-19 health problems.

Synthetic 5-amino-6-D-ribitylaminouracil paired with inflammatory stimuli facilitates MAIT cell expansion in vivo.

Introduction: Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which mediate host immunity to microbial infection by recognizing metabolite antigens derived from microbial riboflavin synthesis presented by the MHC-I-related protein 1 (MR1). Namely, the potent MAIT cell antigens, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), form via the condensation of the riboflavin precursor 5-amino-6-D-ribitylaminouracil (5-A-RU) with the reactive carbonyl species (RCS) methylglyoxal (MG) and glyoxal (G), respectively. Although MAIT cells are abundant in humans, they are rare in mice, and increasing their abundance using expansion protocols with antigen and adjuvant has been shown to facilitate their study in mouse models of infection and disease. Methods: Here, we outline three methods to increase the abundance of MAIT cells in C57BL/6 mice using a combination of inflammatory stimuli, 5-A-RU and MG. Results: Our data demonstrate that the administration of synthetic 5-A-RU in combination with one of three different inflammatory stimuli is sufficient to increase the frequency and absolute numbers of MAIT cells in C57BL/6 mice. The resultant boosted MAIT cells are functional and can provide protection against a lethal infection of Legionella longbeachae. Conclusion: These results provide alternative methods for expanding MAIT cells with high doses of commercially available 5-A-RU (± MG) in the presence of various danger signals.

Histone deacetylase 7: a signalling hub controlling development, inflammation, metabolism and disease.

Histone deacetylases (HDACs) catalyse removal of acetyl groups from lysine residues on both histone and non-histone proteins to control numerous cellular processes. Of the 11 zinc-dependent classical HDACs, HDAC4, 5, 7 and 9 are class IIa HDAC enzymes that regulate cellular and developmental processes through both enzymatic and non-enzymatic mechanisms. Over the last two decades, HDAC7 has been associated with key roles in numerous physiological and pathological processes. Molecular, cellular, in vivo and disease association studies have revealed that HDAC7 acts through multiple mechanisms to control biological processes in immune cells, osteoclasts, muscle, the endothelium and epithelium. This HDAC protein regulates gene expression, cell proliferation, cell differentiation and cell survival and consequently controls development, angiogenesis, immune functions, inflammation and metabolism. This review focuses on the cell biology of HDAC7, including the regulation of its cellular localisation and molecular mechanisms of action, as well as its associative and causal links with cancer and inflammatory, metabolic and fibrotic diseases. We also review the development status of small molecule inhibitors targeting HDAC7 and their potential for intervention in different disease contexts.

Quantitative affinity measurement of small molecule ligand binding to major histocompatibility complex class-I-related protein 1 MR1.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC50 < 1 µM), moderate (1 µM < IC50 < 100 µM), and weak (IC50 > 100 µM). We demonstrated a clear correlation between ligand-binding affinity for MR1, the presence of a covalent bond between MR1 and ligand, and the number of salt bridge and hydrogen bonds formed between MR1 and ligand. Using this newly developed fluorescence polarization-based assay to screen for candidate ligands, we identified the dietary molecules vanillin and ethylvanillin as weak bona fide MR1 ligands. Both upregulated MR1 on the surface of C1R.MR1 cells and the crystal structure of a MAIT cell T cell receptor-MR1-ethylvanillin complex revealed that ethylvanillin formed a Schiff base with K43 of MR1 and was buried within the A'-pocket. Collectively, we developed and validated a method to quantitate the binding affinities of ligands for MR1 that will enable an efficient and rapid screening of candidate MR1 ligands.

A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells.

MR1 is a highly conserved microbial immune-detection system in mammals. It captures vitamin B-related metabolite antigens from diverse microbes and presents them at the cell surface to stimulate MR1-restricted lymphocytes including mucosal-associated invariant T (MAIT) cells. MR1 presentation and MAIT cell recognition mediate homeostasis through host defense and tissue repair. The cellular mechanisms regulating MR1 cell surface expression are critical to its function and MAIT cell recognition, yet they are poorly defined. Here, we report that human MR1 is equipped with a tyrosine-based motif in its cytoplasmic domain that mediates low affinity binding with the endocytic adaptor protein 2 (AP2) complex. This interaction controls the kinetics of MR1 internalization from the cell surface and minimizes recycling. We propose MR1 uses AP2 endocytosis to define the duration of antigen presentation to MAIT cells and the detection of a microbial metabolic signature by the immune system.

CD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells.

Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.

Tuning Electrostatic and Hydrophobic Surfaces of Aromatic Rings to Enhance Membrane Association and Cell Uptake of Peptides.

Aromatic groups are key mediators of protein-membrane association at cell surfaces, contributing to hydrophobic effects and π-membrane interactions. Here we show electrostatic and hydrophobic influences of aromatic ring substituents on membrane affinity and cell uptake of helical, cyclic and cell penetrating peptides. Hydrophobicity is important, but subtle changes in electrostatic surface potential, dipoles and polarizability also enhance association with phospholipid membranes and cell uptake. A combination of fluorine and sulfur substituents on an aromatic ring induces microdipoles that enhance cell uptake of 12-residue peptide inhibitors of p53-HDM2 interaction and of cell-penetrating cyclic peptides. These aromatic motifs can be readily inserted into peptide sidechains to enhance their cell uptake.

Determination of Sample Concentrations by PULCON NMR Spectroscopy.

PULCON (Pulse Length Based Concentration Determination) is a powerful, versatile, non-invasive, and accurate technique for measuring solution concentrations during routine NMR spectroscopy. As solutes are quantified directly by their unique resonances, this technique avoids weight-based errors caused by contaminants (e.g. moisture), allows NMR samples to be directly employed in biological assays, and is particularly useful for quantifying small molecules, peptides, unstable molecules, and other materials that are difficult to weigh or handle. This article provides an introductory guide for biological and medicinal chemists, and highlights the diversity of applications.

Differential location of NKT and MAIT cells within lymphoid tissue.

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αß T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

MAIT cells regulate NK cell-mediated tumor immunity.

The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.

Chemical Modulators of Mucosal Associated Invariant T Cells.

Over the past decade, we have contributed to the chemistry of microbial natural products and synthetic ligands, related to riboflavin and uracils, that modulate immune cells called mucosal associated invariant T cells (MAIT cells). These highly abundant T lymphocytes were only discovered in 2003 and have become recognized for their importance in mammalian immunology. Unlike other T cells, MAIT cells are not activated by peptide or lipid antigens. In collaboration with immunology and structural biology research groups, we discovered that they are instead activated by unstable nitrogen-containing heterocycles synthesized by bacteria. The most potent naturally occurring activating compound (antigen) is 5-(2-oxopropylideneamino)-d-ribitylaminouracil (5-OP-RU). This compound is an imine (Schiff base) formed through condensation between an intermediate in the biosynthesis of riboflavin (vitamin B2) and a metabolic byproduct of mammalian and microbial glycolysis. Although it is very unstable in water due to intramolecular ring closure or hydrolysis, we were able to develop a non-enzymatic synthesis that yields a pure kinetically stable compound in a nonaqueous solvent. This compound has revolutionized the study of MAIT cell immunology due to its potent activation (EC50 = 2 pM) of MAIT cells and its development into immunological reagents for detecting and characterizing MAIT cells in tissues. MAIT cells are now linked to key physiological processes and disease, including antibacterial defense, tissue repair, regulation of graft-vs-host disease, gastritis, inflammatory bowel diseases, and cancer. 5-OP-RU activates MAIT cells and, like a vaccine, has been shown to protect mice from bacterial infections and cancers. Mechanistic studies on the binding of 5-OP-RU to its dual protein targets, the major histocompatibility complex class I related protein (MR1) and the MAIT cell receptor (MAIT TCR), have involved synthetic chemistry, 2D 1H NMR spectroscopy, mass spectrometry, computer modeling and molecular dynamics simulations, biochemical, cellular, and immunological assays, and protein structural biology. These combined studies have revealed structural influences for 5-OP-RU in solution on protein binding and antigen presentation and potency; informed the development of potent (EC50 = 2 nM) and water stable analogues; led to fluorescent analogues for detecting and tracking binding proteins in and on cells; and enabled discovery of drugs and drug-like molecules that bind MR1 and modulate MAIT cell function. MAIT cells offer new opportunities for chemical synthesis to enhance the stability, potency, selectivity, and bioavailability of small molecule ligands for MR1 or MAIT TCR proteins, and to contribute to the understanding of T cell immunity and the development of prospective new immunomodulating medicines.

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

Mucosal-Associated Invariant T Cell Effector Function Is an Intrinsic Cell Property That Can Be Augmented by the Metabolic Cofactor α-Ketoglutarate.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of unconventional T cells that respond rapidly to microbial metabolite Ags or cytokine stimulation. Because of this reactivity and surface expression of CD45RO+, CD45RA-, and CD127+, they are described as effector memory cells. Yet, there is heterogeneity in MAIT cell effector response. It is unclear what factors control MAIT cell effector capacity, whether it is fixed or can be modified and if this differs based on whether activation is TCR dependent or independent. To address this, we have taken a systematic approach to examine human MAIT cell effector capacity across healthy individuals in response to ligand and cytokine stimulation. We demonstrate the heterogenous nature of MAIT cell effector capacity and that the ability to produce an effector response is not directly attributable to TCR clonotype or coreceptor expression. Global gene transcription analysis revealed that the MAIT cell effector capacity produced in response to TCR stimulation is associated with increased expression of the epigenetic regulator lysine demethylase 6B (KDM6B). Addition of a KDM6B inhibitor did not alter MAIT cell effector function to Ag or cytokine stimulation. However, addition of the KDM6B cofactor α-ketoglutarate greatly enhanced MAIT cell effector capacity to TCR-dependent stimulation in a partially KDM6B-dependent manner. These results demonstrate that the TCR-dependent effector response of MAIT cells is epigenetically regulated and dependent on the availability of metabolic cofactors.

HDAC7 Inhibition by Phenacetyl and Phenylbenzoyl Hydroxamates.

The zinc-containing histone deacetylase enzyme HDAC7 is emerging as an important regulator of immunometabolism and cancer. Here, we exploit a cavity in HDAC7, filled by Tyr303 in HDAC1, to derive new inhibitors. Phenacetyl hydroxamates and 2-phenylbenzoyl hydroxamates bind to Zn2+ and are 50-2700-fold more selective inhibitors of HDAC7 than HDAC1. Phenylbenzoyl hydroxamates are 30-70-fold more potent HDAC7 inhibitors than phenacetyl hydroxamates, which is attributed to the benzoyl aromatic group interacting with Phe679 and Phe738. Phthalimide capping groups, including a saccharin analogue, decrease rotational freedom and provide hydrogen bond acceptor carbonyl/sulfonamide oxygens that increase inhibitor potency, liver microsome stability, solubility, and cell activity. Despite being the most potent HDAC7 inhibitors to date, they are not selective among class IIa enzymes. These strategies may help to produce tools for interrogating HDAC7 biology related to its catalytic site.

MR1-Restricted T Cells with MAIT-like Characteristics Are Functionally Conserved in the Pteropid Bat Pteropus alecto.

Bats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

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

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.