Molecule MR1 Research Articles

Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation.

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen presenting cells (APC). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT- TCR stimulatory capacities. Only bacteria species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high-stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T cell subsets can also present riboflavin metabolites to MAIT cells in MR1- restricted fashion. This T-T cell mediated signaling also induced IFN𝛄, TNF and GranzymeB from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.

Insights Into Mucosal-Associated Invariant T Cell Biology From Studies of Invariant Natural Killer T Cells.

Mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cells are innate-like T cells that function at the interface between innate and adaptive immunity. They express semi-invariant T cell receptors (TCRs) and recognize unconventional non-peptide ligands bound to the MHC Class I-like molecules MR1 and CD1d, respectively. MAIT cells and iNKT cells exhibit an effector-memory phenotype and are enriched within the liver and at mucosal sites. In humans, MAIT cell frequencies dwarf those of iNKT cells, while in laboratory mouse strains the opposite is true. Upon activation via TCR- or cytokine-dependent pathways, MAIT cells and iNKT cells rapidly produce cytokines and show direct cytotoxic activity. Consequently, they are essential for effective immunity, and alterations in their frequency and function are associated with numerous infectious, inflammatory, and malignant diseases. Due to their abundance in mice and the earlier development of reagents, iNKT cells have been more extensively studied than MAIT cells. This has led to the routine use of iNKT cells as a reference population for the study of MAIT cells, and such an approach has proven very fruitful. However, MAIT cells and iNKT cells show important phenotypic, functional, and developmental differences that are often overlooked. With the recent availability of new tools, most importantly MR1 tetramers, it is now possible to directly study MAIT cells to understand their biology. Therefore, it is timely to compare the phenotype, development, and function of MAIT cells and iNKT cells. In this review, we highlight key areas where MAIT cells show similarity or difference to iNKT cells. In addition, we discuss important avenues for future research within the MAIT cell field, especially where comparison to iNKT cells has proven less informative.

The Conventional Nature of Non-MHC-Restricted T Cells.

The definition “unconventional T cells” identifies T lymphocytes that recognize non-peptide antigens presented by monomorphic antigen-presenting molecules. Two cell populations recognize lipid antigens and small metabolites presented by CD1 and MR1 molecules, respectively. A third cell population expressing the TCR Vγ9Vδ2 is stimulated by small phosphorylated metabolites. In the recent past, we have learnt a lot about the selection, tissue distribution, gene transcription programs, mode of expansion after antigen recognition, and persistence of these cells. These studies depict their functions in immune homeostasis and diseases. Current investigations are revealing that unconventional T cells include distinct sub-populations, which display unexpected similarities to classical MHC-restricted T cells in terms of TCR repertoire diversity, antigen specificity variety, functional heterogeneity, and naïve-to-memory differentiation dynamic. This review discusses the latest findings with a particular emphasis on these T cells, which appear to be more conventional than previously appreciated, and with the perspective of using CD1 and MR1-restricted T cells in vaccination and immunotherapy.

MR1 antigen presentation to MAIT cells: new ligands, diverse pathways?

The major histocompatibility complex class I-related molecule MR1 is the only antigen (Ag) presenting molecule that captures and displays vitamin B-derived metabolites that are unique to a wide array of microbes. Presentation of these metabolite Ag at the cell surface activates mucosal-associated invariant T (MAIT) cells, a highly abundant innate-like T cell population, and represents a recently-described mechanism used by the mammalian immune system to sense pathogenic or commensal microbes. Our understanding of the cell biology of how MR1 accomplishes this unique task is still evolving, but recent advances are allowing a general picture to emerge. Further, the list of metabolites presented by MR1 is expanding both by identification of natural metabolites and the design of synthetic ligands. Here we review the latest studies contributing to our growing understanding of this rapidly expanding field.

Interleukin-18 Is Critical for Mucosa-Associated Invariant T Cell Gamma Interferon Responses to Francisella SpeciesIn Vitrobut NotIn Vivo

Mucosa-associated invariant T (MAIT) cells are a subset of innate T cells that express a semi-invariant Vα chain paired with limited Vβ chains. MAIT cells are activated by riboflavin metabolite derivatives presented by the nonpolymorphic major histocompatibility complex class I (MHC-I)-like molecule MR1. The precise mechanisms required to activate MAIT cells are an area of intense interest. Here we used two closely related intracellular pathogens with distinct inflammasome activation phenotypes to probe the role of innate cytokines in MAIT cell activation. Using an in vitro assay containing transgenic murine MAIT cells, we show that macrophages infected with Francisella novicida, a strong inflammasome activator, released high levels of interleukin-18 (IL-18) and stimulated high levels of MAIT cell gamma interferon (IFN-γ) through a partially MR1-independent pathway. In contrast, macrophages infected with Francisella tularensis live vaccine strain (LVS), a weak inflammasome activator, generated little IL-18 and stimulated low MAIT cell IFN-γ through an MR1-dependent pathway. By manipulating the quantities of IL-18 in these cultures, we show that the IL-18 concentration is sufficient to influence the magnitude of MAIT cell IFN-γ production. Correspondingly, infected IL-18-deficient macrophages failed to induce substantial MAIT cell IFN-γ. In contrast, we found that MAIT cell IFN-γ production in the lungs of IL-18-deficient mice was not significantly different from that in WT mice during F. tularensis LVS pulmonary infection. Overall, we demonstrate that while IL-18 is essential for the MAIT cell IFN-γ response in vitro, it is not essential for MAIT cell IFN-γ production during in vivo LVS pulmonary infection, suggesting that additional signals can drive MAIT cell IFN-γ production in vivo.

Recipient mucosal-associated invariant T cells control GVHD within the colon.

Mucosal-associated invariant T (MAIT) cells are a unique innate-like T cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC class I-like molecule MR1. Here, we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells likely regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A-/- and MR1-/- mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity that appear to represent pathways controlled by similar, IL-17A-dependent mechanisms. Thus, MAIT cells act to control barrier function to attenuate pathogenic T cell responses in the colon and, given their very high frequency in humans, likely represent an important population in clinical BMT.

Mucosal-associated invariant T cells in autoimmune and immune-mediated diseases.

Autoimmune and inflammatory diseases have complex etiologies not fully understood. Both innate and adaptive immune cells are involved in the pathogenesis of these diseases. Mucosal-associated invariant T (MAIT) cells express an invariant TCRα chain (Vα7.2-Jα33 in humans and Vα19-Jα33 in mice) and recognize the conserved MHC-I-related molecule MR1 presenting bacterial metabolites derived from the synthesis of vitamin B. MAIT cells harbor tissue homing properties and produce inflammatory cytokines, suggesting that MAIT cells may play a key role in autoimmune and inflammatory diseases. In this review, we described the current knowledge on MAIT cells in these pathologies, based on patients analyses as well as mouse models. While most of the studies support a deleterious role of MAIT cells in tissue inflammation and destruction, a few reports suggest a protective role of MAIT cells. MAIT cells could represent a new biomarker of disease progression, and a better knowledge of their function might open new avenues for therapeutic strategies based on their manipulation.

Mucosal-associated invariant T cell receptor recognition of small molecules presented by MR1.

The major histocompatibility complex (MHC) class-I related molecule MR1 is a monomorphic and evolutionary conserved antigen (Ag)-presenting molecule that shares the overall architecture of MHC-I and CD1 proteins. However, in contrast to MHC-I and the CD1 family that present peptides and lipids, respectively, MR1 specifically presents small organic molecules. During microbial infection of mammalian cells, MR1 captures and presents vitamin B precursors, derived from the microbial biosynthesis of riboflavin, on the surface of antigen-presenting cells. These MR1-Ag complexes are recognized by the mucosal-associated invariant T cell receptor (MAIT TCR), which subsequently leads to MAIT cell activation. Recently, MR1 was shown to trap chemical scaffolds including drug and drug-like molecules. Here, we review this metabolite Ag-presenting molecule and further define the key molecular interactions underlying the recognition and reactivity of MAIT TCRs to MR1 in an Ag-dependent manner.

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

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.

Conservation of sequence motifs suggests that the nonclassical MHC class I lineages CD1/PROCR and UT were established before the emergence of tetrapod species.

Humans have a number of nonclassical major histocompatibility complex (MHC) class I molecules that are quite divergent from the classical ones, and that may have separated from the classical lineage in pre-mammalian times. To estimate when in evolution the respective nonclassical lineages separated from the classical lineage, we first identified "phylogenetic marker motifs" within the evolution of classical MHC class I; the selected motifs are rather specific for and rather stably inherited within clades of species. Distribution of these motifs in nonclassical MHC class I molecules indicates that the lineage including the nonclassical MHC class I molecules CD1 and PROCR separated from the classical lineage before the emergence of tetrapod species, and that the human nonclassical MHC class I molecules FCGRT, MIC/ULBP/RAET, HFE, MR1, and ZAG show similarity with classical MHC class I at the avian/reptilian level. An MR1-like α1 exon sequence was identified in turtle. Our system furthermore indicates that the lineage UT, hitherto only found in non-eutherian mammals, predates tetrapod existence, and we identified UT genes in reptiles. If only accepting wide distribution of a lineage among extant species as true evidence for ancientness, the oldest identified nonclassical MHC class I lineage remains the fish-specific lineage Z, which was corroborated in the present study by finding both Z and classical-type MHC class I sequences in a primitive fish, the bichir. In short, we gained important new insights into the evolution of classical MHC class I motifs and the probable time of origin of nonclassical MHC class I lineages.

Activation of Human Mucosal-Associated Invariant T Cells Induces CD40L-Dependent Maturation of Monocyte-Derived and Primary Dendritic Cells.

Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. It remains unclear whether, in addition to their cytolytic activity that is important in antimicrobial defense, MAIT cells have immune-modulatory functions that could enhance dendritic cell (DC) maturation. In this study, we investigated the molecular mechanisms dictating the interactions between human MAIT cells and DCs and demonstrate that human MAIT cells mature monocyte-derived and primary DCs in an MR1- and CD40L-dependent manner. Furthermore, we show that MAIT cell–derived signals synergize with microbial stimuli to induce secretion of bioactive IL-12 by DCs. Activation of human MAIT cells in whole blood leads to MR1- and cytokine-dependent NK cell transactivation. Our results underscore an important property of MAIT cells, which can be of translational relevance to rapidly orchestrate adaptive immunity through DC maturation.

Cytotoxic and regulatory roles of mucosal-associated invariant T cells in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease that results from the destruction of pancreatic β-cells by the immune system that involves innate and adaptive immune cells. Mucosal-associated invariant T cells (MAIT cells) are innate-like T-cells that recognize derivatives of precursors of bacterial riboflavin presented by the major histocompatibility complex (MHC) class I-related molecule MR1. Since T1D is associated with modification of the gut microbiota, we investigated MAIT cells in this pathology. In patients with T1D and mice of the non-obese diabetic (NOD) strain, we detected alterations in MAIT cells, including increased production of granzyme B, which occurred before the onset of diabetes. Analysis of NOD mice that were deficient in MR1, and therefore lacked MAIT cells, revealed a loss of gut integrity and increased anti-islet responses associated with exacerbated diabetes. Together our data highlight the role of MAIT cells in the maintenance of gut integrity and the control of anti-islet autoimmune responses. Monitoring of MAIT cells might represent a new biomarker of T1D, while manipulation of these cells might open new therapeutic strategies.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Multiple layers of heterogeneity and subset diversity in human MAIT cell responses to distinct microorganisms and to innate cytokines

Mucosa-associated invariant T (MAIT) cells are a large innate-like T-cell subset in humans defined by invariant TCR Vα7.2 use and expression of CD161. MAIT cells recognize microbial riboflavin metabolites of bacterial or fungal origin presented by the monomorphic MR1 molecule. The extraordinary level of evolutionary conservation of MR1 and the limited known diversity of riboflavin metabolite antigens have suggested that MAIT cells are relatively homogeneous and uniform in responses against diverse microbes carrying the riboflavin biosynthesis pathway. The ability of MAIT cells to exhibit microbe-specific functional specialization has not been thoroughly investigated. Here, we found that MAIT cell responses against Escherichia coli and Candida albicans displayed microbe-specific polyfunctional response profiles, antigen sensitivity, and response magnitudes. MAIT cell effector responses against E. coli and C. albicans displayed differential MR1 dependency and TCR β-chain bias, consistent with possible divergent antigen subspecificities between these bacterial and fungal organisms. Finally, although the MAIT cell immunoproteome was overall relatively homogenous and consistent with an effector memory-like profile, it still revealed diversity in a set of natural killer cell-associated receptors. Among these, CD56, CD84, and CD94 defined a subset with higher expression of the transcription factors promyelocytic leukemia zinc finger (PLZF), eomesodermin, and T-bet and enhanced capacity to respond to IL-12 and IL-18 stimulation. Thus, the conserved and innate-like MAIT cells harbor multiple layers of functional heterogeneity as they respond to bacterial or fungal organisms or innate cytokines and adapt their antimicrobial response patterns in a stimulus-specific manner.

Functionally diverse human T cells recognize non-microbial antigens presented by MR1.

MHC class I-related molecule MR1 presents riboflavin- and folate-related metabolites to mucosal-associated invariant T cells, but it is unknown whether MR1 can present alternative antigens to other T cell lineages. In healthy individuals we identified MR1-restricted T cells (named MR1T cells) displaying diverse TCRs and reacting to MR1-expressing cells in the absence of microbial ligands. Analysis of MR1T cell clones revealed specificity for distinct cell-derived antigens and alternative transcriptional strategies for metabolic programming, cell cycle control and functional polarization following antigen stimulation. Phenotypic and functional characterization of MR1T cell clones showed multiple chemokine receptor expression profiles and secretion of diverse effector molecules, suggesting functional heterogeneity. Accordingly, MR1T cells exhibited distinct T helper-like capacities upon MR1-dependent recognition of target cells expressing physiological levels of surface MR1. These data extend the role of MR1 beyond microbial antigen presentation and indicate MR1T cells are a normal part of the human T cell repertoire.

Multiple layers of heterogeneity and subset diversity in human MAIT cell responses to distinct microorganisms, and to innate cytokines

Abstract Mucosa-associated invariant T (MAIT) cells are a large innate-like T cell subset in humans defined by invariant TCR Vα7.2 usage and expression of CD161. MAIT cells recognize microbial riboflavin metabolites of bacterial or fungal origin presented by the MR1 molecule. The extraordinary level of evolutionary conservation of MR1 and the limited known diversity of riboflavin metabolite antigens have suggested that MAIT cells are relatively homogeneous and uniform in responses against diverse microbes carrying the riboflavin biosynthesis pathway. The ability of MAIT cells to exhibit microbe-specific functional specialization has not been thoroughly investigated. Here, we found that MAIT cell responses against Escherichia coli and Candida albicans displayed microbe-specific polyfunctional response profiles, antigen-sensitivity and response magnitudes. MAIT cell effector responses against E. coli and C. albicans displayed differential MR1-dependency and TCR β-chain bias, consistent with possible divergent antigen sub-specificities between these microorganisms. Finally, while the MAIT cell immuno-proteome was overall relatively homogenous and consistent with an effector-memory-like profile, it still revealed diversity in a set of NK cell-associated receptors. Among these, CD56, CD84 and CD94 defined a subset with higher expression of the transcription factors promyelocytic leukemia zink finger (PLZF), Eomesodermin and T-bet and enhanced capacity to respond to IL-12 and IL-18 stimulation. Thus, the conserved and innate-like MAIT cells harbor multiple layers of functional heterogeneity as they respond to microorganisms or innate cytokines, and adapt their response patterns in a stimulus-specific manner.

Title: Human mucosal-associated invariant T (MAIT) cells in inflamed tissues

Abstract Mucosal-associated invariant T (MAIT) cells recognize bacterial metabolites via a semi-invariant T cell receptor (TCR) presented by MHC-like molecule MR1. Once activated, MAIT cells are a potent effector population with cytotoxic and pro-inflammatory properties. We recently reported that a TCR signal is not sufficient to elicit effector function, instead pro-inflammatory cytokines in addition to a TCR signal are required for sustained MAIT cell effector functions. We proposed that this requirement allows MAIT cells to distinguish between commensal (TCR signal only) and pathogenic bacteria (TCR signal + inflammation). We next wanted to address how MAIT cells function within an inflamed tissue environment. Specifically, if inflammation resulted in increased MAIT effector function or an exhausted phenotype because of the sustained stimuli. We obtained gingival tissue and matched blood from donors undergoing periodontic surgery for various inflammation-related diseases. MAIT cells within inflamed mucosa showed increased effector function directly ex vivo compared to blood including expression of granzyme B and TNFa. RNAseq analysis of MAIT cells from inflamed mucosal tissue revealed a distinct transcriptional signature compared to MAIT cells in the blood. We observed a significant upregulation of chemotaxis and activation genes as well as inhibitory genes from multiple immune-modulatory pathways. Our data suggest that MAIT cells in inflamed tissues can maintain effector function, but also express immunomodulatory genes that presumably serve to avoid unwanted tissue damage.

Reduced circulating MAIT cells in obesity

Abstract Obesity is a major risk factor for certain types of cancer and the mechanisms by which obesity contributes to tumor metastasis are not well understood. Whereas the immune system is essential for fighting against tumors, in the context of obesity, systemic chronic inflammation with altered immune responses are found. Recent studies have revealed that the gut microbiota also contributes to obesity and the pathology associated with that disease. Mucosal-associated innate T (MAIT) cells are a unique subpopulation of T cells that are characterized by the expression of a semi-invariant TCR a chain (Vα19 in mice; Vα7.2 in humans). Although the development and maturation of MAIT cells require the gut microbiota and antigen presenting molecule MR1, the actual function of MAIT cells has not been well studied. We have found reduced circulating MAIT cells in obese patients as compared to age- and gender-matched healthy donors. Using real-time PCR to determine the expression levels of the invariant TCR of MAIT cells and classical T cell transcription factors, we found that, although no difference was observed in any gene expression analyzed between samples from obese patients or healthy donors, mRNA expression of the Vα7.2 TCR positively correlated with the transcription factor TBX21 (found in T helper 1 cells) and BCL6 (found in follicular helper T cells). Our work suggests that obesity alters the number of MAIT cells. The correlation of MAIT cell TCR gene expression with transcription factors of classical effector T cells, suggests there may be transcriptional pathways linking MAIT cells to the function of conventional T cells. Future work will focus on determining how obesity impacts the function of MAIT cells.

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells.

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.

The role of mucosal‐associated invariant T cells in infectious diseases

Mucosal-associated invariant T (MAIT) cells are donor-unrestricted lymphocytes that are surprisingly abundant in humans, representing 1-10% of circulating T cells and further enriched in mucosal tissues. MAIT cells recognize and are activated by small molecule ligands produced by microbes and presented by MR1, a highly conserved MHC-related antigen-presenting protein that is ubiquitously expressed in human cells. Increasing evidence suggests that MAIT cells play a protective role in anti-bacterial immunity at mucosal interfaces. Some fungi are known to produce MAIT-activating ligands, but the role of MAIT cells in fungal infections has not yet been investigated. In viral infections, specifically HIV, which has received the most study, MAIT cell biology is clearly altered, but the mechanisms explaining these alterations and their clinical significance are not yet understood. Many questions remain unanswered about the potential of MAIT cells for protection or pathogenesis in infectious diseases. Because they interact with the universal, donor-unrestricted ligand-presenting MR1 molecule, MAIT cells may be attractive immunotherapy or vaccine targets. New tools, including the development of MR1-ligand tetramers and next-generation T-cell receptor sequencing, have the potential to accelerate MAIT cell research and lead to new insights into the role of this unique set of lymphocytes in infectious diseases.