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.

The Emerging Role of MAIT Cell Responses in Viral Infections.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells with innate-like antimicrobial responsiveness. MAIT cells are known for MR1 (MHC class I-related protein 1)-restricted recognition of microbial riboflavin metabolites giving them the capacity to respond to a broad range of microbes. However, recent progress has shown that MAIT cells can also respond to several viral infections in humans and in mouse models, ranging from HIV-1 and hepatitis viruses to influenza virus and SARS-CoV-2, in a primarily cognate Ag-independent manner. Depending on the disease context MAIT cells can provide direct or indirect antiviral protection for the host and may help recruit other immune cells, but they may also in some circumstances amplify inflammation and aggravate immunopathology. Furthermore, chronic viral infections are associated with varying degrees of functional and numerical MAIT cell impairment, suggesting secondary consequences for host defense. In this review, we summarize recent progress and highlight outstanding questions regarding the emerging role of MAIT cells in antiviral immunity.

Targeting the MR1-MAIT cell axis improves vaccine efficacy and affords protection against viral pathogens.

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. Additionally, MAIT cells sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unclear. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster virus-specific CD8+ T cell responses, and potentiate heterosubtypic anti-influenza protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency and Toll-like receptor 3 and type I interferon receptor signaling. The observed phenomenon was reproducible in female and male mice, and in both young and old animals. It could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

Role of MAIT cells in gastrointestinal tract bacterial infections in humans: More than a gut feeling.

Mucosa-associated invariant T (MAIT) cells are the largest population of unconventional T cells in humans. These antimicrobial T cells are poised with rapid effector responses following recognition of the cognate riboflavin (vitamin B2)-like metabolite antigens derived from microbial riboflavin biosynthetic pathway. Presentation of this unique class of small molecule metabolite antigens is mediated by the highly evolutionarily conserved major histocompatibility complex class I-related protein. In humans, MAIT cells are widely found along the upper and lower gastrointestinal tracts owing to their high expression of chemokine receptors and homing molecules directing them to these tissue sites. In this review, we discuss recent findings regarding the roles MAIT cells play in various gastrointestinal bacterial infections, and how their roles appear to differ depending on the etiological agents and the anatomical location. We further discuss the potential mechanisms by which MAIT cells contribute to pathogen control, orchestrate adaptive immunity, as well as their potential contribution to inflammation and tissue damage during gastrointestinal bacterial infections, and the ensuing tissue repair following resolution. Finally, we propose and discuss the use of the emerging three-dimensional organoid technology to test different hypotheses regarding the role of MAIT cells in gastrointestinal bacterial infections, inflammation, and immunity.

Relevant mechanisms of MAIT cells involved in the pathogenesis of periodontitis.

Mucosal-associated invariant T (MAIT) cells are a group of unconventional T cells that are abundant in the human body, recognize microbial-derived vitamin B metabolites presented by MHC class I-related protein 1 (MR1), and rapidly produce proinflammatory cytokines, which are widely involved in the immune response to various infectious diseases. In the oral mucosa, MAIT cells tend to accumulate near the mucosal basal lamina and are more inclined to secrete IL-17 when activated. Periodontitis is a group of diseases that manifests mainly as inflammation of the gums and resorption of the alveolar bone due to periodontal tissue invasion by plaque bacteria on the dental surface. The course of periodontitis is often accompanied by a T-cell-mediated immune response. This paper discussed the pathogenesis of periodontitis and the potential contribution of MAIT cells to periodontitis.

Mucosal-Associated Invariant T (MAIT) cells are highly activated in duodenal tissue of humans with Vibrio cholerae O1 infection: A preliminary report.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

MAIT cells in liver inflammation and fibrosis.

Mucosal-associated invariant T cells or MAIT cells are an abundant cell type in humans and especially so in the liver. MAIT cells are a subset of T lymphocytes that sit at a bridge between innate and adaptive immunity, so-called innate-like or "unconventional" T cells. The specificity of their antigen receptor (T cell receptor or TCR) is for the conserved major histocompatibility complex (MHC)-related molecule MR1, which presents a modified bacterial metabolite from the vitamin B2 biosynthesis pathway - this allows them to respond in the presence of many bacteria or yeast. MAIT cells also possess an array of cytokine receptors, which allows triggering independently of the TCR. The combination of such signals drives their functionality - this means they can respond to a range of stimuli and likely play a role not only in infection or inflammation, but also under homeostatic conditions.In this review, we will look at the question of what MAIT cells are doing in the normal liver and how they behave in the setting of disease. These questions are of relevance because MAIT cells are such a distinctive cell type enriched in the liver under normal conditions, and their modulation could be of therapeutic benefit. The recent discovery that they appear to be involved in liver fibrosis is particularly of interest in this context.

Emerging Roles of Mucosal-Associated Invariant T Cells in Rheumatology.

Mucosal-associated invariant T (MAIT) cells are an unconventional T cell subset expressing a semi-invariant TCR and recognize microbial riboflavin metabolites presented by major histocompatibility complex class 1-related molecule (MR1). MAIT cells serve as innate-like T cells bridging innate and adaptive immunity, which have attracted increasing attention in recent years. The involvement of MAIT cells has been described in various infections, autoimmune diseases and malignancies. In this review, we first briefly introduce the biology of MAIT cells, and then summarize their roles in rheumatic diseases including systemic lupus erythematosus, rheumatoid arthritis, primary Sjögren's syndrome, psoriatic arthritis, systemic sclerosis, vasculitis and dermatomyositis. An increased knowledge of MAIT cells will inform the development of novel biomarkers and therapeutic approaches in rheumatology.

Mucosal-associated invariant T cells: A cryptic coordinator in HIV-infected immune reconstitution.

Human immunodeficiency virus type 1 (HIV-1) infection causes considerable morbidity and mortality worldwide. Although antiretroviral therapy (ART) has largely transformed HIV infection from a fatal disease to a chronic condition, approximately 10%-40% of HIV-infected individuals who receive effective ART and sustain long-term viral suppression still cannot achieve optimal immune reconstitution. These patients are called immunological nonresponders, a state associated with poor clinical prognosis. Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved unconventional T-cell subset defined by expression of semi-invariant αß T-cell receptor (TCR), which recognizes metabolites derived from the riboflavin biosynthetic pathway presented on major histocompatibility complex-related protein-1. MAIT cells, which are considered to act as a bridge between innate and adaptive immunity, produce a wide range of cytokines and cytotoxic molecules upon activation through TCR-dependent and TCR-independent mechanisms, which is of major importance in defense against a variety of pathogens. In addition, MAIT cells are involved in autoimmune and immune-mediated diseases. The number of MAIT cells is dramatically and irreversibly decreased in the early stage of HIV infection and is not fully restored even after long-term suppressive ART. In light of the important role of MAIT cells in mucosal immunity and because microbial translocation is inversely associated with CD4+ T-cell counts, we propose that MAIT cells participate in the maintenance of intestinal barrier integrity and microbial homeostasis, thus further affecting immune reconstitution in HIV-infected individuals.

Mucosal-associated invariant T (MAIT) cells, a new source of universal immune cells for chimeric antigen receptor (CAR)-cell therapy.

Treatment of hematological malignancies by autologous T cells expressing a chimeric antigen receptor (CAR) is a breakthrough in the field of cancer immunotherapy. As CAR-T cells are entering advanced phases of clinical development, there is a need to develop universal, ready-to-use products using immune cells from healthy donors, to reduce time to treatment, improve response rate and finally reduce the cost of production. Mucosal-associated invariant T cells (MAIT) are unconventional T cells which recognize microbial-derived riboflavin derivatives presented by the conserved MR1 molecule and are endowed with potent effector functions. Because they are not selected by classical MHC/peptide complexes and express a semi-invariant T cell receptor, MAIT cells do not mediate alloreactivity, prompting their use as a new source of universal effector cells for allogeneic CAR-T cell therapy without the need to inactivate their endogenous TCR. We produced CD19-CAR MAIT cells as proof-of-concept allowing subsequent head-to-head comparison with currently used CD19-CAR T cells. We demonstrated their anti-tumor efficacy in vitro and their capacity to engraft without mediating GVHD in preclinical immunodeficient mouse models. Universal, off-the-shelf CAR-MAIT cells could provide a suitable alternative to current autologous CAR-T cells to treat patients regardless of HLA disparity, without production delay, enabling a cost-effective manufacturing model for large-scale clinical application.

MR1-dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined. METHODS: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD. RESULTS: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin. CONCLUSION: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands.

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.

The Immune Modulating Properties of Mucosal-Associated Invariant T Cells.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes that express a semi-invariant T cell receptor (TCR) recognizing microbial vitamin B metabolites presented by the highly conserved major histocompatibility complex (MHC) class I like molecule, MR1. The vitamin B metabolites are produced by several commensal and pathogenic bacteria and yeast, but not viruses. Nevertheless, viral infections can trigger MAIT cell activation in a TCR-independent manner, through the release of pro-inflammatory cytokines by antigen-presenting cells (APCs). MAIT cells belong to the innate like T family of cells with a memory phenotype, which allows them to rapidly release Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and in some circumstances Interleukin (IL)-17 and IL-10, exerting an immunomodulatory role on the ensuing immune response, akin to iNKT cells and γδ T cells. Recent studies implicate MAIT cells in a variety of inflammatory, autoimmune diseases, and in cancer. In addition, through the analysis of the transcriptome of MAIT cells activated in different experimental conditions, an important function in tissue repair and control of immune homeostasis has emerged, shared with other innate-like T cells. In this review, we discuss these recent findings, focussing on the understanding of the molecular mechanisms underpinning MAIT cell activation and effector function in health and disease, which ultimately will aid in clinically harnessing this unique, not donor-restricted cell subtype.

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

Mucosal-associated Invariant T (MAIT) cells recognize vitamin B-based antigens presented by the non-polymorphic MHC class I related-1 molecule (MR1). Both MAIT T cell receptors (TCR) and MR1 are highly conserved among mammals, suggesting an important, and conserved, immune function. For many years, the antigens they recognize were unknown. The discovery that MR1 presents vitamin B-based small molecule ligands resulted in a rapid expansion of research in this area, which has yielded information on the role of MAIT cells in immune protection, autoimmune disease and recently in homeostasis and cancer. More recently, we have begun to appreciate the diverse nature of the small molecule ligands that can bind MR1, with several less potent antigens and small molecule drugs that can bind MR1 being identified. Complementary structural information has revealed the complex nature of interactions defining antigen recognition. Additionally, we now view MAIT cells (defined here as MR1-riboflavin-Ag reactive, TRAV1-2+ cells) as one subset of a broader family of MR1-reactive T cells (MR1T cells). Despite these advances, we still lack a complete understanding of how MR1 ligands are generated, presented and recognized in vivo. The biological relevance of these MR1 ligands and the function of MR1T cells in infection and disease warrants further investigation with new tools and approaches.

Leveraging Public Single-Cell and Bulk Transcriptomic Datasets to Delineate MAIT Cell Roles and Phenotypic Characteristics in Human Malignancies.

Mucosa-associated invariant T (MAIT) cells are unconventional, innate-like T lymphocytes that recognize vitamin B metabolites of microbial origin among other antigens displayed by the monomorphic molecule MHC class I-related protein 1 (MR1). Abundant in human tissues, reactive to local inflammatory cues, and endowed with immunomodulatory and cytolytic functions, MAIT cells are likely to play key roles in human malignancies. They accumulate in various tumor microenvironments (TMEs) where they often lose some of their functional capacities. However, the potential roles of MAIT cells in anticancer immunity or cancer progression and their significance in shaping clinical outcomes remain largely unknown. In this study, we analyzed publicly available bulk and single-cell tumor transcriptomic datasets to investigate the tissue distribution, phenotype, and prognostic significance of MAIT cells across several human cancers. We found that expanded MAIT cell clonotypes were often shared between the blood, tumor tissue and adjacent healthy tissue of patients with colorectal, hepatocellular, and non-small cell lung carcinomas. Gene expression comparisons between tumor-infiltrating and healthy tissue MAIT cells revealed the presence of activation and/or exhaustion programs within the TMEs of primary hepatocellular and colorectal carcinomas. Interestingly, in basal and squamous cell carcinomas of the skin, programmed cell death-1 (PD-1) blockade upregulated the expression of several effector genes in tumor-infiltrating MAIT cells. We derived a signature comprising stable and specific MAIT cell gene markers across several tissue compartments and cancer types. By applying this signature to estimate MAIT cell abundance in pan-cancer gene expression data, we demonstrate that a heavier intratumoral MAIT cell presence is positively correlated with a favorable prognosis in esophageal carcinoma but predicts poor overall survival in colorectal and squamous cell lung carcinomas. Finally, in colorectal carcinoma and four other cancer types, we found a positive correlation between MR1 expression and estimated MAIT cell abundance. Collectively, our findings indicate that MAIT cells serve important but diverse roles in human cancers. Our work provides useful models and resources that employ gene expression data platforms to enable future studies in the realm of MAIT cell biology.

Manuka honey-derived methylglyoxal enhances microbial sensing by mucosal-associated invariant T cells.

Methylglyoxal (MGO) is the main antimicrobial determinant associated with using Manuka Honey as a topical dressing. While direct mechanisms of Manuka honey MGO's antimicrobial activity have been demonstrated, such as disruption of bacterial fimbria and flagella, no interaction of Manuka honey-derived MGO with antimicrobial effector cells of the immune system, such as mucosal-associated invariant T cells (MAIT cells), has yet been reported. MAIT cells are an abundant subset of human T cells, critical for regulating a diverse range of immune functions, including antimicrobial defense mechanisms but also mucosal barrier integrity. MAIT cells become activated by recognition of an important microbial metabolite, 5-amino-6-d-ribitylaminouracil (5-A-RU), which is produced by a wide range of microbial pathogens and commensals. Recognition is afforded when 5-A-RU condenses with mammalian-cell derived MGO to form the potent MAIT cell activator, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Formation of 5-OP-RU and its subsequent presentation to MAIT cells by major histocompatibility (MHC)-related molecule 1 (MR1) facilitates host-pathogen and host-commensal interactions. While MGO is a metabolite naturally present in mammalian cells, it is unclear whether exogenous dietary MGO sources, such as those obtained from Manuka honey intake, can contribute to 5-OP-RU formation and enhance MAIT cell activation. In this work, we report that endogenous MGO is the rate-limiting substrate for converting microbial 5-A-RU to 5-OP-RU and that Manuka honey-derived MGO significantly enhances MAIT cell activation in vitro. Our findings posit a novel mechanism by which intake of a food item, such as Manuka honey, can potentially support immune homeostasis by enhancing MAIT cell-specific microbial sensing.

Type I interferons are important co-stimulatory signals during T cell receptor mediated human MAIT cell activation.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as TLR agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCR-mediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-d-ribitylaminouracil/methylglyoxal. We found that T1-IFNs were able to boost interferon-γ and granzyme B production in 5-amino-6-d-ribitylaminouracil/methylglyoxal-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was also evident in liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.

Characterization of Human Mucosal-associated Invariant T (MAIT) Cells.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I-like molecule MHC-related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin-producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc. The main protocols include: Basic Protocol 1: Determining the frequency and steady-state surface phenotype of human MAIT cells Basic Protocol 2: Determining the activation phenotype of human MAIT cells in blood Basic Protocol 3: Characterizing MAIT cell TCRs using TCR-positive reporter cell lines Alternate protocols are provided for determining the absolute number, transcription factor phenotype, and TCR usage of human MAIT cells; and determining activation phenotype by staining for intracellular markers, measuring secreted cytokines, and measuring fluorescent dye dilution due to proliferation. Additional methods are provided for determining the capacity of MAIT cells to produce cytokine independently of antigen using plate-bound or bead-immobilized CD3/CD28 stimulation; and determining the MR1-Ag dependence of MAIT cell activation using MR1-blocking antibody or competitive inhibition. For TCR-positive reporter cell lines, methods are also provided for evaluating the MAIT TCR-mediated MR1-Ag response, determining the capacity of the reporter lines to produce cytokine independently of antigen, determining the MR1-Ag dependence of the reporter lines, and evaluating the MR1-Ag response of the reporter lines using IL-2 secretion. Support Protocols describe the preparation of PBMCs from human blood, the preparation of single-cell suspensions from tissue, the isolation of MAIT cells by FACS and MACS, cloning MAIT TCRα and ß chain genes and MR1 genes for transduction, generating stably and transiently transfected cells lines, generating a stable MR1 knockout antigen-presenting cell line, and generating monocyte-derived dendritic cells.

The biology and functional importance of MAIT cells.

In recent years, a population of unconventional T cells called 'mucosal-associated invariant T cells' (MAIT cells) has captured the attention of immunologists and clinicians due to their abundance in humans, their involvement in a broad range of infectious and non-infectious diseases and their unusual specificity for microbial riboflavin-derivative antigens presented by the major histocompatibility complex (MHC) class I-like protein MR1. MAIT cells use a limited T cell antigen receptor (TCR) repertoire with public antigen specificities that are conserved across species. They can be activated by TCR-dependent and TCR-independent mechanisms and exhibit rapid, innate-like effector responses. Here we review evidence showing that MAIT cells are a key component of the immune system and discuss their basic biology, development, role in disease and immunotherapeutic potential.