Mucosal-associated Invariant T Cell Activation Research Articles

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

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes expressing a semi-invariant T-cell receptor (TCR) present as TCR Vα7.2-Jα33 in humans and TCR Vα19-Jα33 in mice. They are activated by ligands produced during microbial biosynthesis of riboflavin that is presented by major histocompatibility complex class I-related (MR1) molecules on antigen-presenting cells. MAIT cells also possess interleukin (IL)-12 and IL-18 receptors and can be activated by the respective cytokines released from microbially stimulated antigen-presenting cells. Therefore, MAIT cells can be involved in bacterial and viral defenses and are a significant part of the human immune system. They are particularly abundant in the liver, an organ serving as the second firewall of gut microbes next to the intestinal barrier. Therefore, the immune functions of MAIT cells are greatly impacted by changes in the gut-microbiota and play important roles in the gut-liver pathogenesis axis. In this review, we discuss the nature and mechanisms of MAIT cell activation and their dynamics during different types of liver pathogenesis conditions. We also share our perspectives on important aspects that should be explored further to reveal the exact roles that MAIT cells play in liver pathogenesis in the context of the gut microbiota.

Notch signaling regulates function of human mucosal-associated invariant T (MAIT) cells.

Notch signaling plays an important role in the development of T lymphocytes and regulates their effector functions. The regulatory roles of Notch signaling on T cells have been intensely investigated, but whether it involves in effector functions of mucosal-associated invariant T (MAIT) cells has never been reported. To elucidate the expression profiles of Notch receptors/ligands and to investigate their roles in human MAIT cell function. Peripheral blood mononuclear cells (PBMCs) from health donors were stimulated with or without anti-CD3/ CD28-coupled beads, recombinant IL-12/IL-18 cytokines, riboflavin- or non-riboflavin-synthesizing bacterial cultured supernatant for 24 hours. The expression profiles of Notch receptors and ligands on MAIT cells were detected by flow cytometry. PBMCs were treated with a Notch signaling inhibitor, gamma secretase inhibitor (GSI), before stimulation to investigate the impact of interfering with Notch signaling on activation and function of MAIT cells. Resting MAIT cells predominantly expressed Notch2 receptor and the ligand, Jagged 2, on their surface. Upon stimulation, MAIT cells further upregulated Notch2 and also Notch1 with its cleaved form, indicating active Notch signaling. Cytokines and cytotoxic molecules which are secreted by activated MAIT cells, were suppressed by treatment with GSI. Moreover, both TCR-dependent MAIT cell activation by microbial-derived riboflavin intermediates and TCR-independent MAIT cell activation driven by IL-18 in synergy with IL-12, were blocked by GSI treatment. Notch signaling is operating in MAIT cells and is involved in their activation both in a TCR-independent and -dependent manners.

The effects of 5-OP-RU stereochemistry on its stability and MAIT-MR1 axis.

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2'- and 3'-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4'-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.

Targeting mitochondrial dysfunction in MAIT cells: Potential immunotherapeutic treatment for colorectal cancer

Mucosal associated invariant T (MAIT) cells have captured the attention of immunologists and clinicians in recent years due to their abundance in humans, especially in human liver and mucosal tissue. Colorectal cancer is one of the most common forms of cancer in mucosal tissue. Recent evidence reveal activated MAIT cells within the microenvironment of colorectal tumors. The increased tumor infiltration with MAIT cells correlates with poor survival in the colorectal cancer patients, suggesting MAIT cells are promising immunotherapeutic targets in colorectal cancer. Besides well-known role in anti-microbial immunity, MAIT cells have been associated with various forms of cancer. The Th1-biased MAIT cells are proposed to mediate anti-tumor immunity, while IL-17-producing subsets have been implicated in promoting malignancy. Reduced IFN-γ production and elevated IL-17 production of MAIT cells have been found in colorectal tumor tissue and shown to promote tumor growth and metastases. Although the mechanism(s) driving the increase in Th17-biased MAIT cells with reduced IFN-γ production in tumor is not fully understood, recent studies have linked IL-17 response to dysfunctional mitochondria and reactive oxygen species (ROS) from the mitochondria. Therefore, we hypothesize that mitochondrial dysfunction contributes to Th17-skewed MAIT cell responses with decreased IFN-γ production. Mitochondrial targeted antioxidants are supposed to be beneficial for recovering Th1-baised antitumor immunity and inhibiting IL-17 production of MAIT by improving mitochondrial function.

Hepatitis B Virus Drives Divergent Adaptive Immunity in Hepatocellular Carcinoma

In hepatitis B virus (HBV)-related hepatocellular carcinoma (HBV+ HCC), the mechanisms by virus-derived antigens shape the T cell immune response remains unclear. Here, we employed bulk and single-cell multi-omics sequencing technologies to study the T cell receptor (TCR) repertoire, antigen specificity and epigenomic profiles in HBV+ HCC patients. We found that HBV is a major driver of T cells, with HBV-specific recurrent and expanding TCR clonotypes from CD8+ T cells, mucosal-associated invariant T (MAIT) cells and regulatory T cells (Tregs). HBV virions can directly activate MAIT cells in a MR1-dependent manner, indicating that these cells are not only sensors of metabolic products, but could participate directly in pathogen defense. And increased numbers of intratumoral Tregs, many of which are HBV specific, correlated with both increased viral titers and tumor size, while more a robust expansion of virus specific CD8+ T cells had better clinical prognosis after surgery. Integrating TCR and ATAC sequences also allowed us to track the epigenomic changes of specific T cell clonal lineages, with HBV-specific conventional CD8+ T cells expressing an effector phenotype when adjacent to the tumor, but cells of the same lineage exhibiting an “exhausted” phenotype in the tumor itself.

Alternative splicing of MR1 regulates antigen presentation to MAIT cells

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full-length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are incompletely understood. In this report, we sought to characterize the expression and function of these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. However only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A decreases MAIT cell activation following bacterial infection. Additionally, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Here, we find that relative expression of MR1A/MR1B transcript is associated with the prevalence of MR1 + CD4/CD8 cells in the thymus. Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand(s).

Evasion of MAIT cell recognition by the African Salmonella Typhimurium ST313 pathovar that causes invasive disease

Mucosal-associated invariant T (MAIT) cells are innate T lymphocytes activated by bacteria that produce vitamin B2 metabolites. Mouse models of infection have demonstrated a role for MAIT cells in antimicrobial defense. However, proposed protective roles of MAIT cells in human infections remain unproven and clinical conditions associated with selective absence of MAIT cells have not been identified. We report that typhoidal and nontyphoidal Salmonella enterica strains activate MAIT cells. However, S. Typhimurium sequence type 313 (ST313) lineage 2 strains, which are responsible for the burden of multidrug-resistant nontyphoidal invasive disease in Africa, escape MAIT cell recognition through overexpression of ribB This bacterial gene encodes the 4-dihydroxy-2-butanone-4-phosphate synthase enzyme of the riboflavin biosynthetic pathway. The MAIT cell-specific phenotype did not extend to other innate lymphocytes. We propose that ribB overexpression is an evolved trait that facilitates evasion from immune recognition by MAIT cells and contributes to the invasive pathogenesis of S. Typhimurium ST313 lineage 2.

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.

MAIT Cell Activation and Functions.

Mucosal associated invariant T (MAIT) cells are striking in their abundance and their strict conservation across 150 million years of mammalian evolution, implying they must fulfill critical immunological function(s). MAIT cells are defined by their expression of a semi-invariant αβ TCR which recognizes biosynthetic derivatives of riboflavin synthesis presented on MR1. Initial studies focused on their role in detecting predominantly intracellular bacterial and mycobacterial infections. However, it is now recognized that there are several modes of MAIT cell activation and these are related to activation of distinct transcriptional programmes, each associated with distinct functional roles. In this minireview, we summarize current knowledge from human and animal studies of MAIT cell activation induced (1) in an MR1-TCR dependent manner in the context of inflammatory danger signals and associated with antibacterial host defense; (2) in an MR1-TCR independent manner by the cytokines interleukin(IL)-12/-15/-18 and type I interferon, which is associated with antiviral responses; and (3) a recently-described TCR-dependent “tissue repair” programme which is associated with accelerated wound healing in the context of commensal microbiota. Because of this capability for diverse functional responses in diverse immunological contexts, these intriguing cells now appear to be multifunctional effectors central to the interface of innate and adaptive immunity.

Ligand-dependent downregulation of MR1 cell surface expression

The antigen-presenting molecule MR1 presents riboflavin-based metabolites to Mucosal-Associated Invariant T (MAIT) cells. While MR1 egress to the cell surface is ligand-dependent, the ability of small-molecule ligands to impact on MR1 cellular trafficking remains unknown. Arising from an in silico screen of the MR1 ligand-binding pocket, we identify one ligand, 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoic acid, DB28, as well as an analog, methyl 3-([2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl]formamido)propanoate, NV18.1, that down-regulate MR1 from the cell surface and retain MR1 molecules in the endoplasmic reticulum (ER) in an immature form. DB28 and NV18.1 compete with the known MR1 ligands, 5-OP-RU and acetyl-6-FP, for MR1 binding and inhibit MR1-dependent MAIT cell activation. Crystal structures of the MAIT T cell receptor (TCR) complexed with MR1-DB28 and MR1-NV18.1, show that these two ligands reside within the A'-pocket of MR1. Neither ligand forms a Schiff base with MR1 molecules; both are nevertheless sequestered by a network of hydrophobic and polar contacts. Accordingly, we define a class of compounds that inhibits MR1 cellular trafficking.

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro.

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand. We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota.

Association of mucosal-associated invariant T cells with different disease phases of polymyalgia rheumatica.

Although T cells are thought to be involved in the pathogenesis of PMR, whether innate-like T cells are involved in the process remains unknown. The serum levels of 27 cytokines/chemokines in patients with PMR were measured by a multiplex immunoassay (Bio-Plex Assay). The cytokine-producing capacity of T and innate-like T cells was assessed by intracellular cytokine staining and flow cytometry. The frequency and activated status of T and innate-like T cells were investigated by flow cytometry and their associations with clinical parameters were assessed. The levels of inflammatory cytokines were associated with disease activity in PMR. The cytokine-producing capacity by CD8+ T and innate-like T cells was associated with disease activity. The frequency of HLA-DR+ CD38+ cells among CD8+ T cells was increased in patients with active disease. The frequencies of HLA-DR+ CD38+ cells among CD4+ T, mucosal-associated invariant T (MAIT) and γδ T cells were higher in patients with inactive disease. The frequency of HLA-DR+ CD38+ MAIT cells was associated with the PMR activity score and CRP levels in patients in remission. The inflammatory cytokine-producing capacity and expression of activation markers of CD8+ T and innate-like T cells were associated with the disease activity of PMR. MAIT cell activation in patients in remission may contribute to the subclinical activity of the disease.

The molecular basis underpinning the potency and specificity of MAIT cell antigens.

Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.

Altered distribution and enhanced osteoclastogenesis of mucosal-associated invariant T cells in gouty arthritis.

This study was designed to investigate the role of mucosal-associated invariant T (MAIT) cells in gouty arthritis (GA) and their effects on osteoclastogenesis. Patients with GA (n = 61), subjects with hyperuricaemia (n = 11) and healthy controls (n = 30) were enrolled in this study. MAIT cells, cytokines, CD69, programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) levels were measured by flow cytometry. In vitro osteoclastogenesis experiments were performed using peripheral blood mononuclear cells in the presence of M-CSF and RANK ligand. Circulating MAIT cell levels were significantly reduced in GA patients. However, their capacities for IFN-γ, IL-17 and TNF-α production were preserved. Expression levels of CD69, PD-1 and LAG-3 in MAIT cells were found to be elevated in GA patients. In particular, CD69 expression in circulating MAIT cells was increased by stimulation with MSU crystals, suggesting that deposition of MSU crystals might contribute to MAIT cell activation. Interestingly, MAIT cells were found to be accumulated in synovial fluid and infiltrated into gouty tophus tissues within joints. Furthermore, activated MAIT cells secreted pro-resorptive cytokines (i.e. IL-6, IL-17 and TNF-α) and facilitated osteoclastogenesis. This study demonstrates that circulating MAIT cells are activated and numerically deficient in GA patients. In addition, MAIT cells have the potential to migrate to inflamed tissues and induce osteoclastogenesis. These findings provide an important role of MAIT cells in the pathogenesis of inflammation and bone destruction in GA patients.

Sustained IFN-I stimulation impairs MAIT cell responses to bacteria by inducing IL-10 during chronic HIV-1 infection.

Mucosal-associated invariant T (MAIT) cells in HIV-1-infected individuals are functionally impaired by poorly understood mechanisms. Single-cell transcriptional and surface protein analyses revealed that peripheral MAIT cells from HIV-1-infected subjects were highly activated with the up-regulation of interferon (IFN)-stimulated genes as compared to healthy individuals. Sustained IFN-α treatment suppressed MAIT cell responses to Escherichia coli by triggering high-level interleukin-10 (IL-10) production by monocytes, which subsequently inhibited the secretion of IL-12, a crucial costimulatory cytokine for MAIT cell activation. Blocking IFN-α or IL-10 receptors prevented MAIT cell dysfunction induced by HIV-1 exposure in vitro. Moreover, blocking the IL-10 receptor significantly improved anti-Mycobacterium tuberculosis responses of MAIT cells from HIV-1-infected patients. Our findings demonstrate the central role of the IFN-I/IL-10 axis in MAIT cell dysfunction during HIV-1 infection, which has implications for the development of anti-IFN-I/IL-10 strategies against bacterial coinfections in HIV-1-infected patients.

Neutrophils suppress mucosal-associated invariant Tcells in humans.

Mucosal-associated invariant T(MAIT)cells are innate-like Tlymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a nonredundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and hydrogen peroxide dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF-α that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent overactivation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

The Chemical Synthesis, Stability, and Activity of MAIT Cell Prodrug Agonists That Access MR1 in Recycling Endosomes.

Mucosal-associated invariant T (MAIT) cells are antibacterial effector T cells that react to pyrimidines derived from bacterial riboflavin synthesis presented by the monomorphic molecule MR1. A major challenge in MAIT cell research is that the commonly used MAIT agonist precursor, 5-amino-6-d-ribitylaminouracil (5-A-RU), is labile to autoxidation, resulting in a loss of biological activity. Here, we characterize two independent autoxidation processes by LCMS. To overcome the marked instability, we report the synthesis of a 5-A-RU prodrug generated by modification of the 5-amino group with a cleavable valine-citrulline-p-aminobenzyl carbamate. The compound is stable in prodrug form, with the parent amine (i.e., 5-A-RU) released only after enzymatic cleavage. Analysis of the prodrug in vitro and in vivo showed an enhanced MAIT cell activation profile compared to 5-A-RU, which was associated with preferential loading within recycling endosomes, a route used by some natural agonists. This prodrug design therefore overcomes the difficulties associated with 5-A-RU in biological studies and provides an opportunity to explore different presentation pathways.

Activation, Deficiency, and Reduced IFN-γ Production of Mucosal-Associated Invariant T Cells in Patients with Inflammatory Bowel Disease

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can activate either in response to T-cell receptor (TCR) engagement or through activating cytokines and play an important role in autoimmune disorders. The study examined the level and function of MAIT cells in patients with inflammatory bowel disease (IBD). Circulating MAIT cell levels were significantly reduced in IBD patients. This MAIT cell deficiency was correlated with IBD disease activity grades, hemoglobin, and CRP. IFN-γ production of circulating MAIT cells in response to both MHC class 1b-like related protein (MR1)-dependent and -independent stimulations was decreased in IBD patients, which was partially associated with reduced activation of nuclear factor of activated T cells 1 (NFAT1) transcription factor, a main regulator of IFN-γ production. Expression levels of CD69, programmed death-1 (PD-1), and annexin V in MAIT cells were elevated in IBD patients. CCL20, CXCL10, CXCL16, and CCL25 were expressed higher in inflamed intestinal tissues than in noninflamed tissues. This study demonstrates that circulating MAIT cells are activated and numerically and functionally deficient in IBD patients. Furthermore, activated MAIT cells have the potential to migrate to inflamed tissues. These findings suggest an important role of MAIT cells in mucosal immunity in IBD.

Human MAIT Cell Activation In Vitro.

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T cell subset in humans, enriched in mucosal tissues and the liver. MAIT cells express a semi-invariant T cell receptor (TCR) and recognize microbial-derived riboflavin metabolites presented on the MHC Class I-like molecule MR1. In addition to activation via the TCR, MAIT cells can also be activated in response to cytokines such as IL-12 and IL-18, in contrast to conventional T cells. Here we describe TCR-dependent and -independent methods for MAIT cell activation. The TCR-dependent approaches include stimulation with microbead- or plate-bound anti-CD3/anti-CD28 antibodies, and with 5-OP-RU or paraformaldehyde (PFA)-fixed E. coli in the presence of antigen-presenting cells (APCs). The latter method includes a combination of TCR- and cytokine-mediated stimulation. The TCR-independent methods include direct stimulation with the recombinant cytokines IL-12 and IL-18, and indirect stimulation with TLR-4/TLR-8 agonists or influenza A virus in the presence of APCs. Finally, we outline a protocol to analyze activated MAIT cells using flow cytometry.

MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria.

Tuberculosis (TB) is the leading cause of mortality from a single infectious agent, Mycobacterium tuberculosis Relevant immune targets of the partially efficacious TB vaccine bacille Calmette-Guérin (BCG) remain poorly defined. Mucosal-associated invariant T (MAIT) cells are MHC-related protein 1 (MR1)-restricted T cells, which are reactive against M. tuberculosis, and underexplored as potential TB vaccine targets. We sought to determine whether BCG vaccination activated mycobacteria-specific MAIT cell responses in humans. We analyzed whole blood samples from M. tuberculosis-infected South African adults who were revaccinated with BCG after a six-month course of isoniazid preventative therapy. In vitro BCG stimulation potently induced IFN-γ expression by phenotypic (CD8+CD26+CD161+) MAIT cells, which constituted the majority (75%) of BCG-reactive IFN-γ-producing CD8+ T cells. BCG revaccination transiently expanded peripheral blood frequencies of BCG-reactive IFN-γ+ MAIT cells, which returned to baseline frequencies a year following vaccination. In another cohort of healthy adults who received BCG at birth, 53% of mycobacteria-reactive-activated CD8 T cells expressed CDR3α TCRs, previously reported as MAIT TCRs, expressing the canonical TRAV1-2-TRAJ33 MAIT TCRα rearrangement. CD26 and CD161 coexpression correlated with TRAV1-2+CD161+ phenotype more accurately in CD8+ than CD4-CD8- MAIT cells. Interestingly, BCG-induced IFN-γ expression by MAIT cells in vitro was mediated by the innate cytokines IL-12 and IL-18 more than MR1-induced TCR signaling, suggesting TCR-independent activation. Collectively, the data suggest that activation of blood MAIT cells by innate inflammatory cytokines is a major mechanism of responsiveness to vaccination with whole cell vaccines against TB or in vitro stimulation with mycobacteria (Clinical trial registration: NCT01119521).