T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Mucosal associated invariant T (MAIT) cells recognise conserved microbial metabolites from riboflavin synthesis. Striking evolutionary conservation and pulmonary abundance implicate them in antibacterial host defence, yet their functions in protection against clinically important pathogens are unknown. Here we show that mouse Legionella longbeachae infection induces MR1-dependent MAIT cell activation and rapid pulmonary accumulation of MAIT cells associated with immune protection detectable in immunocompetent host animals. MAIT cell protection is more evident in mice lacking CD4+ cells, and adoptive transfer of MAIT cells rescues immunodeficient Rag2−/−γC−/− mice from lethal Legionella infection. Protection is dependent on MR1, IFN-γ and GM-CSF, but not IL-17A, TNF or perforin, and enhanced protection is detected earlier after infection of mice antigen-primed to boost MAIT cell numbers before infection. Our findings define a function for MAIT cells in protection against a major human pathogen and indicate a potential role for vaccination to enhance MAIT cell immunity.

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Mucosal-associated invariant T (MAIT) cells are activated by unstable antigens formed by reactions of 5-amino-6-D-ribitylaminouracil (a vitamin B2 biosynthetic intermediate) with glycolysis metabolites such as methylglyoxal. Here we show superior preparations of antigens in dimethylsulfoxide, avoiding their rapid decomposition in water (t1/2 1.5 h, 37 °C). Antigen solution structures, MAIT cell activation potencies (EC50 3–500 pM), and chemical stabilities are described. Computer analyses of antigen structures reveal stereochemical and energetic influences on MAIT cell activation, enabling design of a water stable synthetic antigen (EC50 2 nM). Like native antigens, this antigen preparation induces MR1 refolding and upregulates surface expression of human MR1, forms MR1 tetramers that detect MAIT cells in human PBMCs, and stimulates cytokine expression (IFNγ, TNF) by human MAIT cells. These antigens also induce MAIT cell accumulation in mouse lungs after administration with a co-stimulant. These chemical and immunological findings provide new insights into antigen properties and MAIT cell activation.

Mucosal-associated invariant T (MAIT) cells are activated in a TCR-dependent manner by antigens derived from the riboflavin synthesis pathway, including 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), bound to MHC-related protein-1 (MR1). However, MAIT cell activation in vivo has not been studied in detail. Here, we have found and characterized additional molecular signals required for optimal activation and expansion of MAIT cells after pulmonary Legionella or Salmonella infection in mice. We show that either bone marrow–derived APCs or non–bone marrow–derived cells can activate MAIT cells in vivo, depending on the pathogen. Optimal MAIT cell activation in vivo requires signaling through the inducible T cell costimulator (ICOS), which is highly expressed on MAIT cells. Subsequent expansion and maintenance of MAIT-17/1-type responses are dependent on IL-23. Vaccination with IL-23 plus 5-OP-RU augments MAIT cell–mediated control of pulmonary Legionella infection. These findings reveal cellular and molecular targets for manipulating MAIT cell function under physiological conditions.

Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast, and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as mucosal associated invariant T (MAIT) cells. The major histocompatibility class I-like antigen-presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (␣␤ T cell receptors (TCRs)), and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here, we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs. MAIT CellsT cells are lymphocytes that mediate a range of immune functions such as killing infected host cells and producing cytokines and other factors that regulate immunity and inflammation. T cells generally recognize peptide antigens (Ags) 6 or lipid-based Ags complexed to specialized Ag-presenting molecules, MHC and CD1, respectively, that interact with an Agspecific ␣␤ T cell receptor (TCR) (1, 2). An expansive repertoire of TCRs is generated by the random rearrangement of V, D, and J gene segments and by pairing of TCR ␣-and ␤-chains, allowing specific recognition of a diverse range of Ags. Mucosal associated invariant T (MAIT) cells are a specialized subset of ␣␤ T cells originally identified in CD4 Ϫ CD8 Ϫ (double negative) human blood lymphocytes expressing a dominant invariant TCR ␣-chain gene rearrangement, TRAV1-2-TRAJ33 (based on IMGT, or V␣7.2-J␣33 based on Arden nomenclature (3)) (4, 5) with less frequent usage of TRAJ12 and TRAJ20 (6 -9). The MAIT TCR ␣-chain contains a complementarity-determining region (CDR) 3 loop of constant length with two variable amino acids in the V-J junction (4) located at the base of this loop (10, 11). MAIT cells are evolutionarily conserved in mammals with the canonical TCR ␣-chain composed of homologous TRAV and TRAJ elements also found in mice, cattle (5) and sheep (12). TRAV1-2 is not exclusive to MAIT cells, with some MHC-Irestricted T cells (13,14) and CD1b-restricted germline-encoded, mycolyl lipid-reactive (GEM) T cells also using this TRAV gene segment (15).The ␤-chain of MAIT cells has no apparent restrictions in J␤ usage, but there is a bias toward the use of human TRBV20 and TRBV6 segments (or V␤2 and V␤13 based on Arden nomenclature) and, similarly in mice, to TRBV19 and TRBV13 (or V␤6 and V␤8 based on Arden nomenclature), both being the murine orthologous segments of human TRBV6 (4, 5, 9). Furthermore, recent TCR sequencing revealed a marked oligoclonality of MAIT TCR CDR3␤ regions and a bias in the length of most...

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that require MHC class I-related protein 1 (MR1) for their development. The role of MAIT cells in cancer is unclear, and to date no study has evaluated these cells in vivo in this context. Here, we demonstrated that tumor initiation, growth, and experimental lung metastasis were signifi cantly reduced in Mr1 −/− mice, compared with wild-type mice. The antitumor activity observed in Mr1 −/− mice required natural killer (NK) and/or CD8 + T cells and IFNγ. Adoptive transfer of MAIT cells into Mr1 −/− mice reversed metastasis reduction. Similarly, MR1-blocking antibodies decreased lung metastases and suppressed tumor growth. Following MR1 ligand exposure, some, but not all, mouse and human tumor cell lines upregulated MR1. Pretreatment of tumor cells with the stimulatory ligand 5-OP-RU or inhibitory ligand Ac-6-FP increased or decreased lung metastases, respectively. MR1-deleted tumors resulted in fewer metastases compared with parental tumor cells. MAIT cell suppression of NK-cell effector function was tumor-MR1-dependent and partially required IL17A. Our studies indicate that MAIT cells display tumor-promoting function by suppressing T and/or NK cells and that blocking MR1 may represent a new therapeutic strategy for cancer immunotherapy. SIGNIFICANCE: Contradicting the perception that MAIT cells kill tumor cells, here MAIT cells promoted tumor initiation, growth, and metastasis. MR1-expressing tumor cells activated MAIT cells to reduce NK-cell effector function, partly in a host IL17A-dependent manner. MR1-blocking antibodies reduced tumor metastases and growth, and may represent a new class of cancer therapeutics.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing–based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 “mature” MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ–producing MAIT1 cells and interleukin-17–producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)–deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.