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Engineering immune cells with chimeric Ag receptors (CARs) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8+ T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. In this study, we devised an approach to program a nonclassical cytotoxic T cell subset called mucosal-associated invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8+ T cells, expanded them in vitro, and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs that CAR-MAIT cells exhibit high levels of cytotoxicity toward target cells, comparable to CD8+ CAR-T cells, but interestingly expressed lower levels of IFN-γ than conventional CAR CD8+ T cells. Additionally, in the presence of vitamin B2 metabolite 5-ARU (5-amino-4-d-ribitylaminouracil dihydrochloride), which is a conserved compound that activates MAIT cells through MHC class I–related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose-dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity.

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF. In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

Mucosal-associated invariant T (MAIT) cells are important for immune responses against microbial infections. Although known to undergo marked numerical changes with age in humans, our understanding of how MAIT cells are altered during different phases across the human life span is largely unknown. Although also abundant in the tissues, our study focuses on MAIT cell analyses in blood. Across the human life span, we show that naive-like MAIT cells in umbilical cord blood switch to a central/effector memory-like profile that is sustained into older age. Whereas low-grade levels of plasma cytokine/chemokine were apparent in older donors (>65 y old), surprisingly, they did not correlate with the ex vivo MAIT hyperinflammatory cytokine profile observed in older adults. Removal of MAIT cells from older individuals and an aged environment resulted in the reversal of the baseline effector molecule profile comparable with MAIT cells from younger adults. An upregulated basal inflammatory profile accounted for reducedEscherichia coli–specific responses in aged MAIT cells compared with their young adult counterparts when fold change in expression levels of GzmB, CD107a, IFN-γ, and TNF was examined. However, the magnitude of antimicrobial MR1-dependent activation remained as potent and polyfunctional as with younger adults. Paired TCRαβ analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαβ repertoire diversity of virus-specific CD8+T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαβ expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity.

Key Points MAIT cells comprise half the CD8 T cell IFN-γ response to BCG in blood. Frequencies of MAIT cells were not sustainably modulated by BCG vaccination. Innate cytokines mediate BCG-induced MAIT cell responses in whole blood., 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).

Mucosal-associated invariant T (MAIT) cells play a key role in local and systemic immune responses. Studies suggest that type 2 diabetes (T2D) is associated with alterations in the human MAIT cell response. However, the mechanisms that regulate the survival and homeostasis of human MAIT cells are poorly defined. In this study, we demonstrate that the costimulatory TNF superfamily receptor OX40 was highly expressed in MAIT cells of patients with T2D. Compared with OX40-negative MAIT cells, OX40-positive MAIT cells showed a high activation and a memory phenotype. Surprisingly, OX40 expression was negatively correlated with the frequency of MAIT cells in the peripheral blood of T2D patients. Increased cleaved caspase-3 levels were observed in OX40+-expressing MAIT cells in T2D patients. In vitro, activated OX40 signaling by recombinant OX40L protein promoted caspase-3 activation and apoptosis of MAIT cells. Inhibition of caspase-3 restored apoptosis of MAIT cells induced by OX40 signaling. These results identify OX40 as an amplifier of activation-induced cell death of human blood MAIT cells and shed new light on the regulation of MAIT cells in the phase of immune responses in T2D.

Humans have populations of innate-like T lymphocytes with an invariant TCR α-chain that recognize nonpeptide Ags, including invariant NKT (iNKT) cells and mucosal-associated invariant T (MAIT) cells. iNKT cell involvement in human asthma is controversial, whereas there has been little analysis of MAIT cells. Using peripheral blood cells from 110 participants from the Urban Environment and Childhood Asthma (URECA) birth cohort study, these cells were analyzed for number and function. We determined whether iNKT cell or MAIT cell frequency at 1 y is correlated with the cytokine polarization of mainstream CD4+ T cells and/or the development of asthma by age 7 y. Dust samples from 300 houses were tested for iNKT cell antigenic activity. Our results show that a higher MAIT cell frequency at 1 y of age was associated with a decreased risk of asthma by age 7 y. The frequency of MAIT cells was associated with increased production of IFN-γ by activated CD4+ T cells from the URECA cohort. iNKT cell antigenic activity in bedroom dust samples was associated with higher endotoxin concentration and also with reduced risk of asthma. In conclusion, MAIT cell frequency at 1 y may reflect the tendency of the immune system toward Th1 responses and is associated with protection from asthma. Additionally, iNKT cell antigenic activity may be a marker of houses with increased microbial exposures and therefore also with protection from asthma.

Animal model studies highlight the role of innate-like lymphocyte populations in the early inflammatory response and subsequent parasite control following Plasmodium infection. IFN-γ production by these lymphocytes likely plays a key role in the early control of the parasite and disease severity. Analyzing human innate-like T cell and NK cell responses following infection with Plasmodium has been challenging because the early stages of infection are clinically silent. To overcome this limitation, we examined blood samples from a controlled human malaria infection (CHMI) study in a Tanzanian cohort, in which volunteers underwent CHMI with a low or high dose of Plasmodium falciparum sporozoites. The CHMI differentially affected NK, NKT (invariant NKT), and mucosal-associated invariant T cell populations in a dose-dependent manner, resulting in an altered composition of this innate-like lymphocyte compartment. Although these innate-like responses are typically thought of as short-lived, we found that changes persisted for months after the infection was cleared, leading to significantly increased frequencies of mucosal-associated invariant T cells 6 mo postinfection. We used single-cell RNA sequencing and TCR αβ-chain usage analysis to define potential mechanisms for this expansion. These single-cell data suggest that this increase was mediated by homeostatic expansion-like mechanisms. Together, these data demonstrate that CHMI leads to previously unappreciated long-lasting alterations in the human innate-like lymphocyte compartment. We discuss the consequences of these changes for recurrent parasite infection and infection-associated pathologies and highlight the importance of considering host immunity and infection history for vaccine design.

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.

Innate-like T cells display characteristics of both innate lymphoid cells (ILCs) and mainstream αβ T cells, leading to overlapping functions of innate-like T cells with both subsets. In this review, we show that although innate-like T cells are probably present in all vertebrates, their main characteristics are much better known in amphibians and mammals. Innate-like T cells encompass both γδ and αβ T cells. In mammals, γδ TCRs likely coevolved with molecules of the butyrophilin family they interact with, whereas the semi-invariant TCRs of iNKT and mucosal-associated invariant T cells are evolutionarily locked with their restricting MH1b molecules, CD1d and MR1, respectively. The strong conservation of the Ag recognition systems of innate-like T cell subsets despite similar effector potentialities supports that each one fulfills nonredundant roles related to their Ag specificity.