MAIT cells monitor intestinal dysbiosis and contribute to host protection during colitis.

Intestinal inflammation shifts microbiota composition and metabolism. How the host monitors and responds to such changes remains unclear. Here, we describe a protective mechanism by which mucosal-associated invariant T (MAIT) cells detect microbiota metabolites produced upon intestinal inflammation and promote tissue repair. At steady state, MAIT ligands derived from the riboflavin biosynthesis pathway were produced by aerotolerant bacteria residing in the colonic mucosa. Experimental colitis triggered luminal expansion of riboflavin-producing bacteria, leading to increased production of MAIT ligands. Modulation of intestinal oxygen levels suggested a role for oxygen in inducing MAIT ligand production. MAIT ligands produced in the colon rapidly crossed the intestinal barrier and activated MAIT cells, which expressed tissue-repair genes and produced barrier-promoting mediators during colitis. Mice lacking MAIT cells were more susceptible to colitis and colitis-driven colorectal cancer. Thus, MAIT cells are sensitive to a bacterial metabolic pathway indicative of intestinal inflammation.

Divergent local and systemic antitumor response in primary uveal melanomas.

Uveal melanoma (UM) is the most common cancer of the eye. The loss of chromosome 3 (M3) is associated with a high risk of metastases. M3 tumors are more infiltrated by T-lymphocytes than low-risk disomic-3 (D3) tumors, contrasting with other tumor types in which T cell infiltration correlates with better prognosis. Whether these T cells represent an antitumor response and how these T cells would be primed in the eye are both unknown. Herein, we characterized the T cells infiltrating primary UMs. CD8+ and Treg cells were more abundant in M3 than in D3 tumors. CD39+PD-1+CD8+ T cells were enriched in M3 tumors, suggesting specific responses to tumor antigen (Ag) as confirmed using HLA-A2:Melan-A tetramers. scRNAseq-VDJ analysis of T cells evidenced high numbers of proliferating CD39+PD1+CD8+ clonal expansions, suggesting in situ antitumor Ag responses. TCRseq and tumor-Ag tetramer staining characterized the recirculation pattern of the antitumor responses in M3 and D3 tumors. Thus, tumor-Ag responses occur in localized UMs, raising the question of the priming mechanisms in the absence of known lymphatic drainage.

Human MAIT cells inhibit alloreactive T cell responses and protect against acute graft-versus-host disease.

Adoptive transfer of immunoregulatory cells can prevent or ameliorate graft-versus-host disease (GVHD), which remains the main cause of nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Mucosal-associated invariant T (MAIT) cells were recently associated with tissue repair capacities and with lower rates of GVHD in humans. Here, we analyzed the immunosuppressive effect of MAIT cells in an in vitro model of alloreactivity and explored their adoptive transfer in a preclinical xenogeneic GVHD model. We found that MAIT cells, whether freshly purified or short-term expanded, dose-dependently inhibited proliferation and activation of alloreactive T cells. In immunodeficient mice injected with human PBMCs, MAIT cells greatly delayed GVHD onset and decreased severity when transferred early after PBMC injection but could also control ongoing GVHD when transferred at delayed time points. This effect was associated with decreased proliferation and effector function of human T cells infiltrating tissues of diseased mice and was correlated with lower circulating IFN-γ and TNF-α levels and increased IL-10 levels. MAIT cells acted partly in a contact-dependent manner, which likely required direct interaction of their T cell receptor with MHC class I-related molecule (MR1) induced on host-reactive T cells. These results support the setup of clinical trials using MAIT cells as universal therapeutic tools to control severe GVHD or mucosal inflammatory disorders.

Multimodal profiling reveals site-specific adaptation and tissue residency hallmarks of γδ T cells across organs in mice.

γδ T cells perform heterogeneous functions in homeostasis and disease across tissues. However, it is unclear whether these roles correspond to distinct γδ subsets or to a homogeneous population of cells exerting context-dependent functions. Here, by cross-organ multimodal single-cell profiling, we reveal that various mouse tissues harbor unique site-adapted γδ subsets. Epidermal and intestinal intraepithelial γδ T cells are transcriptionally homogeneous and exhibit epigenetic hallmarks of functional diversity. Through parabiosis experiments, we uncovered cellular states associated with cytotoxicity, innate-like rapid interferon-γ production and tissue repair functions displaying tissue residency hallmarks. Notably, our observations add nuance to the link between interleukin-17-producing γδ T cells and tissue residency. Moreover, transcriptional programs associated with tissue-resident γδ T cells are analogous to those of CD8+ tissue-resident memory T cells. Altogether, this study provides a multimodal landscape of tissue-adapted γδ T cells, revealing heterogeneity, lineage relationships and their tissue residency program.

A conserved transcriptional program for MAIT cells across mammalian evolution.

Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.

Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε-/- mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).1.

Role of MR1-driven signals and amphiregulin on the recruitment and repair function of MAIT cells during skin wound healing.

Tissue repair processes maintain proper organ function following mechanical or infection-related damage. In addition to antibacterial properties, mucosal associated invariant T (MAIT) cells express a tissue repair transcriptomic program and promote skin wound healing when expanded. Herein, we use a human-like mouse model of full-thickness skin excision to assess the underlying mechanisms of MAIT cell tissue repair function. Single-cell RNA sequencing analysis suggested that skin MAIT cells already express a repair program at steady state. Following skin excision, MAIT cells promoted keratinocyte proliferation, thereby accelerating healing. Using skin grafts, parabiosis, and adoptive transfer experiments, we show that MAIT cells migrated into the wound in a T cell receptor (TCR)-independent but CXCR6 chemokine receptor-dependent manner. Amphiregulin secreted by MAIT cells following excision promoted wound healing. Expression of the repair function was probably independent of sustained TCR stimulation. Overall, our study provides mechanistic insights into MAIT cell wound healing function in the skin.

MAIT cell development in mice and humans.

MAIT cells arise in the thymus following rearrangement of a T cell receptor (TCR) reactive against microbial vitamin B2-derived metabolites presented by the MHC-Ib molecule, MR1. Mechanisms that are conserved in mammals ensure the frequent production of MR1-restricted TCRs and the intra-thymic differentiation of MR1-restricted thymocytes into effector cells. Upon thymic egress and migration into non-lymphoid tissues, additional signals modulate MAIT cell functions according to each local tissue environment. Here, we review the recent progress made towards a better understanding of the establishment of this major immune cell subset.

MAIT Cell Development and Functions: the Microbial Connection.

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved T cell subset, which reacts to most bacteria through T cell receptor (TCR)-mediated recognition of metabolites derived from the vitamin B2 biosynthetic pathway. Microbiota-derived signals affect all stages of MAIT cell biology including intra-thymic development, peripheral expansion, and functions in specific organs. In tissues, MAIT cells can integrate multiple signals and display effector functions involved in the defense against infectious pathogens. In addition to anti-bacterial activity, MAIT cells improve wound healing in the skin, suggesting a role in epithelium homeostasis through bi-directional interactions with the local microbiota. In humans, blood MAIT cell frequency is modified during several auto-immune diseases, which are often associated with microbiota dysbiosis, further emphasizing the potential interplay of MAIT cells with the microbiota. Here, we will review how microbes interact with MAIT cells, from initial intra-thymic development to tissue colonization and functions.

A TCR-Dependent Tissue Repair Potential of MAIT Cells.

Three studies published in Cell Reports (Hinks et al., Lamichhane et al., and Leng et al.) describe the transcriptome of human and mouse mucosal-associated invariant T (MAIT) cells after cognate and noncognate stimulation. The results confirm the variability of MAIT cell effector functions and provide evidence of a new tissue-repair gene signature expressed upon T cell receptor (TCR) stimulation.

Microbial metabolites control the thymic development of mucosal-associated invariant T cells.

How the microbiota modulate immune functions remains poorly understood. Mucosal-associated invariant T (MAIT) cells are implicated in mucosal homeostasis and absent in germ-free mice. Here, we show that commensal bacteria govern murine MAIT intrathymic development, as MAIT cells did not recirculate to the thymus. MAIT development required RibD expression in bacteria, indicating that production of the MAIT antigen 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) was necessary. 5-OP-RU rapidly traveled from mucosal surfaces to the thymus, where it was captured by the major histocompatibility complex class Ib molecule MR1. This led to increased numbers of the earliest MAIT precursors and the expansion of more mature receptor-related, orphan receptor γt-positive MAIT cells. Thus, a microbiota-derived metabolite controls the development of mucosally targeted T cells in a process blurring the distinction between exogenous antigens and self-antigens.

A common transcriptomic program acquired in the thymus defines tissue residency of MAIT and NKT subsets.

Mucosal-associated invariant T (MAIT) cells are abundant T cells with unique specificity for microbial metabolites. MAIT conservation along evolution indicates important functions, but their low frequency in mice has hampered their detailed characterization. Here, we performed the first transcriptomic analysis of murine MAIT cells. MAIT1 (RORγtneg) and MAIT17 (RORγt+) subsets were markedly distinct from mainstream T cells, but quasi-identical to NKT1 and NKT17 subsets. The expression of similar programs was further supported by strong correlations of MAIT and NKT frequencies in various organs. In both mice and humans, MAIT subsets expressed gene signatures associated with tissue residency. Accordingly, parabiosis experiments demonstrated that MAIT and NKT cells are resident in the spleen, liver, and lungs, with LFA1/ICAM1 interactions controlling MAIT1 and NKT1 retention in spleen and liver. The transcriptional program associated with tissue residency was already expressed in thymus, as confirmed by adoptive transfer experiments. Altogether, shared thymic differentiation processes generate "preset" NKT and MAIT subsets with defined effector functions, associated with specific positioning into tissues.

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

In the version of this Article originally published, the asterisks indicating statistical significance were missing from Supplementary Figure 6; the file with the correct figure is now available.

Induction of anergic or regulatory tumor-specific CD4+ T cells in the tumor-draining lymph node.

CD4+ T cell antitumor responses have mostly been studied in transplanted tumors expressing secreted model antigens (Ags), while most mutated proteins in human cancers are not secreted. The fate of Ag-specific CD4+ T cells recognizing a cytoplasmic Ag in mice bearing autochthonous tumors is still unclear. Here we show, using a genetically engineered lung adenocarcinoma mouse model, that naive tumor-specific CD4+ T cells are activated and proliferate in the tumor-draining lymph node (TdLN) but do not differentiate into effectors or accumulate in tumors. Instead, these CD4+ T cells are driven toward anergy or peripherally-induced Treg (pTreg) differentiation, from the early stage of tumor development. This bias toward immune suppression is restricted to the TdLN, and is maintained by Tregs enriched in the tumor Ag-specific cell population. Thus, tumors may enforce a dominant inhibition of the anti-tumor CD4 response in the TdLN by recapitulating peripheral self-tolerance mechanisms.

Ontogeny of human mucosal-associated invariant T cells and related T cell subsets.

Mucosal-associated invariant T (MAIT) cells are semi-invariant Vα7.2+ CD161highCD4- T cells that recognize microbial riboflavin precursor derivatives such as 5-OP-RU presented by MR1. Human MAIT cells are abundant in adult blood, but there are very few in cord blood. We longitudinally studied Vα7.2+ CD161high T cell and related subset levels in infancy and after cord blood transplantation. We show that Vα7.2+ and Vα7.2- CD161high T cells are generated early during gestation and likely share a common prenatal developmental program. Among cord blood Vα7.2+ CD161high T cells, the minority recognizing MR1:5-OP-RU display a TRAV/TRBV repertoire very similar to adult MAIT cells. Within a few weeks of life, only the MR1:5-OP-RU reactive Vα7.2+ CD161high T cells acquire a memory phenotype. Only these cells expand to form the adult MAIT pool, diluting out other Vα7.2+ CD161high and Vα7.2- CD161high populations, in a process requiring at least 6 years to reach adult levels. Thus, the high clonal size of adult MAIT cells is antigen-driven and likely due to the fine specificity of the TCRαß chains recognizing MR1-restricted microbial antigens.

An intermediate level of CD161 expression defines a novel activated, inflammatory, and pathogenic subset of CD8+ T cells involved in multiple sclerosis.

Several lines of evidence support a key role for CD8+ T cells in central nervous system tissue damage of patients with multiple sclerosis. However, the precise phenotype of the circulating CD8+ T cells that may be recruited from the peripheral blood to invade the CNS remains largely undefined to date. It has been suggested that IL-17 secreting CD8 (Tc17) T cells may be involved, and in humans these cells are characterized by the expression of CD161. We focused our study on a unique and recently described subset of CD8 T cells characterized by an intermediate expression of CD161 as its role in neuroinflammation has not been investigated to date. The frequency, phenotype, and function of CD8+ T cells with an intermediate CD161 expression level were characterized ex-vivo, in vitro, and in situ using RNAseq, RT-PCR, flow cytometry, TCR sequencing, and immunohistofluorescence of cells derived from healthy volunteers (n = 61), MS subjects (n = 90), as well as inflammatory (n = 15) and non-inflammatory controls (n = 6). We report here that CD8+CD161int T cells present characteristics of effector cells, up-regulate cell-adhesion molecules and have an increased ability to cross the blood-brain barrier and to secrete IL-17, IFNγ, GM-CSF, and IL-22. We further demonstrate that these cells are recruited and enriched in the CNS of MS subjects where they produce IL-17. In the peripheral blood, RNAseq, RT-PCR, high-throughput TCR repertoire analyses, and flow cytometry confirmed an increased effector and transmigration pattern of these cells in MS patients, with the presence of supernumerary clones compared to healthy controls. Our data demonstrate that intermediate levels of CD161 expression identifies activated and effector CD8+ T cells with pathogenic properties that are recruited to MS lesions. This suggests that CD161 may represent a biomarker and a valid target for the treatment of neuroinflammation.

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 ß-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 in infectious diseases.

In humans, MAIT cells represent the most abundant T cell subset reacting against bacteria. Their frequency in the blood is decreased in a large variety of infectious diseases of either bacterial or viral origin. MAIT cells accumulate at the site of bacterial infection and are protective in experimental infection models. Recent epidemiological evidence supports an implication of MAIT cells in protecting against tuberculosis. MAIT cells can be activated either through direct recognition of microbial ligands or by inflammatory cytokines such as IL-12 and IL-18. MAIT cells secrete IFN-γ, IL-17 and/or other effector molecules according to the context of triggering. MAIT cells can kill bacterially infected epithelial cells in vitro. Herein, we summarize and discuss the data suggesting a role for MAIT cells in infectious diseases.

Unconventional or Preset αß T Cells: Evolutionarily Conserved Tissue-Resident T Cells Recognizing Nonpeptidic Ligands.

A majority of T cells bearing the αß T cell receptor (TCR) are specific for peptides bound to polymorphic classical major histocompatibility complex (MHC) molecules. Smaller subsets of T cells are reactive toward various nonpeptidic ligands associated with nonpolymorphic MHC class-Ib (MHC-Ib) molecules. These cells have been termed unconventional for decades, even though only the composite antigen is different from the one seen by classical T cells. Herein, we discuss the identity of these particular T cells in light of the coevolution of their TCR and MHC-Ib restricting elements. We examine their original thymic development: selection on hematopoietic cells leading to the acquisition of an original differentiation program. Most of these cells acquire memory cell features during thymic maturation and exhibit unique patterns of migration into peripheral nonlymphoid tissues to become tissue resident. Thus, these cells are termed preset T cells, as they also display a variety of effector functions. They may act as microbial or danger sentinels, fight microbes, or regulate tissue homeostasis.

MHC class I-related molecule, MR1, and mucosal-associated invariant T cells.

The MHC-related 1, MR1, molecule presents a new class of microbial antigens (derivatives of the riboflavin [Vitamin B2] biosynthesis pathway) to mucosal-associated invariant T (MAIT) cells. This raises many questions regarding antigens loading and intracellular trafficking of the MR1/ligand complexes. The MR1/MAIT field is also important because MAIT cells are very abundant in humans and their frequency is modified in many infectious and non-infectious diseases. Both MR1 and the invariant TCRα chain expressed by MAIT cells are strikingly conserved among species, indicating important functions. Riboflavin is synthesized by plants and most bacteria and yeasts but not animals, and its precursor derivatives activating MAIT cells are short-lived unless bound to MR1. The recognition of MR1 loaded with these compounds is therefore an exquisite manner to detect invasive bacteria. Herein, we provide an historical perspective of the field before describing the main characteristics of MR1, its ligands, and the few available data regarding its cellular biology. We then summarize the current knowledge of MAIT cell differentiation and discuss the definition of MAIT cells in comparison to related subsets. Finally, we describe the phenotype and effector activities of MAIT cells.