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.

Molecular mechanisms of lineage decisions in metabolite-specific T cells.

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44- naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions.

CD1 and MR1: An update after a long-awaited reunion.

CD1 molecules and the MHC-related protein 1 (MR1) present lipid and small molecule antigens, respectively, for T cell surveillance. The biology of these molecules, the antigens they present, and the T cells that respond to them were recently discussed during the 12th International CD1-MR1 Meeting held in Gothenburg, Sweden.

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.

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.

Innate-like T cell subset commitment in the murine thymus is independent of TCR characteristics and occurs during proliferation.

How T-cell receptor (TCR) characteristics determine subset commitment during T-cell development is still unclear. Here, we addressed this question for innate-like T cells, mucosal-associated invariant T (MAIT) cells, and invariant natural killer T (iNKT) cells. MAIT and iNKT cells have similar developmental paths, leading in mice to two effector subsets, cytotoxic (MAIT1/iNKT1) and IL17-secreting (MAIT17/iNKT17). For iNKT1 vs iNKT17 fate choice, an instructive role for TCR affinity was proposed but recent data argue against this model. Herein, we examined TCR role in MAIT and iNKT subset commitment through scRNAseq and TCR repertoire analysis. In our dataset of thymic MAIT cells, we found pairs of T-cell clones with identical amino acid TCR sequences originating from distinct precursors, one of which committed to MAIT1 and the other to MAIT17 fates. Quantitative in silico simulations indicated that the number of such cases is best explained by lineage choice being independent of TCR characteristics. Comparison of TCR features of MAIT1 and MAIT17 clonotypes demonstrated that the subsets cannot be distinguished based on TCR sequence. To pinpoint the developmental stage associated with MAIT sublineage choice, we demonstrated that proliferation takes place both before and after MAIT fate commitment. Altogether, we propose a model of MAIT cell development in which noncommitted, intermediate-stage MAIT cells undergo a first round of proliferation, followed by TCR characteristics-independent commitment to MAIT1 or MAIT17 lineage, followed by an additional round of proliferation. Reanalyzing a published iNKT TCR dataset, we showed that this model is also relevant for iNKT cell development.

CD4+ T Cell Tolerance to Tissue-Restricted Self Antigens Is Mediated by Antigen-Specific Regulatory T Cells Rather Than Deletion.

Deletion of self-antigen-specific T cells during thymic development provides protection from autoimmunity. However, it is unclear how efficiently this occurs for tissue-restricted self antigens, or how immune tolerance is maintained for self-antigen-specific T cells that routinely escape deletion. Here we show that endogenous CD4+ T cells with specificity for a set of tissue-restricted self antigens were not deleted at all. For pancreatic self antigen, this resulted in an absence of steady-state tolerance, while for the lung and intestine, tolerance was maintained by the enhanced presence of thymically-derived antigen-specific Foxp3+ regulatory T (Treg) cells. Unlike deletional tolerance, Treg cell-mediated tolerance was broken by successive antigen challenges. These findings reveal that for some tissue-restricted self antigens, tolerance relies entirely on nondeletional mechanisms that are less durable than T cell deletion. This might explain why autoimmunity is often tissue-specific, and it offers a rationale for cancer vaccine strategies targeting tissue-restricted tumor antigens.

γδ T, NKT, and MAIT Cells During Evolution: Redundancy or Specialized Functions?

Innate-like T cells display characteristics of both innate lymphoid cells (ILCs) and mainstream αß 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 αß 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.

Opposing peripheral fates of tissue-restricted self antigen-specific conventional and regulatory CD4+ T cells.

The development of self antigen-specific T cells is influenced by how the self antigen is expressed. Here, we created a mouse in which a model self antigen is conditionally expressed in different tissue environments. Using peptide:MHCII tetramer-based cell enrichment methods, we examined the development of corresponding endogenous self antigen-specific CD4+ T cell populations. While ubiquitous self antigen expression resulted in efficient deletion of self antigen-specific T cells in the thymus, some tissue-restricted expression patterns resulted in partial deletion of the population in peripheral lymphoid organs. Deletion specifically affected Foxp3- conventional T cells (Tconv) with a bias towards high avidity TCR expressing cells in the case of thymic, but not peripheral deletion. In contrast, Foxp3+ Treg exhibited elevated frequencies with increased TCR avidity. T cells surviving deletion were functionally impaired, with Tconv cells exhibiting more impairment than Tregs. Collectively, our results illustrate how postthymic recognition of tissue-restricted self antigens results in opposing developmental fates for Tconv and Treg cell subsets.

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.

MAIT cells: an historical and evolutionary perspective.

In humans, MAIT cells represent the most abundant T-cell subset reacting against bacteria. MAIT cells belong to the evolutionarily conserved family of "preset" T cells that includes also NKT cells. Both subsets are selected by double positive thymocytes leading to common features such as PLZF expression. Preset T cells correspond to subsets prepositioned in specific tissue locations with preprogrammed versatile effector functions such as antimicrobial functions and possibly also metabolic control and tissue repair activity. Herein, we recall how several groups studying human samples discovered MAIT cells as T cells expressing either a restricted T-cell receptors (TCR) repertoire or homogeneous and singular phenotypic and functional characteristics. We then highlight the main evolutionary features of this subset and its restricting element, MR1 (MHC-related protein (1) with a striking coevolution of TRAV1 and MR1. We introduce another evolutionarily conserved invariant TCRalpha chain coevolving with another MHC class Ib molecule, called MHX, sharing phylogenetic features with MR1. We finally discuss the relationship between MAIT cells and other subsets reacting to microbial antigens or to compounds presented by MR1 in light of confounding experimental issues.

Role of the MHC restriction during maturation of antigen-specific human T cells in the thymus.

In the thymus, a T-cell repertoire able to confer protection against infectious and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is selected. Direct detection of Ag-specific thymocytes, and analysis of the impact of the expression of the MHC-restricting allele on their frequency or function has never been studied in humans because of the extremely low precursor frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex vivo frequency and activation-phenotype of human thymocytes specific for self, viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 double or single-positive compartments in every donor. Our data indicate that the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific fashion, whereas their alloreactive counterparts showed off-target activity. This first ex vivo analysis of human antigen-specific thymocytes at different stages of human T-cell development should open new perspectives in the understanding of the human thymic selection process.

Analysis of relationships between peptide/MHC structural features and naive T cell frequency in humans.

The structural rules governing peptide/MHC (pMHC) recognition by T cells remain unclear. To address this question, we performed a structural characterization of several HLA-A2/peptide complexes and assessed in parallel their antigenicity, by analyzing the frequency of the corresponding Ag-specific naive T cells in A2(+) and A2(-) individuals, as well as within CD4(+) and CD8(+) subsets. We were able to find a correlation between specific naive T cell frequency and peptide solvent accessibility and/or mobility for a subset of moderately prominent peptides. However, one single structural parameter of the pMHC complexes could not be identified to explain each peptide antigenicity. Enhanced pMHC antigenicity was associated with both highly biased TRAV usage, possibly reflecting favored interaction between particular pMHC complexes and germline TRAV loops, and peptide structural features allowing interactions with a broad range of permissive CDR3 loops. In this context of constrained TCR docking mode, an optimal peptide solvent exposed surface leading to an optimal complementarity with TCR interface may constitute one of the key features leading to high frequency of specific T cells. Altogether our results suggest that frequency of specific T cells depends on the fine-tuning of several parameters, the structural determinants governing TCR-pMHC interaction being just one of them.

Characterization of the human CD4(+) T-cell repertoire specific for major histocompatibility class I-restricted antigens.

While CD4(+) T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class-I restricted CD4(+) T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer-based enrichment approach allowing detection and isolation of scarce Ag-specific T cells, we performed a systematic comparative analysis of HLA-A*0201-restricted CD4(+) and CD8(+) T-cell lines directed against several immunodominant viral or tumoral antigens. CD4(+) T cells directed against every peptide-MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA-A2(+) target cells carrying the relevant epitopes. HLA-A2-restricted CD4(+) T cells were seldom expanded in immune HLA-A2(+) donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide-MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class-I restricted T helper cells and high affinity TCR that could be used for adoptive T-cell transfer- or TCR gene transfer-based immunotherapies.

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.

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.

Impact of TCR reactivity and HLA phenotype on naive CD8 T cell frequency in humans.

The impact of MHC phenotype on the shaping of the peripheral naive T cell repertoire in humans remains unknown. To address this, we compared the frequency and antigenic avidity of naive T cells specific for immunodominant self-, viral, and tumor Ags presented by a human MHC class I allele (HLA-A*02, referred to as A2) in individuals expressing or not this allele. Naive T cell frequencies varied from one Ag specificity to another but were restrained for a given specificity. Although A2-restricted T cells showed similar repertoire features and antigenic avidities in A2+ and A2- donors, A2 expression had either a positive, neutral, or negative impact on the frequency of A2-restricted naive CD8 T cells, depending on their fine specificity. We also identified in all donors CD4 T cells specific for A2/peptide complexes, whose frequencies were not affected by MHC class I expression, but nevertheless correlated with those of their naive CD8 T cell counterparts. Therefore, both selection by self-MHC and inherent TCR reactivity regulate the frequency of human naive T cell precursors. Moreover this study also suggests that T cell repertoire shaping by a given self-MHC allele is dispensable for generation of immunodominant T cell responses restricted by this particular allele.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.