MR1-restricted T Cells Research Articles

Conserved allomorphs of MR1 drive the specificity of MR1-restricted TCRs.

Major histocompatibility complex class-1-related protein (MR1), unlike human leukocyte antigen (HLA) class-1, was until recently considered to be monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs specific to tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment. The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity. The TCR from a MR1-restricted T-cell clone, MC.7.G5, with reported cancer specificity and pan-cancer activity, was cloned and expressed in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells. Functional activity of 7G5.TCR-T was demonstrated using cytotoxicity assays and by measuring cytokine release after co-culture with cancer cell lines with or without loading of previously described MR1 ligands. MR1 allele sequencing was undertaken after the amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI, USA) or Epistem Ltd (Manchester, UK). The TCR cloned from MC.7.G5 retained MR1-restricted functional cytotoxicity as 7G5.TCR-T. However, activity was not pan-cancer, as initially reported with the clone MC.7.G5. Recognition was restricted to cells expressing a SNV of MR1 (MR1*04) and was not cancer-specific. 7G5.TCR-T and 7G5-like TCR-T cells reacted to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity could be overcome by expressing supraphysiological levels of the wild-type MR1 (MR1*01) in cell lines. Healthy individuals harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described "cancer-specific" MR1-restricted TCRs need further validation, covering conserved allomorphs of MR1. Ligands require identification to ensure targeting MR1 is restricted to those specific to cancer and not normal tissues. For the wider field of immunology and transplant biology, the observation that MR1*04 may behave as an alloantigen warrants further study. .

MR1-restricted T cell clonotypes are associated with "resistance" to Mycobacterium tuberculosis infection.

T cells are required for protective immunity against Mycobacterium tuberculosis. We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to "resist" M. tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ-independent T cell responses to M. tuberculosis-specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and "resistance" to M. tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M. tuberculosis infection.

MR1 antigen presentation to MAIT cells and other MR1-restricted T cells.

MHC antigen presentation plays a fundamental role in adaptive and semi-invariant T cell immunity. Distinct MHC molecules bind antigens that differ in chemical structure, origin and location and present them to specialized T cells. MHC class I-related protein1 (MR1) presents a range of small molecule antigens to MR1-restricted T (MR1T) lymphocytes. The best studied MR1 ligands are derived from microbial metabolism and are recognized by a major class of MR1T cells known as mucosal-associated invariant T (MAIT) cells. Here, we describe the MR1 antigen presentation pathway: the known types of antigens presented by MR1, the location where MR1-antigen complexes form, the route followed by the complexes to the cell surface, the mechanisms involved in termination of MR1 antigen presentation and the accessory cellular proteins that comprise the MR1 antigen presentation machinery. The current road map of the MR1 antigen presentation pathway reveals potential strategies for therapeutic manipulation of MR1T cell function and provides a foundation for further studies that will lead to a deeper understanding of MR1-mediated immunity.

MR1-restricted T cell clonotypes are associated with resistance to Mycobacterium tuberculosisinfection

Abstract T-cells are a critical facet of the adaptive immune system and have been shown in both human and animal studies to be essential for control of Mycobacterium tuberculosis (Mtb) infection. T-cells recognize foreign antigens presented by infected cells via a unique T-cell receptor (TCR). Understanding the specific TCRs that mediate recognition and clearance of Mtb-infected cells is essential to inform vaccine design. We recently described a cohort of Ugandan household contacts of tuberculosis cases that appear to ‘resist’ Mtb infection (RSTRs) and showed that these individuals harbor IFN-γ independent T-cell responses to Mtb peptide antigens. The role of unconventional T-cells that recognize non-peptide antigens, such as gd T-cells, CD1-restricted and MR1-restricted T-cells (MR1T), in “resistance” is unknown. In this study, we aimed to characterize the frequencies, functional programs, and repertoire of unconventional T-cells in RSTRs in comparison with latently infected (LTBI) controls. We used multi-parameter flow cytometry, single cell RNA and TCR sequencing and immunosequencing to address these gaps. We observed a 1.65-fold increase in frequency of circulating MR1T cells among RSTRs in comparison with LTBI (p=0.03). Single-cell RNA-sequencing of 18,251 sorted MR1T cells revealed 5,150 clonotypes expressing a common transcriptional program, the majority of which were private. Immunosequencing of the TCR-α/d repertoire revealed several TCRα clonotypes that were expanded in RSTRs (p <0.01), including at least two MR1T clonotypes. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between MR1 clonotypes and ‘resistance’ to Mtb infection. This work was supported by grants from the NIH (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri) and the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn and GH-VAP-IS-ID5 to Seshadri).

Deaza-modification of MR1 ligands modulates recognition by MR1-restricted T cells

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8-d-ribityllumazine (DMRL) to generate 6,7-dimethyl-8-d-ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-γ response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-γ production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells.

The burgeoning role of MR1-restricted T-cells in infection, cancer and autoimmune disease.

MR1 is a ubiquitously expressed, monomorphic antigen presenting molecule that has been largely preserved throughout mammalian evolution. The primary role of MR1 is to present conserved microbial metabolites to highly abundant mucosal-associated invariant T (MAIT) cells. The role of MAIT cells and other MR1-restricted T cells (MR1T) has been recently extended to immunomodulation during cancer. MR1Ts have also been implicated in autoimmune disease. The highly conserved nature of MR1 across the human population is in stark contrast to the MHC molecules recognised by conventional αβ T-cells, therefore MR1Ts may form fertile ground for the development of pan-population T-cell immunotherapeutics for a wide range of important morbidities.

T cell receptor diversity, specificity and promiscuity of functionally heterogeneous human MR1-restricted T cells

The monomorphic MHC-class I-like molecule, MR1, presents small metabolites to T cells. MR1 is the restriction element for microbe-reactive mucosal-associated invariant T (MAIT) cells. MAIT cells have limited TCR usage, including a semi-invariant TCR alpha chain and express high levels of CD161 and CD26. In addition to microbial lumazine metabolites, recent studies have demonstrated that MR1 is able to capture a variety of diverse chemical entities including folate-derivatives, a number of drug-like and other synthetic small molecules, and as yet undefined compounds of self-origin. This capacity of MR1 to bind distinct ligands likely accounts for the recent identification of additional, non-canonical, subsets of MR1-restricted T (MR1T) cells. These subsets can be defined based on their ability to recognize diverse microbes as well as their reactivity to non-microbial cell-endogenous ligands, including tumor-associated antigens. Herein, we will discuss our current understanding of MR1T cell diversity in terms of TCR usage, ligand recognition and functional attributes.

MR1-Restricted T Cells with MAIT-like Characteristics Are Functionally Conserved in the Pteropid Bat Pteropus alecto.

SummaryBats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

MR1-restricted T cells: the new dawn of cancer immunotherapy.

Cancer immunotherapy has recently undergone rapid development into a validated therapy for clinical use. The adoptive transfer of engineered autologous T cells, such as chimeric antigen receptor (CAR) T cells, has been remarkably successful in patients with leukemia and lymphoma with cluster of differentiation (CD)19 expression. Because of the higher number of antigen choices and reduced incidence of cytokine release syndrome (CRS) than CAR-T cells, T cell receptor (TCR)-T cells are also considered a promising immunotherapy. More therapeutic targets for other cancers need to be explored due to the human leukocyte antigen (HLA)-restricted recognition of TCR-T. Major histocompatibility complex (MHC), class I-related (MR1)-restricted T cells can recognize metabolites presented by MR1 in the context of host cells infected with pathogens. MR1 is expressed by all types of human cells. Recent studies have shown that one clone of a MR1-restricted T (MR1-T) cell can recognize many types of cancer cells without HLA-restriction. These studies provide additional information on MR1-T cells for cancer immunotherapy. This review describes the complexity of MR1-T cell TCR in diseases and the future of cancer immunotherapy.

Postnatal Expansion, Maturation, and Functionality of MR1T Cells in Humans.

MR1-restricted T (MR1T) cells are defined by their recognition of metabolite antigens presented by the monomorphic MHC class 1-related molecule, MR1, the most highly conserved MHC class I related molecule in mammalian species. Mucosal-associated invariant T (MAIT) cells are the predominant subset of MR1T cells expressing an invariant TCR α-chain, TRAV1-2. These cells comprise a T cell subset that recognizes and mediates host immune responses to a broad array of microbial pathogens, including Mycobacterium tuberculosis. Here, we sought to characterize development of circulating human MR1T cells as defined by MR1-5-OP-RU tetramer labeling and of the TRAV1-2+ MAIT cells defined by expression of TRAV1-2 and high expression of CD26 and CD161 (TRAV1-2+CD161++CD26++ cells). We analyzed postnatal expansion, maturation, and functionality of peripheral blood MR1-5-OP-RU tetramer+ MR1T cells in cohorts from three different geographic settings with different tuberculosis (TB) vaccination practices, levels of exposure to and infection with M. tuberculosis. Early after birth, frequencies of MR1-5-OP-RU tetramer+ MR1T cells increased rapidly by several fold. This coincided with the transition from a predominantly CD4+ and TRAV1-2− population in neonates, to a predominantly TRAV1-2+CD161++CD26++ CD8+ population. We also observed that tetramer+ MR1T cells that expressed TNF upon mycobacterial stimulation were very low in neonates, but increased ~10-fold in the first year of life. These functional MR1T cells in all age groups were MR1-5-OP-RU tetramer+TRAV1-2+ and highly expressed CD161 and CD26, markers that appeared to signal phenotypic and functional maturation of this cell subset. This age-associated maturation was also marked by the loss of naïve T cell markers on tetramer+ TRAV1-2+ MR1T cells more rapidly than tetramer+TRAV1-2− MR1T cells and non-MR1T cells. These data suggest that neonates have infrequent populations of MR1T cells with diverse phenotypic attributes; and that exposure to the environment rapidly and preferentially expands the MR1-5-OP-RU tetramer+TRAV1-2+ population of MR1T cells, which becomes the predominant population of functional MR1T cells early during childhood.

MR1-Restricted T Cells Are Unprecedented Cancer Fighters.

Non-polymorphic MHC class I-related molecule MR1 presents antigenic bacterial metabolites to mucosal-associated invariant T (MAIT) cells and self-antigens to MR1-restricted T (MR1T) cells. Both MR1-restricted T cell populations are readily identified in healthy individuals, with MAIT cells accounting for 1–10% of circulating T cells, while MR1T cells have frequencies comparable to peptide-specific T cells (<0.1%). Self-reactive MR1T cells display a heterogeneous phenotype, and are capable of releasing both TH1 and TH2 cytokines, supporting not only activation of inflammation but also contributing to its regulation. Importantly, MR1T cells recognize and kill a diverse range of MR1-expressing tumor cells. On the other hand, evidence suggests MAIT cells augment cancer growth and metastases. This review addresses the potential role of MR1-restricted T cells in controlling tumor cells, facilitating their elimination and regulating cancer immunity. We also discuss therapeutic opportunities surrounding MR1-restricted T cells in cancer.

Identification of MAIT Cells in Human Term Decidua

Abstract Mucosal-associated invariant T (MAIT) cells are a CD161+Vα7+ MR1-restricted T-cell subset, that has a complex relationship in regulating the microbiome and defense from invasive bacteria. Recent discovery of a placental microbiome, and genital mucosa MAIT cells, raises the possibility that MAIT are present at the maternal fetal interface and perform regulatory functions in this setting. As a first step, we analyzed human term decidua to detect MAIT cells and analyze their transcriptional programming. Decidua was dissected from human term placentas and mononuclear cells (MCs) were isolated by mechanical (GentleMACS) and enzymatic (Collagenase V, DNAse I) disruption. MCs labeled by flurochrome-conjugated antibodies against CD3, 4, 8, 19, 27, 45RO, 161, and TCR Vα7.2 and intracellular Eomes, RORγt, and T-bet. Data was acquired on a 5 laser (355, 405, 488, 562, 633nm), 18-color BD Fortessa cytometer and analyzed with FlowJo 10.1r7. Data revealed MAIT (CD3+CD161+Vα7.2+) cells in human term decidua, particularly of a memory CD8+ phenotype (CD27+CD45R0+), while fewer of CD4-CD8- and scant CD4+ cells were seen. Transcription factor expression analysis demonstrated uniform expression of PLZF, RORγt, and EOMES in phenotypic MAIT cells, confirming MAIT programming. MAIT cells are present in human term decidua and add a new dimension to the immunological complex regulating the maternal-fetal interface. Mucosal immune dysregulation is implicated in pregnancy pathology, and our findings open a novel area to mechanistic investigation.

Human TRAV1-2-negative MR1-restricted T cells detect S. pyogenes and alternatives to MAIT riboflavin-based antigens.

Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2+ MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2+ clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.

Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in chronic HIV-1 infection

AbstractMucosal-associated invariant T (MAIT) cells are an evolutionarily conserved antimicrobial MR1-restricted T-cell subset. MAIT cells are CD161+, express a Vα7.2 TCR, are primarily CD8+ and numerous in blood and mucosal tissues. However, their role in HIV-1 infection is unknown. In this study, we found levels of MAIT cells to be severely reduced in circulation in patients with chronic HIV-1 infection. Residual MAIT cells were highly activated and functionally exhausted. Their decline was associated with time since diagnosis, activation levels, and the concomitant expansion of a subset of functionally impaired CD161− Vα7.2+ T cells. Such cells were generated in vitro by exposure of MAIT cells to Escherichia coli. Notably, whereas the function of residual MAIT cells was at least partly restored by effective antiretroviral therapy, levels of MAIT cells in peripheral blood were not restored. Interestingly, MAIT cells in rectal mucosa were relatively preserved, although some of the changes seen in blood were recapitulated in the mucosa. These findings are consistent with a model in which the MAIT-cell compartment, possibly as a result of persistent exposure to microbial material, is engaged, activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.