MAIT cell-MR1 reactivity is highly conserved across multiple divergent species.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Potent Immunomodulators Developed from an Unstable Bacterial Metabolite of Vitamin B2 Biosynthesis.

Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17 nM), and their potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.

Mouse mucosal-associated invariant T cell receptor recognition of MR1 presenting the vitamin B metabolite, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil.

Mucosal-associated invariant T (MAIT) cells can elicit immune responses against riboflavin-based antigens presented by the evolutionary conserved MHC class I related protein, MR1. While we have an understanding of the structural basis of human MAIT cell receptor (TCR) recognition of human MR1 presenting a variety of ligands, how the semi-invariant mouse MAIT TCR binds mouse MR1-ligand remains unknown. Here, we determine the crystal structures of 2 mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3ß loops. These mouse MAIT TCRs mediate high affinity interactions with mouse MR1-5-OP-RU and cross-recognize human MR1-5-OP-RU. Similarly, a human MAIT TCR could bind mouse MR1-5-OP-RU with high affinity. This cross-species recognition indicates the evolutionary conserved nature of this MAIT TCR-MR1 axis. Comparing crystal structures of the mouse versus human MAIT TCR-MR1-5-OP-RU complexes provides structural insight into the conserved nature of this MAIT TCR-MR1 interaction and conserved specificity for the microbial antigens, whereby key germline-encoded interactions required for MAIT activation are maintained. This is an important consideration for the development of MAIT cell-based therapeutics that will rely on preclinical mouse models of disease.

Synthetic 5-amino-6-D-ribitylaminouracil paired with inflammatory stimuli facilitates MAIT cell expansion in vivo.

Introduction: Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells, which mediate host immunity to microbial infection by recognizing metabolite antigens derived from microbial riboflavin synthesis presented by the MHC-I-related protein 1 (MR1). Namely, the potent MAIT cell antigens, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU), form via the condensation of the riboflavin precursor 5-amino-6-D-ribitylaminouracil (5-A-RU) with the reactive carbonyl species (RCS) methylglyoxal (MG) and glyoxal (G), respectively. Although MAIT cells are abundant in humans, they are rare in mice, and increasing their abundance using expansion protocols with antigen and adjuvant has been shown to facilitate their study in mouse models of infection and disease. Methods: Here, we outline three methods to increase the abundance of MAIT cells in C57BL/6 mice using a combination of inflammatory stimuli, 5-A-RU and MG. Results: Our data demonstrate that the administration of synthetic 5-A-RU in combination with one of three different inflammatory stimuli is sufficient to increase the frequency and absolute numbers of MAIT cells in C57BL/6 mice. The resultant boosted MAIT cells are functional and can provide protection against a lethal infection of Legionella longbeachae. Conclusion: These results provide alternative methods for expanding MAIT cells with high doses of commercially available 5-A-RU (± MG) in the presence of various danger signals.

Varicella Zoster Virus infects mucosal associated Invariant T cells.

Introduction: Mucosal Associated Invariant T (MAIT) cells are innate-like T cells that respond to conserved pathogen-derived vitamin B metabolites presented by the MHC class I related-1 molecule (MR1) antigen presentation pathway. Whilst viruses do not synthesize these metabolites, we have reported that varicella zoster virus (VZV) profoundly suppresses MR1 expression, implicating this virus in manipulation of the MR1:MAIT cell axis. During primary infection, the lymphotropism of VZV is likely to be instrumental in hematogenous dissemination of virus to gain access to cutaneous sites where it clinically manifests as varicella (chickenpox). However, MAIT cells, which are found in the blood and at mucosal and other organ sites, have yet to be examined in the context of VZV infection. The goal of this study was to examine any direct impact of VZV on MAIT cells. Methods: Using flow cytometry, we interrogated whether primary blood derived MAIT cells are permissive to infection by VZV whilst further analysing differential levels of infection between various MAIT cell subpopulations. Changes in cell surface extravasation, skin homing, activation and proliferation markers after VZV infection of MAIT cells was also assessed via flow cytometry. Finally the capacity of MAIT cells to transfer infectious virus was tested through an infectious center assay and imaged via fluorescence microscopy. Results: We identify primary blood-derived MAIT cells as being permissive to VZV infection. A consequence of VZV infection of MAIT cells was their capacity to transfer infectious virus to other permissive cells, consistent with MAIT cells supporting productive infection. When subgrouping MAIT cells by their co- expression of a variety cell surface markers, there was a higher proportion of VZV infected MAIT cells co-expressing CD4+ and CD4+/CD8+ MAIT cells compared to the more phenotypically dominant CD8+ MAIT cells, whereas infection was not associated with differences in co-expression of CD56 (MAIT cell subset with enhanced responsiveness to innate cytokine stimulation), CD27 (co-stimulatory) or PD-1 (immune checkpoint). Infected MAIT cells retained high expression of CCR2, CCR5, CCR6, CLA and CCR4, indicating a potentially intact capacity for transendothelial migration, extravasation and trafficking to skin sites. Infected MAIT cells also displayed increased expression of CD69 (early activation) and CD71 (proliferation) markers. Discussion: These data identify MAIT cells as being permissive to VZV infection and identify impacts of such infection on co- expressed functional markers.

RIPK3 controls MAIT cell accumulation during development but not during infection.

Cell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.

T-cell receptor αß+ double-negative T cells in the kidney are predominantly extravascular and increase in abundance in response to ischemia-reperfusion injury.

T-cell receptor+ CD4- CD8- double-negative (DN) T cells are a population of T cells present in low abundance in blood and lymphoid organs, but enriched in various organs including the kidney. Despite burgeoning interest in these cells, studies examining their abundance in the kidney have reported conflicting results. Here we developed a flow cytometry strategy to clearly segregate DN T cells from other immune cells in the mouse kidney and used it to characterize their phenotype and response in renal ischemia-reperfusion injury (IRI). These experiments revealed that in the healthy kidney, most DN T cells are located within the renal parenchyma and exhibit an effector memory phenotype. In response to IRI, the number of renal DN T cells is unaltered after 24 h, but significantly increased by 72 h. This increase is not related to alterations in proliferation or apoptosis. By contrast, adoptive transfer studies indicate that circulating DN T cells undergo preferential recruitment to the postischemic kidney. Furthermore, DN T cells show the capacity to upregulate CD8, both in vivo following adoptive transfer and in response to ex vivo activation. Together, these findings provide novel insights regarding the phenotype of DN T cells in the kidney, including their predominant extravascular location, and show that increases in their abundance in the kidney following IRI occur in part as a result of increased recruitment from the circulation. Furthermore, the observation that DN T cells can upregulate CD8 in vivo has important implications for detection and characterization of DN T cells in future studies.

The establishment of a cytomegalovirus -specific CD8+ T-cell threshold by kinetic modeling for the prediction of post-hemopoietic stem cell transplant reactivation.

The dynamic interaction between the CMV virus and host immune response remains obscure, thus hindering the diagnosis and therapeutic management of patients with HSCT. The current diagnosis of CMV viremia depends on viral load estimation. Medical intervention based on viral load, can be unnecessary or poorly timed for many patients. Here we examined the clinical features and blood samples of patients with HSCT and assessed the CMV reactivation kinetics and corresponding CMV antigen-specific T-cell response in individual patients based on a peptide pool stimulation T-cell assay, which showed that CMV-specific CD8+ T cells were more suitable to be a diagnosis indicator for suppressing CMV reactivation. Using ROC analysis, we defined and verified a CMV-specific CD8+ T-cell counts threshold (925 cells/106 PBMCs) as an indicator of CMV reactivation post-HSCT, and suggested that use of this threshold would provide more accurate guidance for prompt medication and better management of CMV infection post-HSCT.

CD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells.

Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.

Human mucosal Vα7.2+ CD161hi T cell distribution at physiologic state and in Helicobacter pylori infection.

Mucosal-associated invariant T (MAIT) cells are innate-like, unconventional T cells that are present in peripheral blood and mucosal surfaces. A clear understanding of how MAIT cells in the mucosae function and their role in host immunity is still lacking. Therefore, our aim was to investigate MAIT cell distribution and their characteristics in the gastrointestinal (GI) mucosal tissue based on Vα7.2+ CD161hi identification. We showed that Vα7.2+ CD161hi T cells are present in both intraepithelial layer and lamina propriae of the GI mucosa, but have different abundance at each GI site. Vα7.2+ CD161hi T cells were most abundant in the duodenum, but had the lowest reactivity to MR1-5-OP-RU tetramers when compared with Vα7.2+ CD161hi T cells at other GI tissue sites. Striking discrepancies between MR1-5-OP-RU tetramer reactive cells and Vα7.2+ CD161hi T cells were observed along each GI tissue sites. Vα7.2+ CD161hi TCR repertoire was most diverse in the ileum. Similar dominant profiles of TRBV usage were observed among peripheral blood, duodenum, ileum, and colon. Some TRBV chains were detected at certain intestinal sites and not elsewhere. The frequency of peripheral blood Vα7.2+ CD161hi T cells correlated with mucosal Vα7.2+ CD161hi T cells in lamina propriae ileum and lamina propriae colon. The frequency of peripheral blood Vα7.2+ CD161hi T cells in Helicobacter pylori-infected individuals was significantly lower than uninfected individuals, but this was not observed with gastric Vα7.2+ CD161hi T cells. This study illustrates the biology of Vα7.2+ CD161hi T cells in the GI mucosa and provides a basis for understanding MAIT cells in the mucosa and MAIT-related GI diseases.

Unconventional T Cell Immunity in the Lungs of Young Children with Cystic Fibrosis.

BACKGROUND: People with Cystic Fibrosis (CF) develop pulmonary inflammation, chronic infection and structural lung damage early in life, with these manifestations being prevalent among preschool children and infants. While early immune events are believed to play critical roles in shaping the progression, severity and disease burden later in life, T cells and their subsets are poorly studied in the CF lung, particularly during the formative early stages of disease. METHODS: Using flow cytometry, we analyzed Mucosal Associated Invariant T (MAIT) cells, γδ T cells, and Natural Killer T (NKT)-like cells in bronchoalveolar lavage (BAL) samples from seventeen children with CF, aged two to six years old. The effect of age, sex and lung infections on the frequencies of these cells in BAL samples was analysed (grouped data were tested for normality and compared by t-test or Kruskal-Wallis analysis). RESULTS: No difference was noted in the proportions of unconventional T cells related to the sex or age of the children. The frequency of γδ T cells and MAIT cells appeared unchanged by infection status. However, viral infections were associated with a significant increase in the proportion of NKT-like cells. CONCLUSIONS: By evaluating T cells in the lungs of children during the early formative stages of CF, this study identified potentially important interactions between these cells and viral pathogens.

The balance of interleukin-12 and interleukin-23 determines the bias of MAIT1 versus MAIT17 responses during bacterial infection.

Mucosal-associated invariant T (MAIT) cells are a major subset of innate-like T cells mediating protection against bacterial infection through recognition of microbial metabolites derived from riboflavin biosynthesis. Mouse MAIT cells egress from the thymus as two main subpopulations with distinct functions, namely, T-bet-expressing MAIT1 and RORγt-expressing MAIT17 cells. Previously, we reported that inducible T-cell costimulator and interleukin (IL)-23 provide essential signals for optimal MHC-related protein 1 (MR1)-dependent activation and expansion of MAIT17 cells in vivo. Here, in a model of tularemia, in which MAIT1 responses predominate, we demonstrate that IL-12 and IL-23 promote MAIT1 cell expansion during acute infection and that IL-12 is indispensable for MAIT1 phenotype and function. Furthermore, we showed that the bias toward MAIT1 or MAIT17 responses we observed during different bacterial infections was determined and modulated by the balance between IL-12 and IL-23 and that these responses could be recapitulated by cytokine coadministration with antigen. Our results indicate a potential for tailored immunotherapeutic interventions via MAIT cell manipulation.

Dual TCR-α Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRß-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRß-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous ß-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.

Exposure to SARS-CoV-2 generates T-cell memory in the absence of a detectable viral infection.

T-cell immunity is important for recovery from COVID-19 and provides heightened immunity for re-infection. However, little is known about the SARS-CoV-2-specific T-cell immunity in virus-exposed individuals. Here we report virus-specific CD4+ and CD8+ T-cell memory in recovered COVID-19 patients and close contacts. We also demonstrate the size and quality of the memory T-cell pool of COVID-19 patients are larger and better than those of close contacts. However, the proliferation capacity, size and quality of T-cell responses in close contacts are readily distinguishable from healthy donors, suggesting close contacts are able to gain T-cell immunity against SARS-CoV-2 despite lacking a detectable infection. Additionally, asymptomatic and symptomatic COVID-19 patients contain similar levels of SARS-CoV-2-specific T-cell memory. Overall, this study demonstrates the versatility and potential of memory T cells from COVID-19 patients and close contacts, which may be important for host protection.

Mouse models illuminate MAIT cell biology.

The field of mucosal-associated invariant T cell (MAIT) biology has grown rapidly since the identification of the vitamin-B-based antigens recognised by these specialised T cells. Over the past few years, our understanding of the complexities of MAIT cell function has developed, as they find their place among the other better known cells of the immune system. Key questions relate to understanding when MAIT cells help, when they hinder or cause harm, and when they do not matter. Exploiting mouse strains that differ in MAIT cell numbers, leveraged by specific detection of MAIT cells using MR1-tetramers, it has now been shown that MAIT cells play important immune roles in settings that include bacterial and viral infections, autoimmune diseases and cancer. We have also learnt much about their development, modes of activation and response to commensal microbiota, and begun to try ways to manipulate MAIT cells to improve disease outcomes. Here we review recent studies that have assessed MAIT cells in models of disease.

When it's good to have MAITs.

This article discusses mucosal-associated invariant T-cell prophylactic and therapeutic vaccination schemes tested by Sakai et al. in mouse models of Mycobacterium tuberculosis infection together with the outcomes and observations.

Antigen Recognition by MR1-Reactive T Cells; MAIT Cells, Metabolites, and Remaining Mysteries.

Mucosal-associated Invariant T (MAIT) cells recognize vitamin B-based antigens presented by the non-polymorphic MHC class I related-1 molecule (MR1). Both MAIT T cell receptors (TCR) and MR1 are highly conserved among mammals, suggesting an important, and conserved, immune function. For many years, the antigens they recognize were unknown. The discovery that MR1 presents vitamin B-based small molecule ligands resulted in a rapid expansion of research in this area, which has yielded information on the role of MAIT cells in immune protection, autoimmune disease and recently in homeostasis and cancer. More recently, we have begun to appreciate the diverse nature of the small molecule ligands that can bind MR1, with several less potent antigens and small molecule drugs that can bind MR1 being identified. Complementary structural information has revealed the complex nature of interactions defining antigen recognition. Additionally, we now view MAIT cells (defined here as MR1-riboflavin-Ag reactive, TRAV1-2+ cells) as one subset of a broader family of MR1-reactive T cells (MR1T cells). Despite these advances, we still lack a complete understanding of how MR1 ligands are generated, presented and recognized in vivo. The biological relevance of these MR1 ligands and the function of MR1T cells in infection and disease warrants further investigation with new tools and approaches.