Semi-invariant TCR Research Articles

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 2mouse TRAV1-TRBV13-2+ MAIT TCR-MR1-5-OP-RU ternary complexes, whose TCRs differ only in the composition of their CDR3β 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.

Abstract 6708: MAIT engagers: Bispecific antibody-mediated redirection of mucosal associated invariant T cells to treat solid tumors

Abstract Background. Using bispecific antibodies (BiXAb) to redirect T cells to engage and kill cancer cells is an efficacious modality in the treatment of haematological cancers but has had limited success in the treatment of solid tumors. Mucosal-Associated Invariant T cells (MAITs) are an abundant subset of non-conventional T-cells with potent cytotoxic capacity (up to 20% of circulating T-cells) that are naturally resident in many tissues and solid tumors. MAIT cells utilize a semi-invariant TCR and recognize bacterial metabolites presented in the context of the MR1 protein. Biomunex has generated bispecific antibodies that bind the MAIT semi-invariant TCR (iTCR) and the HER2 receptor tyrosine kinase expressed on tumor cells. At difference to classical T-cell engagers that activate all T-cell subsets, MAIT engagers only modulate the activity of the cytotoxic MAIT cells leading to efficient tumor control and greater safety profile. Methods. Using the Biomunex proprietary BiXAb platform, bispecific, tetravalent antibodies were generated that target the MAIT iTCR and the HER2. MAIT-cell activation, proliferation and degranulation were followed by gating on MAIT cells within a purified CD8 cell population. Tumor cell lines (varying in [HER2] expression) were co-cultured with MAIT cells and the BiXAbs in several cytotoxic assays (evaluated by Chromium release). Cytokine release was assessed by Legend Plex assays or ELISA. Cytotoxicity of tumor-resident MAITs, from fresh patient samples, was determined by impedence measurements and in patient-derived 3D organoids by The Hub organoids (imaging). For in vivo analysis, sub-cutaneous tumor growth was measured with callipers. Results. MAIT engagers induce the rapid activation, proliferation and degranulation of MAIT cells leading to efficient killing of engaged HER2+ cancer cells with a potency similar to that of a classical HER2-directed TCE (in clinical development). The MAIT engager did not activate the regulatory T cells or other CD4/CD8 subsets. In addition, using PBMCs, total cytokine release was 1000-fold less with the MAIT engager compared to a classical TCE, opening up a large therapeutic window, which should permit effective treatment of solid tumors. Ex vivo cytotoxicity of tumor-resident MAIT cells using dissociated ovarian cancer samples at “real E:T ratios”, MAIT engager-mediated cytotoxicity of patient-derived organoids (CRC) and in vivo evaluation of tumor control will be shown. Conclusions. MAIT cell redirection is a promising approach for the treatment of solid tumors that has the potency and efficacy of a classical TCE but with a significantly improved cytokine safety profile leading to a larger therapeutic window. Citation Format: Simon Edward Plyte, Marie Fraudeau, Dorothee Winterberg, Claire Germain, Camille Rousseau, Gaetano Sodaro, Lise Fenou, Maxime Audin, Alexandre Ivagnes, Han-Heinrich Oberg, Pierre Emmanuel Gerard, Isabelle Navarro-Teulon, Daniela Wesch, Matthias Peipp, Julie Prigent. MAIT engagers: Bispecific antibody-mediated redirection of mucosal associated invariant T cells to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6708.

Abstract 1332: Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies

Abstract Invariant Natural Killer T (iNKT) cells are a unique subset of innate lymphocytes, constituting <1% of human T cells. iNKT cells express a semi-invariant TCR (iTCR) recognizing glycolipids presented by the monomorphic, MHC-like molecule CD1d. Previous studies indicate that due to distinctive TCR constitution and antigen recognition properties, iNKT cells do not induce acute graft-versus-host disease. Based on the CD4 and CD8 expression, mature human iNKT cells can be classified into CD4+CD8-, CD4-CD8- & CD4-CD8+ subsets with overlapping and distinct functions. The functional profiling of CAR19-iNKT cell subsets serves the dual purpose of ensuring their safety and efficacy as a therapeutic intervention. Here we characterized the phenotypic and functional profile of CD4+ and CD4- CAR19-iNKT cells. iNKT cells were isolated with >99% purity from healthy donors' peripheral blood and were transduced with a CD19 CAR-encoding 3rd generation lentivirus. After expansion, the CAR19-iNKT cells were cryopreserved and later analyzed post-thawing. Differential expression of CD27 and PD1 was noted in CAR+CD4+ and CAR+CD4- iNKT cells compared to other markers. For functional profiling, CAR+CD4+ iNKT cells were positively selected, and 24h cytotoxicity assays were performed using several tumor cell lines including SEM, Ramos and C1R-CD1d (+/- α-GalCer). The CAR+CD4- iNKT cells exhibited superior cytotoxicity to multiple tumor cell lines compared to that of CAR+CD4+ iNKT cells from two donors. However, both subsets lysed α-GalCer pulsed C1R-CD1d cells in a comparable manner indicating the potential of these subsets to recognize antigen through the iTCR. Subsequently, we assessed the proliferation ability of the CAR+CD4+ and CAR+CD4- subsets by exposing the cells to three rounds of stimulation, 24h apart, using SEM (CD19+CD1d-) or K562 (CD19-CD1d-) tumor cells with or without IL-15. Proliferation was assessed seven days following the first stimulation. The CAR+CD4+ subset of CAR19-iNKT cells demonstrated faster proliferation than CAR+CD4- cells in the presence of IL-15 after repeated exposure to tumor cells. Ongoing RNA seq analysis of CAR+CD4+/CAR+CD4- cells will further elucidate differences between these subsets. The outcomes of these studies have shown encouraging results, indicating the potential benefit of having diverse subsets among CAR19-iNKT cells for treating CD19+ cancers. The therapeutic potential of CAR+CD4+ iNKT cell subset could be enhanced to match that of CAR+CD4- subset with the use of α-GalCer. In summary, inclusion of both CD4+ and CD4- iNKT cells is critical for the functionality of allogeneic CAR-iNKT cell therapy. Extensive characterization is crucial for clinical translation, but additional research, including preclinical and clinical trials, is essential to determine the safety and effectiveness of CAR19-iNKT cell therapy in real-world applications. Citation Format: Kanagaraju Ponnusamy, Simon Poon, Nicole van der Weerden, Robson Dossa, Michael J. Baker, Mini Bharathan, Anastasios Karadimitris. Allogeneic CD19-directed CAR-iNKT cells and their phenotypic subsets for the treatment of CD19+ hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1332.

Abstract B030: Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models

Abstract Background: Mucosal-associated invariant T cells (MAIT) are unconventional αβT cells with a semi-invariant TCR that recognizes microbially-derived riboflavin metabolites presented by MHC-related protein 1 (MR1). Upon TCR engagement or cytokine stimulation, MAIT cells can rapidly secrete pro-inflammatory cytokines and cytotoxic molecules. The precise role of MAIT cells in the tumor-immune microenvironment remains unclear, as different reports have described them as either beneficial or detrimental to patient prognosis. Considering that MAIT cells constitute a notable proportion (2-4%) of the lung T cell repertoire in both humans and mice, the lung serves as an appropriate setting for ex vivo and in vivo MAIT cell characterization. Objective: To characterize the phenotype of MAIT cells in lung tumor and uninvolved tissue from non-small cell lung cancer (NSCLC) specimens and orthotopic murine models. Results: Surgical resections of primary tumor and normal adjacent tissue from 29 treatment-naïve NSCLC patients were profiled using a comprehensive 31-marker spectral flow cytometry panel. MAIT cells expressing activating and inhibitory receptors including CD69, PD-1, CTLA-4, and CD39, were enriched in tumor tissue. Conversely, cytotoxic MAIT cells expressing granzyme B, granulysin, and CD57, were enriched in normal adjacent tissue. For in vivo studies, lungs of B6-MAITCAST mice, which harbor higher frequencies of MAIT cells compared to conventional mouse strains, were orthotopically injected with CMT167 tumor cells or vehicle control. MAIT cells from lungs injected with CMT167 tumors also expressed higher levels of activating and inhibitory receptors, and decreased levels of granzyme B. Additionally, there was a slight decrease in IFNγ and IL-17A production upon stimulation in vitro with PMA/ionomycin compared to vehicle controls. Conclusion: In the context of lung cancer, MAIT cells are activated but may lose functionality within the tumor-immune microenvironment. Rationale: There is growing interest in the immunotherapeutic potential of unconventional T cells due to their rapid effector functions and non-MHC-restricted nature. By investigating the role of MAIT cells in the tumor microenvironment, we can comprehensively identify their biological advantages and limitations. Citation Format: Stephanie WY Wong, Carolina de Amat Herbozo, Adriana Vukosich-Pennell, Ben X Wang, Pamela S Ohashi, Adrian G Sacher. Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B030.

Perturbation of Invariant Natural Killer T cells by the Clonal Hematopoiesis-related inflammatory environment

Abstract Invariant Natural Killer T (iNKT) cells are T cells characterized by a semi-invariant TCR and NK markers. They respond to glycolipid antigens presented by the major histocompatibility complex (MHC)-like molecule CD1d. Our single-cell transcriptomic profiling of several tissues identified marrow iNKT as a unique subset, possibly modulating marrow homeostasis. However, little is known about their role in hematological malignancies and how the disease-related environment impacts iNKT cells. For example, clonal hematopoiesis of indeterminate potential (CHIP) corresponds to a condition in which mutated hematopoietic cells have an enhanced fitness and expansion, increasing the risk of leukemia and other diseases. Loss of TET2 in CHIP promotes myeloid skewing and inflammation. TET2 is a methycytosine dioxygenase catalyzing DNA demethylation of genes involved in proliferation, differentiation and oncogenesis. However, the impact that TET2 loss has on iNKT lineage output and function is yet to be defined. In our chimeric mouse model of CHIP, only 15% of TET2 mutants differentiate into iNKT. We also observe a decrease of non-transformed iNKT cells in the marrow of our mice, suggesting environmental inhibition. We expect these bystander iNKT cells to be functionally impaired where inflammation leads to their chromatin rewiring potentially contributing to disease. This is addressed by studying CHIP-exposed iNKT transcriptomes and chromatin accessibility. Understanding the molecular basis of the functional reprogramming by the disease-related inflammation will improve clinical outcomes for marrow transplantation and for the development of cellular therapies using iNKT cells.

Ets1 represses a T-bet dependent migration program and stabilizes the iNKT1 adhesome

Abstract iNKT cells are innate-like T cells that recognize glycolipids presented on MHCI-like CD1d using a semi-invariant TCR. Upon activation, iNKTs rapidly produce cytokines to direct the adaptive response towards a T H1, T H2 or T H17 response. The transcription factor ETS1 is important for iNKT cell development, but its roles in iNKT function and homeostasis are not well characterized. Utilizing a novel mouse model for targeted deletion of Ets1 in iNKT1s, we show that ETS1 limits expression of T-bet, the signature transcription factor of the iNKT1 lineage. Ets1 Δ/Δhepatic iNKT1 cells upregulated a gene program associated with migration and had reduced expression of LFA-1 and ICAM-1, receptors critical for retention in the liver. Remarkably, Ets1 Δ/Δliver iNKT1s remained tissue resident but no longer required LFA-1/ICAM-1 for retention. Heterozygous deletion of T-bet abrogated expression of multiple migration-associated genes, restored LFA-1 and ICAM-1, and re-established reliance on LFA-1/ICAM-1 for retention. Our data demonstrate that ETS1 controls the mechanisms of hepatic iNKT1 cell residency by limiting a T-bet-dependent migration and adhesion program. Supported by NIH/NIAID grant R01 AI106352

Abstract 2954: MAIT engagers: An efficacious novel modality in the field of T-cell engagers for the treatment of solid tumors

Abstract Background: Mucosal-Associated Invariant T cells (MAITs) are an abundant subset of non-conventional T-cells with potent cytotoxic capacity (up to 20% of circulating T-cells) that are naturally resident in many tissues and solid tumors. T-cell redirection is a clinically validated approach to treating haematological cancers but has limited success in solid tumors. Classical T-cell engagers (TCE) bind the epsilon chain of the TCR leading to activation of all T-cells. MAIT cells utilize a semi-invariant TCR and recognize bacterial metabolites presented in the context of the MR1 protein. Biomunex has generated bispecific antibodies that bind the MAIT semi-invariant TCR (iTCR) and the HER2 receptor tyrosine kinase expressed on tumor cells. This enables the formation of an efficient immunological synapse and exclusive redirection of MAIT cells to directly kill cancer cells. Methods: Using the Biomunex proprietary BiXAb® platform, bispecific, tetravalent antibodies were generated that target the MAIT iTCR and HER2. Specific binding to the two targets was demonstrated by ELISA, DUAL ELISA and in FACS. In general, BiXAb®-mediated MAIT-cell activation, proliferation and degranulation were followed by gating on MAIT cells within a purified CD8 cell population. Tumor cell lines expressing varying amounts of HER2 were co-cultured with MAIT cells (peripheral and tumor-resident) and the BiXAb®s in several cytotoxic assays which were evaluated by measuring Chromium release, LDH release and FACS. Results: The iTCR x HER2 BiXAb® efficiently binds both target proteins with similar affinities to the parental Mabs and can bind both simultaneously, as judged by Dual ELISA. The iTCR x HER2 BiXAb® binds the MAIT-cell TCR and can bind cancer cells over a wide range of HER2 expression. BiXAb® engagement of MAIT cells and cancer cells leads to rapid activation, proliferation and degranulation of MAIT cells. In a population of PBMCs, only MAIT cells are activated by the BiXAb®. Even at low effector to target ratios (E:T = 2:1), MAIT cells efficiently kill engaged cancer cells in a HER2-dependent manner (over 50% cytotoxicity in 18 hrs). The MAIT-directed BiXAb® does not activate other T-cell subsets and hence has significantly reduced cytokine release when compared to a classical T-cell engager. Conclusions: BiXAb® mediated MAIT cell redirection leads to efficient killing of cancer cells and is a promising new approach for the treatment of solid tumors. The iTCR x HER2 BiXAb® has no impact on the general CD8 or CD4 T-cell population which may address some of the clinical limitations of classical TCEs. Citation Format: Simon Edward Plyte, Marie Fraudeau, Jonathan Grivel, Paloma Hougron, Katja Klausz, Dorothee Winterberg, Britta von Below, Alexandre Ivagnes, Claire Germain, Sebastian Amigorena, Eugene Zhukovsky, Matthias Peipp, Pierre-Emmanuel Gerard, Olivier Lantz, Julie Prigent. MAIT engagers: An efficacious novel modality in the field of T-cell engagers for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2954.

Adoptive Immunotherapy With Engineered iNKT Cells to Target Cancer Cells and the Suppressive Microenvironment.

Invariant Natural Killer T (iNKT) cells are T lymphocytes expressing a conserved semi-invariant TCR specific for lipid antigens (Ags) restricted for the monomorphic MHC class I-related molecule CD1d. iNKT cells infiltrate mouse and human tumors and play an important role in the immune surveillance against solid and hematological malignancies. Because of unique functional features, they are attractive platforms for adoptive cells immunotherapy of cancer compared to conventional T cells. iNKT cells can directly kill CD1d-expressing cancer cells, but also restrict immunosuppressive myelomonocytic populations in the tumor microenvironment (TME) via CD1d-cognate recognition, promoting anti-tumor responses irrespective of the CD1d expression by cancer cells. Moreover, iNKT cells can be adoptively transferred across MHC barriers without risk of alloreaction because CD1d molecules are identical in all individuals, in addition to their ability to suppress graft vs. host disease (GvHD) without impairing the anti-tumor responses. Within this functional framework, iNKT cells are successfully engineered to acquire a second antigen-specificity by expressing recombinant TCRs or Chimeric Antigen Receptor (CAR) specific for tumor-associated antigens, enabling the direct targeting of antigen-expressing cancer cells, while maintaining their CD1d-dependent functions. These new evidences support the exploitation of iNKT cells for donor unrestricted, and possibly off the shelf, adoptive cell therapies enabling the concurrent targeting of cancer cells and suppressive microenvironment.

Emerging Roles of Mucosal-Associated Invariant T Cells in Rheumatology.

Mucosal-associated invariant T (MAIT) cells are an unconventional T cell subset expressing a semi-invariant TCR and recognize microbial riboflavin metabolites presented by major histocompatibility complex class 1-related molecule (MR1). MAIT cells serve as innate-like T cells bridging innate and adaptive immunity, which have attracted increasing attention in recent years. The involvement of MAIT cells has been described in various infections, autoimmune diseases and malignancies. In this review, we first briefly introduce the biology of MAIT cells, and then summarize their roles in rheumatic diseases including systemic lupus erythematosus, rheumatoid arthritis, primary Sjögren’s syndrome, psoriatic arthritis, systemic sclerosis, vasculitis and dermatomyositis. An increased knowledge of MAIT cells will inform the development of novel biomarkers and therapeutic approaches in rheumatology.

Single-Cell Transcriptional Profiling Reveals Signatures of Helper, Effector, and Regulatory MAIT Cells during Homeostasis and Activation.

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

Brief homogeneous TCR signals instruct common iNKT progenitors whose effector diversification is characterized by subsequent cytokine signaling

Innate-like Tcell populations expressing conserved TCRs play critical roles in immunity through diverse developmentally acquired effector functions. Focusing on the prototypical lineage of invariant natural killer T (iNKT) cells, we sought to dissect the mechanisms and timing of fate decisions and functional effector differentiation. Utilizing induced expression of the semi-invariant NKT cell TCR on double positive thymocytes, an initially highly synchronous wave of iNKT cell development was triggered by brief homogeneous TCR signaling. After reaching a uniform progenitor state characterized by IL-4 production potential and proliferation, effector subsets emerged simultaneously, but then diverged toward different fates. While NKT17 specification was quickly completed, NKT1 cells slowly differentiated and expanded. NKT2 cells resembled maturing progenitors, which gradually diminished in numbers. Thus, iNKT subset diversification occurs in dividing progenitor cells without acute TCR input but utilizes multiple active cytokine signaling pathways. These data imply a two-step model of iNKT effector differentiation.

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.

MAIT Cell Activation and Functions.

Mucosal associated invariant T (MAIT) cells are striking in their abundance and their strict conservation across 150 million years of mammalian evolution, implying they must fulfill critical immunological function(s). MAIT cells are defined by their expression of a semi-invariant αβ TCR which recognizes biosynthetic derivatives of riboflavin synthesis presented on MR1. Initial studies focused on their role in detecting predominantly intracellular bacterial and mycobacterial infections. However, it is now recognized that there are several modes of MAIT cell activation and these are related to activation of distinct transcriptional programmes, each associated with distinct functional roles. In this minireview, we summarize current knowledge from human and animal studies of MAIT cell activation induced (1) in an MR1-TCR dependent manner in the context of inflammatory danger signals and associated with antibacterial host defense; (2) in an MR1-TCR independent manner by the cytokines interleukin(IL)-12/-15/-18 and type I interferon, which is associated with antiviral responses; and (3) a recently-described TCR-dependent “tissue repair” programme which is associated with accelerated wound healing in the context of commensal microbiota. Because of this capability for diverse functional responses in diverse immunological contexts, these intriguing cells now appear to be multifunctional effectors central to the interface of innate and adaptive immunity.

Targeting MAIT cells by vaccination with S. enterica serotype Typhimurium to prevent lung infections

Abstract Nearly all currently licensed vaccines elicit antibody responses to induce long-lasting immunity. For intracellular pathogens, to some extent out of reach for antibodies, protective vaccination will require not just antibody production, but also T cell-mediated responses. Mucosal Associated Invariant T (MAIT) cells recognize microbial vitamin B metabolites presented by the MHC-I-like molecule, MR1, via a semi-invariant TCR. These innate-like T cells are present at low levels in mice in mucosal tissues, especially the lung, as well as other sites such as blood, liver and spleen. Previous studies have demonstrated that Salmonella enterica serotype Typhimurium potently expands long-lived MAIT cells to nearly 40% of lung αβ T cells. Using this model, we determined that expansion of MAIT cells requires infection with intracellular bacteria, as an invasion mutant of S. enterica does not induce MAIT cell expansion. Following treatment with an attenuated strain of S. enterica, MAIT cells expand into short-term effector memory (CD127−KLRG1+) and memory precursor (CD127+KLRG1−) populations that differ in cytokine, transcription factor, and metabolic profiles. Following adoptive transfer, CD127+ MAIT cells, but not KLRG1+ cells, protect mice from infection with a different bacterium, Streptococcus pneumoniae, a leading cause of pneumonia and other diseases. Further, vaccination with S. enterica limits S. pneumoniae-induced mortality in mice. These studies demonstrate that some intracellular bacteria induce distinct, long-lived MAIT cell populations, and that these cells are a promising target for vaccines against lung pathogens.

Peripheral blood MAIT cell response to enterotoxigenic E. coli strains H10407 and B7A after controlled human infection models (CHIMs)

Abstract Mucosal associated invariant T (MAIT) cells are an unconventional T cell subset with a semi-invariant TCR that recognizes microbially-derived riboflavin metabolites and elicit broad antimicrobial activity. MAIT cells are implicated in the control of bacterial infections, however, their role in enterotoxigenic E. coli (ETEC) infection, a common cause of traveler’s diarrhea, is unknown. Using multi-color polychromatic flow cytometry and RNA-seq on sorted peripheral MAIT cells of volunteers orally challenged with ETEC strains H10407 (CFA/I+LT+ST+; median time to first loose stool, mtFLS=47h; n=8) or B7A (CS6+ LT+ST+; mtFLS=13h; n=12), we observed that MAIT cells express higher levels of gut-homing molecules a4b7 and CCR9 7-days post-challenge. Moreover, MAIT cells also have an increase in expression of markers of activation CD38 (p=0.003) and cell proliferation Ki-67 (p=0.01) 7-days post-challenge, despite no change in overall frequency. RNA-seq revealed distinct transcriptional signatures 7-days post challenge in both challenge models. Gene set enrichment analysis (GSEA) showed that challenge with both ETEC strains elicit an upregulation of pathways involved in MAIT cell proliferation and division. Several pathways involving IL-1 secretion and the inflammasome were also upregulated after H10406 challenge. Meanwhile, B7A challenge upregulated innate-like pathways such as Natural Killer Cell Activation. These data suggest that MAIT cells are responding during ETEC challenge with upregulation markers of gut homing, activation and proliferation, but undergo differential means of activation after CHIMs with H10407 compared to the more fact-acting B7A ETEC strain.

Detection, Expansion, and Isolation of Human MAIT Cells.

Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like T cells that recognize vitamin B metabolites from a range of microbes presented by MHC class I-related molecules (MR1). The term mucosal-associated invariant T cells derives from the fact that MAIT cells are abundant in the liver and mucosal tissues, and human MAIT cells use a semi-invariant TCR Vα7.2 Jα33 paired with Vβ2 or Vβ13. Here, based on the interaction between MAIT cell and its ligand 5-OP-RU/MR1, we describe the protocols for identification, rapid expansion, and isolation of human MAIT cells.

MAIT Cells in Type 1 Diabetes Mouse Models.

MAIT cells are unconventional T cells expressing a semi-invariant αβ TCR, and they recognize bacterial metabolites via the highly conserved MR1 protein. MAIT cells interact with gut microbiota and literature reports alterations of gut homeostasis in type 1 diabetes (T1D), suggesting the involvement of MAIT cells in T1D. Since NOD mice is a well-established mouse model of T1D, MAIT cells were studied in these mice to evaluate their potential involvement in disease development. This chapter describes the material and methods required to characterize MAIT cells and to determine their function in T1D mouse models.

Circulating Mucosal-Associated Invariant T Cells in a Large Cohort of Healthy Chinese Individuals From Newborn to Elderly.

Mucosal-associated invariant T (MAIT) cells, which are enriched in human blood and express a semi-invariant TCR chain, play important roles in conditions such as infectious diseases and cancer. The influence of age on levels and functional characteristics of circulating MAIT cells have not been fully addressed. Here we have collected blood samples from a large cohort of healthy Chinese individuals from newborn (cord blood) to the elderly and assessed the levels of circulating MAIT cells as well as their phenotype, activation and apoptosis status, and cytokine expression profiles after in vitro stimulation. We found that the frequencies of circulating MAIT cells gradually increased in blood from newborns as they progressed into adulthood (20–40 years old) but then decreased during further progression toward old age (>60 years old). The lowered numbers of circulating MAIT cells in the elderly was correlated with a gradual increase of apoptosis. A majority of circulating MAIT cells expressed the chemokine receptors CCR5 and CCR6, and most also expressed CD8 and CD45RO. Few expressed CD69 in cord blood, but the frequency increased with age. Upon in vitro activation with PMA plus ionomycin or IL12 plus IL18, fewer MAIT cells isolated from the young adult group expressed IFN-γ, IL17A and Granzyme B then cells from other age groups while the proportion of cells that expressed TNF-α was similar. Taken together, our data provide information for guiding the assessment of normal levels and phenotypes of MAIT cells at different ages in healthy individuals and patients.

The Diverse Family of MR1-Restricted T Cells

Mucosal-associated invariant T (MAIT) cells are characterized by a semi-invariant TCR that recognizes vitamin B metabolite Ags presented by the MHC-related molecule MR1. Their Ag restriction determines a unique developmental lineage, imbuing a tissue-homing, preprimed phenotype with antimicrobial function. A growing body of literature indicates that MR1-restricted T cells are more diverse than the MAIT term implies. Namely, it is increasingly clear that TCR α- and TCR β-chain diversity within the MR1-restricted repertoire provides a potential mechanism of Ag discrimination, and context-dependent functional variation suggests a role for MR1-restricted T cells in diverse physiological settings. In this paper, we summarize MR1-restricted T cell biology, with an emphasis on TCR diversity, Ag discrimination, and functional heterogeneity.

Functionality of lung type II NKT cells developing in CD1d−/− animals

Abstract NKT cells are lymphocytes that recognize lipid antigens presented by CD1d. Type I NKT cells with semi-invariant TCR recognize α-galactosylceramide while many type II NKT cells with a more diverse TCR repertoire recognize sulfatide. We developed a stable sulfatide-loaded CD1d tetramer that allows the specific identification of type II NKT cells and found that they are enriched in the lung, a major site of tumor metastasis. An in-depth phenotypical analysis revealed that lung type II NKT cells were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells were either CD4+CD8- or CD4-CD8-. We showed that type II NKT cells expressed markers of myeloid cells, c-Kit, CD11b and Ly6C, even though morphological analysis revealed lymphocytic morphology. In contrast to type I NKT cells, type II NKT existed in PLZF−/− and CD1d−/− mice. Thus, we asked the importance of CD1d in the in vivo and in vitro activation of type II NKT cells. In the steady state, lung type II NKT cells expressed granzyme A but not granzyme B or perforin. By injecting sulfatide in vivo, we observed that WT but not CD1d−/− lung type II NKT cells upregulated granzyme A and Ki67 expression. Using syngeneic WT bone marrow-derived dendritic cells (BMDC) as APCs, ex-vivo stimulation of WT but not CD1d−/− lung type II NKT cells with sulfatide induced the secretion of IFN-γ, IL-13 and IL-17. This was consistent with the expression of specific transcription factors, T-bet and RORγt, by lung type II NKT cells. In conclusion type II NKT cells still develop in CD1d−/− mice but are not functional. The ability to study responses of type II NKT cells in vitro can now help elucidate the mechanism by which these regulatory cells interact with other cells and modulate tumor immunity in the lung.