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1.
mBio ; 13(1): e0386521, 2021 02 22.
Article in English | MEDLINE | ID: mdl-35164552

ABSTRACT

Mucosa-associated invariant T (MAIT) cells play a critical role in antimicrobial defense. Despite increased understanding of their mycobacterial ligands and the clinical association of MAIT cells with tuberculosis (TB), their function in protection against Mycobacterium tuberculosis infection remains unclear. Here, we show that overexpressing key genes of the riboflavin-biosynthetic pathway potentiates MAIT cell activation and results in attenuation of M. tuberculosis virulence in vivo. Further, we observed greater control of M. tuberculosis infection in MAIThi CAST/EiJ mice than in MAITlo C57BL/6J mice, highlighting the protective role of MAIT cells against TB. We also endogenously adjuvanted Mycobacterium bovis BCG with MR1 ligands via overexpression of the lumazine synthase gene ribH and evaluated its protective efficacy in the mouse model of M. tuberculosis infection. Altogether, our findings demonstrate that MAIT cells confer host protection against TB and that overexpression of genes in the riboflavin-biosynthetic pathway attenuates M. tuberculosis virulence. Enhancing MAIT cell-mediated immunity may also offer a novel approach toward improved vaccines against TB. IMPORTANCE Mucosa-associated invariant T (MAIT) cells are an important subset of innate lymphocytes that recognize microbial ligands derived from the riboflavin biosynthesis pathway and mediate antimicrobial immune responses. Modulated MAIT cell responses have been noted in different forms of tuberculosis. However, it has been unclear if increased MAIT cell abundance is protective against TB disease. In this study, we show that augmentation of the mycobacterial MAIT cell ligands leads to higher MAIT cell activation with reduced M. tuberculosis virulence and that elevated MAIT cell abundance confers greater control of M. tuberculosis infection. Our study also highlights the potential of endogenously adjuvanting the traditional BCG vaccine with MR1 ligands to augment MAIT cell activation. This study increases current knowledge on the roles of the riboflavin-biosynthetic pathway and MAIT cell activation in M. tuberculosis virulence and host immunity against TB.


Subject(s)
Mucosal-Associated Invariant T Cells , Mycobacterium tuberculosis , Tuberculosis , Mice , Animals , Mycobacterium tuberculosis/genetics , Ligands , Biosynthetic Pathways , Virulence , Mice, Inbred C57BL , Tuberculosis/microbiology , Mucous Membrane , Riboflavin
2.
Sci Rep ; 7(1): 4622, 2017 07 04.
Article in English | MEDLINE | ID: mdl-28676677

ABSTRACT

Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8+ T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8+ T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8+ T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8+ T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8+ T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and may have practical impact in the development of vaccines and diagnostics.


Subject(s)
Antigens, Bacterial/immunology , CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/immunology , Mycobacterium tuberculosis/metabolism , A549 Cells , Antigen Presentation , Epitopes, T-Lymphocyte/immunology , Glycopeptides/immunology , HEK293 Cells , Humans , Mannose/metabolism , Mycobacterium tuberculosis/immunology , Protein Processing, Post-Translational , Tuberculosis/immunology , HLA-E Antigens
3.
PLoS Pathog ; 12(3): e1005524, 2016 Mar.
Article in English | MEDLINE | ID: mdl-27031111

ABSTRACT

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.


Subject(s)
Antigen Presentation/immunology , Endosomes/metabolism , Histocompatibility Antigens Class I/metabolism , Lymphocyte Activation/immunology , Mycobacterium tuberculosis/immunology , Protein Transport/physiology , Histocompatibility Antigens Class I/immunology , Humans , Minor Histocompatibility Antigens , Mucous Membrane/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocyte Subsets/immunology
4.
PLoS One ; 9(5): e97515, 2014.
Article in English | MEDLINE | ID: mdl-24828674

ABSTRACT

Mycobacterium tuberculosis (Mtb) is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells). Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Endosomes/immunology , Epithelial Cells/immunology , Mycobacterium tuberculosis/immunology , Respiratory Mucosa/immunology , Vacuoles/immunology , CD8-Positive T-Lymphocytes/microbiology , CD8-Positive T-Lymphocytes/pathology , Cell Line , Coculture Techniques , Endosomes/microbiology , Endosomes/pathology , Epithelial Cells/microbiology , Epithelial Cells/pathology , Gene Expression , HLA-B Antigens/genetics , HLA-B Antigens/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Lysosomal Membrane Proteins/genetics , Lysosomal Membrane Proteins/immunology , Primary Cell Culture , Respiratory Mucosa/microbiology , Respiratory Mucosa/pathology , Vacuoles/microbiology , Vacuoles/pathology , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/immunology , rab7 GTP-Binding Proteins , HLA-E Antigens
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