Chronic Obstructive Pulmonary Disease and Cigarette Smoke Lead to Dysregulated Mucosal-associated Invariant T-Cell Activation.

Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. Although cigarette smoke is the leading risk factor for COPD, the mechanisms driving the development of COPD in only a subset of smokers are incompletely understood. Lung-resident mucosal-associated invariant T (MAIT) cells play a role in microbial infections and inflammatory diseases. The role of MAIT cells in COPD pathology is unknown. Here, we examined MAIT cell activation in response to cigarette smoke-exposed primary human bronchial epithelial cells (BECs) from healthy, COPD, or smoker donors. We observed significantly higher baseline MAIT cell responses to COPD BECs than healthy BECs. However, infected COPD BECs stimulated a smaller fold increase in MAIT cell response despite increased microbial infection. For all donor groups, cigarette smoke-exposed BECs elicited reduced MAIT cell responses; conversely, cigarette smoke exposure increased ligand-mediated MR1 surface translocation in healthy and COPD BECs. Our data demonstrate that MAIT cell activation is dysregulated in the context of cigarette smoke and COPD. MAIT cells could contribute to cigarette smoke- and COPD-associated inflammation through inappropriate activation and reduced early recognition of bacterial infection, contributing to microbial persistence and COPD exacerbations.

MR1-dependent antigen presentation.

MR1 is a non-classical class I molecule that is highly conserved among mammals. Though discovered in 1995, only recently have MR1 ligands and antigens for MR1-restricted T cells been described. Unlike the traditional class I molecules HLA-A, -B, and -C, little MR1 is on the cell surface. Rather, MR1 resides in discrete intracellular vesicles and the endoplasmic reticulum, and can present non-peptidic small molecules such as those found in the riboflavin biosynthesis pathway. Since mammals do not synthesize riboflavin, MR1 can serve as a sensor of the microbial metabolome and could be key to the early detection of intracellular infection. This review will summarize the current understanding of MR1-dependent antigen presentation.

Riboflavin Metabolism Variation among Clinical Isolates of Streptococcus pneumoniae Results in Differential Activation of Mucosal-associated Invariant T Cells.

Streptococcus pneumoniae is an important bacterial pathogen that causes a range of noninvasive and invasive diseases. The mechanisms underlying variability in the ability of S. pneumoniae to transition from nasopharyngeal colonization to disease-causing pathogen are not well defined. Mucosal-associated invariant T (MAIT) cells are prevalent in mucosal tissues such as the airways and are believed to play an important role in the early response to infection with bacterial pathogens. The ability of MAIT cells to recognize and contain infection with S. pneumoniae is not known. In the present study, we analyzed MAIT-cell responses to infection with clinical isolates of S. pneumoniae serotype 19A, a serotype linked to invasive pneumococcal disease. We found that although MAIT cells were capable of responding to human dendritic and airway epithelial cells infected with S. pneumoniae, the magnitude of response to different serotype 19A isolates was determined by genetic differences in the expression of the riboflavin biosynthesis pathway. MAIT-cell release of cytokines correlated with differences in the ability of MAIT cells to respond to and control S. pneumoniae in vitro and in vivo in a mouse challenge model. Together, these results demonstrate first that there are genetic differences in riboflavin metabolism among clinical isolates of the same serotype and second that these likely determine MAIT-cell function in response to infection with S. pneumoniae. These differences are critical when considering the role that MAIT cells play in early responses to pneumococcal infection and determining whether invasive disease will develop.

MAIT cells and microbial immunity.

Mucosal-associated invariant T (MAIT) cells, the most abundant T-cell subset in humans, are increasingly being recognized for their importance in microbial immunity. MAIT cells accumulate in almost every mucosal tissue examined, including the lung, liver and intestinal tract, where they can be activated through T-cell receptor (TCR) triggering as well as cytokine stimulation in response to a host of microbial products. In this review, we specifically discuss MAIT cell responses to bacterial and fungal infections, with a focus on responses that are both MR1-dependent and -independent, the evidence for diversity in MAIT TCR usage in response to discrete microbial products, protective immunity induced by MAIT cells, and MAIT cell antimicrobial functions in the context of these infections.

Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells.

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.

The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence.

The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosismmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity.

Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

Human mucosal associated invariant T cells detect bacterially infected cells.

Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.

Phagocytosis is a primary determinant of pulmonary clearance of clinical Klebsiella pneumoniae isolates.

Introduction: Klebsiella pneumoniae (Kp) is a common cause of hospital-acquired pneumonia. Although previous studies have suggested that evasion of phagocytic uptake is a virulence determinant of Kp, few studies have examined phagocytosis sensitivity in clinical Kp isolates. Methods: We screened 19 clinical respiratory Kp isolates that were previously assessed for mucoviscosity for their sensitivity to macrophage phagocytic uptake, and evaluated phagocytosis as a functional correlate of in vivo Kp pathogenicity. Results: The respiratory Kp isolates displayed heterogeneity in the susceptibility to macrophage phagocytic uptake, with 14 out of 19 Kp isolates displaying relative phagocytosis-sensitivity compared to the reference Kp strain ATCC 43816, and 5 out of 19 Kp isolates displaying relative phagocytosis-resistance. Intratracheal infection with the non-mucoviscous phagocytosis-sensitive isolate S17 resulted in a significantly lower bacterial burden compared to infection with the mucoviscous phagocytosis-resistant isolate W42. In addition, infection with S17 was associated with a reduced inflammatory response, including reduced bronchoalveolar lavage fluid (BAL) polymorphonuclear (PMN) cell count, and reduced BAL TNF, IL-1ß, and IL-12p40 levels. Importantly, host control of infection with the phagocytosis-sensitive S17 isolate was impaired in alveolar macrophage (AM)-depleted mice, whereas AM-depletion had no significant impact on host defense against infection with the phagocytosis-resistant W42 isolate. Conclusion: Altogether, these findings show that phagocytosis is a primary determinant of pulmonary clearance of clinical Kp isolates.

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 Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle.

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-gamma release by CD8(+) T cell clones, we examined the processing and presentation pathway for two Mtb-derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER-golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER-golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER-localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb-derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens.

Construction and validation of an ultraviolet germicidal irradiation system using locally available components.

Coronavirus disease (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is responsible for a global pandemic characterized by high transmissibility and morbidity. Healthcare workers (HCWs) are at risk of contracting COVID-19, but this risk has been mitigated through the use of personal protective equipment such as N95 Filtering Facepiece Respirators (FFRs). At times the high demand for FFRs has exceeded supply, placing HCWs at increased exposure risk. Effective FFR decontamination of many FFR models using ultraviolet-C germicidal irradiation (UVGI) has been well-described, and could maintain respiratory protection for HCWs in the face of supply line shortages. Here, we detail the construction of an ultraviolet-C germicidal irradiation (UVGI) device using previously existing components available at our institution. We provide data on UV-C dosage delivered with our version of this device, provide information on how users can validate the UV-C dose delivered in similarly constructed systems, and describe a simple, novel methodology to test its germicidal effectiveness using in-house reagents and equipment. As similar components are readily available in many hospitals and industrial facilities, we provide recommendations on the local construction of these systems, as well as guidance and strategies towards successful institutional implementation of FFR decontamination.

Augmentation of the Riboflavin-Biosynthetic Pathway Enhances Mucosa-Associated Invariant T (MAIT) Cell Activation and Diminishes Mycobacterium tuberculosis Virulence.

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.

Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection.

The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.

Covering All the Bases: Complementary MR1 Antigen Presentation Pathways Sample Diverse Antigens and Intracellular Compartments.

The ubiquitously expressed, monomorphic MHC class Ib molecule MHC class I-related protein 1 (MR1) presents microbial metabolites to mucosal-associated invariant T (MAIT) cells. However, recent work demonstrates that both the ligands bound by MR1 and the T cells restricted by it are more diverse than originally thought. It is becoming increasingly clear that MR1 is capable of presenting a remarkable variety of both microbial and non-microbial small molecule antigens to a diverse group of MR1-restricted T cells (MR1Ts) and that the antigen presentation pathway differs between exogenously delivered antigen and intracellular microbial infection. These distinct antigen presentation pathways suggest that MR1 shares features of both MHC class I and MHC class II antigen presentation, enabling it to sample diverse intracellular compartments and capture antigen of both intracellular and extracellular origin. Here, we review recent developments and new insights into the cellular mechanisms of MR1-dependent antigen presentation with a focus on microbial MR1T cell antigens.

Generation of MR1-Restricted T Cell Clones by Limiting Dilution Cloning of MR1 Tetramer+ Cells.

Tetramers are a powerful tool for identification of T cell subsets that are restricted by specific antigen presenting molecules and their cognate antigens. The generation of T cell clones from specific T cell subsets allows for further investigation of the phenotype and function of these cells. Here, we describe a method for sorting and cloning of MR1-restricted T cells using the MR1/5-OP-RU tetramer. This protocol can be easily modified to enrich for expansion of specific or unique subsets of MR1-restricted T cell clones from any tissue to further characterize the phenotype and function of those cells.

Rab6 regulates recycling and retrograde trafficking of MR1 molecules.

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset important in the early response to bacterial and viral lung pathogens. MAIT cells recognize bacterial small molecule metabolites presented on the Class I-like molecule MR1. As with other Class I and Class II molecules, MR1 can likely sample ligands in the intracellular environment through multiple cellular pathways. Rab6, a small GTPase that regulates a number of endosomal trafficking pathways including retrograde transport to the trans-Golgi network (TGN), is involved in the presentation of ligands from Mycobacterium tuberculosis (Mtb) to MAIT cells. The Rab6-mediated trafficking pathway contains endosomal compartments that share features with the Mtb intracellular compartment. Using inducible expression of MR1, this study demonstrates that Rab6 regulates the recycling of MR1 molecules from the cell surface through endosomal trafficking compartments to the TGN. This Rab6-dependent pool of recycled MR1, which is available for reloading with ligands from bacterial pathogens like Mtb, may be important for early recognition of infected cells by MAIT cells in the lung.

Alternative splicing of MR1 regulates antigen presentation to MAIT cells.

Mucosal Associated Invariant T (MAIT) cells can sense intracellular infection by a broad array of pathogens. These cells are activated upon encountering microbial antigen(s) displayed by MR1 on the surface of an infected cell. Human MR1 undergoes alternative splicing. The full-length isoform, MR1A, can activate MAIT cells, while the function of the isoforms, MR1B and MR1C, are incompletely understood. In this report, we sought to characterize the expression and function of these splice variants. Using a transcriptomic analysis in conjunction with qPCR, we find that that MR1A and MR1B transcripts are widely expressed. However only MR1A can present mycobacterial antigen to MAIT cells. Coexpression of MR1B with MR1A decreases MAIT cell activation following bacterial infection. Additionally, expression of MR1B prior to MR1A lowers total MR1A abundance, suggesting competition between MR1A and MR1B for either ligands or chaperones required for folding and/or trafficking. Finally, we evaluated CD4/CD8 double positive thymocytes expressing surface MR1. Here, we find that relative expression of MR1A/MR1B transcript is associated with the prevalence of MR1 + CD4/CD8 cells in the thymus. Our results suggest alternative splicing of MR1 represents a means of regulating MAIT activation in response to microbial ligand(s).

Homegrown Ultraviolet Germicidal Irradiation for Hospital-Based N95 Decontamination during the COVID-19 Pandemic.

Coronavirus disease (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is responsible for the 2020 global pandemic and characterized by high transmissibility and morbidity. Healthcare workers (HCWs) are at risk of contracting COVID-19, and this risk is mitigated through the use of personal protective equipment such as N95 Filtering Facepiece Respirators (FFRs). The high demand for FFRs is not currently met by global supply chains, potentially placing HCWs at increased exposure risk. Effective FFR decontamination modalities exist, which could maintain respiratory protection for HCWs in the midst of the current pandemic, through the decontamination and re-use of FFRs. Here, we present a locally-implemented ultraviolet-C germicidal irradiation (UVGI)-based FFR decontamination pathway, utilizing a home-built UVGI array assembled entirely with previously existing components available at our institution. We provide recommendations on the construction of similar systems, as well as guidance and strategies towards successful institutional implementation of FFR decontamination.