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.

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.

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.

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.

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.

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.

MR1 recycling and blockade of endosomal trafficking reveal distinguishable antigen presentation pathways between Mycobacterium tuberculosis infection and exogenously delivered antigens.

The MHC-Ib molecule MR1 presents microbial metabolites to MR1-restricted T cells (MR1Ts). Given the ubiquitous expression of MR1 and the high prevalence of human MR1Ts, it is important to understand the mechanisms of MR1-dependent antigen presentation. Here, we show that MR1-dependent antigen presentation can be distinguished between intracellular Mycobacterium tuberculosis (Mtb) infection and exogenously added antigens. Although both Mtb infection and exogenously added antigens are presented by preformed MR1, only exogenously added antigens are capable of reusing MR1 that had been bound to the folic acid metabolite 6-formylpterin (6-FP). In addition, we identify an endosomal trafficking protein, Syntaxin 4, which is specifically involved in the presentation of exogenously delivered antigens but not Mtb-dependent antigen presentation. These data reveal there are multiple ways that MR1 can sample antigens and that MR1-mediated sampling of intracellular Mtb infection is distinguishable from the sampling of exogenously added antigens.

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.

MR1 displays the microbial metabolome driving selective MR1-restricted T cell receptor usage.

MR1-restricted T cells (MR1Ts) are a T cell subset that recognize and mediate host defense to a broad array of microbial pathogens, including respiratory pathogens (e.g., Mycobacterium tuberculosis, Streptococcus pyogenes, and Francisella tularensis) and enteric pathogens (e.g., Escherichia coli and Salmonella species). Mucosal-associated invariant T (MAIT) cells, a subset of MR1Ts, were historically defined by the use of a semi-invariant T cell receptor (TCR) and recognition of small molecules derived from the riboflavin biosynthesis pathway presented on MR1. We used mass spectrometry to identify the repertoire of ligands presented by MR1 from the microbes E. coli and Mycobacterium smegmatis We found that the MR1 ligandome is unexpectedly broad, revealing functionally distinct ligands derived from E. coli and M. smegmatis The identification, synthesis, and functional analysis of mycobacterial ligands reveal that MR1T ligands can be distinguished by MR1Ts with diverse TCR usage. These data demonstrate that MR1 can serve as an immune sensor of the microbial ligandome.

Role of MAIT cells in pulmonary bacterial infection.

Mucosal-associated invariant T (MAIT) cells represent a population of innate T cells that is highly abundant in humans. MAIT cells recognize metabolites of the microbial vitamin B pathway that are presented by the major histocompatibility complex (MHC) class I-related protein MR1. Upon bacterial infection, activated MAIT cells produce diverse cytokines and cytotoxic effector molecules and accumulate at the site of infection, thus, MAIT cells have been shown to be protective against various bacterial infections. Here, we summarize the current knowledge of the role of MAIT cells in bacterial pulmonary infection models.

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.

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.

T cell recognition of Mycobacterium tuberculosis peptides presented by HLA-E derived from infected human cells.

HLA-E is a non-conventional MHC Class I molecule that has been recently demonstrated to present pathogen-derived ligands, resulting in the TCR-dependent activation of αß CD8+ T cells. The goal of this study was to characterize the ligandome displayed by HLA-E following infection with Mycobacterium tuberculosis (Mtb) using an in-depth mass spectrometry approach. Here we identified 28 Mtb ligands derived from 13 different source proteins, including the Esx family of proteins. When tested for activity with CD8+ T cells isolated from sixteen donors, nine of the ligands elicited an IFN-γ response from at least one donor, with fourteen of 16 donors responding to the Rv0634A19-29 peptide. Further evaluation of this immunodominant peptide response confirmed HLA-E restriction and the presence of Rv0634A19-29-reactive CD8+ T cells in the peripheral blood of human donors. The identification of an Mtb HLA-E ligand that is commonly recognized may provide a target for a non-traditional vaccine strategy.

HLA-E Presents Glycopeptides from the Mycobacterium tuberculosis Protein MPT32 to Human CD8+ T cells.

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.

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.

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

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

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.