MR1- and HLA-E-Dependent Antigen Presentation of Mycobacterium tuberculosis.

MR1 and HLA-E are highly conserved nonclassical antigen-presenting molecules. They can present antigens derived from Mycobacterium tuberculosis to a distinct subset of MR1-restricted or HLA-restricted CD8+ T cells. MR1 presents small microbial metabolites, and HLA-E presents peptides and glycopeptides. In this review, we will discuss the current understanding of MR1 and HLA-E antigen presentation in the context of Mycobacterium tuberculosis infection.

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.

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.

Prevalence of pulmonary artery dilation in non-cystic fibrosis bronchiectasis: a computed tomography analysis from a cohort of the US Bronchiectasis and Nontuberculous Mycobacteria Research Registry.

Although pulmonary artery (PA) dilation is independently associated with significant morbidity and mortality in patients with pulmonary diseases irrespective of diagnosed pulmonary hypertension, its relationship with nontuberculous mycobacteria (NTM) is unknown. The Bronchiectasis and NTM Research Registry is a multicenter registry created to foster research in non-cystic fibrosis (CF) bronchiectasis and NTM lung disease. The majority of patients with non-CF bronchiectasis at Oregon Health & Science University have NTM infections. To determine the prevalence of PA dilation in these patients and its association with supplemental oxygen use, severity of bronchiectasis, tobacco use, and NTM in the sputum culture, we evaluated the chest computed tomography (CT) scans from 321 patients in a cross-sectional analysis. We measured the severity of bronchiectasis by applying modified Reiff criteria and measured the diameters of the PA and aorta (Ao), with PA dilation defined as a PA:Ao ratio >0.9. In our cohort, the mean age was 67.3 years and 83.2% were female. The mean modified Reiff score was 7.1, indicating moderate disease severity. Forty-two patients (13.1%) were found to have PA dilation. PA dilation was positively associated with the use of supplemental oxygen (P<0.001), but there was no association between PA dilation and NTM infection.

Prevalence of pulmonary artery dilation in non-cystic fibrosis bronchiectasis: A CT analysis from a cohort of the US Bronchiectasis and Nontuberculous Mycobacteria Research Registry.

Although pulmonary artery (PA) dilation is independently associated with significant morbidity and mortality in patients with pulmonary diseases irrespective of diagnosed pulmonary hypertension, its relationship to nontuberculous mycobacteria (NTM) is unknown. To determine the prevalence of PA dilation in patients with NTM-predominant non-CF bronchiectasis, we evaluated the chest computed tomography (CT) scans from 321 patient in the United States based Bronchiectasis and NTM Research Registry. The majority of our cohort had NTM infection. We measured the severity of bronchiectasis using modified Reiff criteria and measured the diameters of the PA and aorta (Ao), with PA dilation defined as a PA:Ao ratio > 0.9. Forty-two patients (13%) were found to have PA dilation. PA dilation was positively associated with the use of supplemental oxygen (p < 0.001), but there was no association between PA dilation and NTM infection.

Calcium Signaling in MR1-Dependent Antigen Presentation of Mycobacterium tuberculosis.

MR1 is a ubiquitously expressed MHC-Ib molecule that presents microbial metabolites to MR1-restricted T cells, but there are differences in the antigen presentation pathway of an intracellular microbe compared to exogenous antigen. We have shown the importance of endosomal trafficking proteins in MR1-dependent presentation of Mycobacterium tuberculosis (Mtb). Two pore channels (TPCs) are endosomal calcium channels that regulate endosomal trafficking. Due to their location on endosomes, we hypothesized that TPCs could be required for MR1-dependent presentation of antigens derived from the intracellular microbe Mtb. We found that TPCs are critical for the presentation of Mtb by MR1; inhibition of TPCs had no effect on MR1 presentation of extracellular (exogenous) antigens, HLA-B presentation, or HLA-II presentation. Finally, we found that the calcium sensitive trafficking protein Synaptotagmin 7 was also key in the presentation of Mtb by MR1. This calcium-dependent endosomal pathway is a novel mechanism by which the immune system can sample intracellular antigens.

Human lung-resident mucosal-associated invariant T cells are abundant, express antimicrobial proteins, and are cytokine responsive.

Mucosal-associated Invariant T (MAIT) cells are an innate-like T cell subset that recognize a broad array of microbial pathogens, including respiratory pathogens. Here we investigate the transcriptional profile of MAIT cells localized to the human lung, and postulate that MAIT cells may play a role in maintaining homeostasis at this mucosal barrier. Using the MR1/5-OP-RU tetramer, we identified MAIT cells and non-MAIT CD8+ T cells in lung tissue not suitable for transplant from human donors. We used RNA-sequencing of MAIT cells compared to non-MAIT CD8+ T cells to define the transcriptome of MAIT cells in the human lung. We show that, as a population, lung MAIT cells are polycytotoxic, secrete the directly antimicrobial molecule IL-26, express genes associated with persistence, and selectively express cytokine and chemokine- related molecules distinct from other lung-resident CD8+ T cells, such as interferon-γ- and IL-12- receptors. These data highlight MAIT cells' predisposition to rapid pro-inflammatory cytokine responsiveness and antimicrobial mechanisms in human lung tissue, concordant with findings of blood-derived counterparts, and support a function for MAIT cells as early sensors in the defense of respiratory barrier function.

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.

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.

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.

Bordetella bronchiseptica in non-cystic fibrosis bronchiectasis.

Bordetella bronchiseptica is a rare pulmonary infection, often associated with zoonotic transmission. It has been described in immunocompromised patients and those with underlying pulmonary disease. However, there are no case series describing the spectrum of disease caused by Bordetella bronchiseptica in patients with non-cystic fibrosis bronchiectasis. Here, we report three cases of Bordetella bronchiseptica infection in patients with non-cystic fibrosis bronchiectasis and highlight the pathophysiology of the microbe. While the clinical presentation can be quite variable, it is important to note that Bordetella bronchiseptica can be a cause of pulmonary exacerbations and can be difficult to eradicate.

Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism.

The mitochondrial RNA binding proteins MRP1 and MRP2 form a heteromeric complex that functions in kinetoplastid RNA editing. In this process, MRP1/MRP2 serves as a matchmaker by binding to guide RNAs and facilitating their hybridization with cognate preedited mRNAs. To understand the mechanism by which this complex performs RNA matchmaking, we determined structures of Trypanosoma brucei apoMRP1/MRP2 and an MRP1/MRP2-gRNA complex. The structures show that MRP1/MRP2 is a heterotetramer and, despite little sequence homology, each MRP subunit exhibits the same "Whirly" transcription-factor fold. The gRNA molecule binds to the highly basic beta sheet surface of the MRP complex via nonspecific, electrostatic contacts. Strikingly, while the gRNA stem/loop II base is anchored to the basic surface, stem/loop I (the anchor sequence) is unfolded and its bases exposed to solvent. Thus, MRP1/MRP2 acts as an RNA matchmaker by stabilizing the RNA molecule in an unfolded conformation suitable for RNA-RNA hybridization.