Post-infectious Pulmonary Complications: Establishing Research Priorities to Advance the Field. An Official American Thoracic Society Workshop Report.

Continued improvements in the treatment of pulmonary infections have paradoxically resulted in a growing challenge of individuals with post-infectious pulmonary complications (PIPCs). PIPCs have been long recognized after tuberculosis but recent experiences, such as the SARS-CoV-2 pandemic, have underscored the importance of PIPCs following other lower respiratory tract infections. Independent of the causative pathogen, most available studies of pulmonary infections focus on short-term outcomes rather than long-term morbidity among survivors. In this document, we establish a conceptual scope for PIPCs with discussion of globally significant pulmonary pathogens and an examination of how these pathogens can damage different components of the lung, resulting in a spectrum of PIPCs. We also review potential mechanisms for the transition from acute infection to PIPC, including the interplay between pathogen-mediated injury and aberrant host responses, which together result in PIPCs. Finally, we identify cross-cutting research priorities for the field to facilitate future studies to establish the incidence of PIPCs, define common mechanisms, identify therapeutic strategies, and ultimately reduce the burden of morbidity in survivors of pulmonary infections.

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCE: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.

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.

Multiple Isomers of Photolumazine V Bind MR1 and Differentially Activate MAIT Cells.

In response to microbial infection, the nonclassical Ag-presenting molecule MHC class I-related protein 1 (MR1) presents secondary microbial metabolites to mucosal-associated invariant T (MAIT) cells. In this study, we further characterize the repertoire of ligands captured by MR1 produced in Hi5 (Trichoplusia ni) cells from Mycobacterium smegmatis via mass spectrometry. We describe the (to our knowledge) novel MR1 ligand photolumazine (PL)V, a hydroxyindolyl-ribityllumazine with four isomers differing in the positioning of a hydroxyl group. We show that all four isomers are produced by M. smegmatis in culture and that at least three can induce MR1 surface translocation. Furthermore, human MAIT cell clones expressing distinct TCR ß-chains differentially responded to the PLV isomers, demonstrating that the subtle positioning of a single hydroxyl group modulates TCR recognition. This study emphasizes structural microheterogeneity within the MR1 Ag repertoire and the remarkable selectivity of MAIT cell TCRs.

Production of Proinflammatory Cytokines by CD4+ and CD8+ T Cells in Response to Mycobacterial Antigens among Children and Adults with Tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of pediatric morbidity and mortality. Young children are at high risk of TB following Mtb exposure, and this vulnerability is secondary to insufficient host immunity during early life. Our primary objective was to compare CD4+ and CD8+ T-cell production of proinflammatory cytokines IFN-gamma, IL-2, and TNF-alpha in response to six mycobacterial antigens and superantigen staphylococcal enterotoxin B (SEB) between Ugandan adults with confirmed TB (n = 41) and young Ugandan children with confirmed (n = 12) and unconfirmed TB (n = 41), as well as non-TB lower respiratory tract infection (n = 39). Flow cytometry was utilized to identify and quantify CD4+ and CD8+ T-cell cytokine production in response to each mycobacterial antigen and SEB. We found that the frequency of CD4+ and CD8+ T-cell production of cytokines in response to SEB was reduced in all pediatric cohorts when compared to adults. However, T-cell responses to Mtb-specific antigens ESAT6 and CFP10 were equivalent between children and adults with confirmed TB. In contrast, cytokine production in response to ESAT6 and CFP10 was limited in children with unconfirmed TB and absent in children with non-TB lower respiratory tract infection. Of the five additional mycobacterial antigens tested, PE3 and PPE15 were broadly recognized regardless of TB disease classification and age. Children with confirmed TB exhibited robust proinflammatory CD4+ and CD8+ T-cell responses to Mtb-specific antigens prior to the initiation of TB treatment. Our findings suggest that adaptive proinflammatory immune responses to Mtb, characterized by T-cell production of IFN-gamma, IL-2, and TNF-alpha, are not impaired during early life.

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.

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.

Mycobacterial-specific secretion of cytokines and chemokines in healthcare workers with apparent resistance to infection with Mycobacterium tuberculosis.

Background: Currently, diagnosis of latent TB infection (LTBI) is based on the secretion of IFN-γ in response to Mycobacterium tuberculosis (Mtb) antigens, the absence of which is regarded as no infection. Some individuals appear to resist Mtb infection despite sustained exposure (resisters). In this study, we aimed to assess cytokines, chemokines and antibodies that may be associated with resistance to Mtb infection. We hypothesized that there may be an alternative immune response to Mtb exposure in the absence of IFN-γ in resisters. Methods: We enrolled HIV-uninfected healthcare workers who had worked in high TB-exposure environments for 5 years or longer. We screened them for LTBI using the tuberculin skin test and the QuantiFERON-TB Gold Plus assay. We performed multiplex Luminex to measure concentrations of T cell-associated cytokines and chemokines as well as total antibodies in plasma collected from unstimulated fresh whole blood and supernatants from QuantiFERON-TB Gold Plus tubes following incubation of whole blood for 16-24 hours with ESAT6/CFP10 peptides. Results: Samples from 78 individuals were analyzed: 33 resisters (TST<10mm; IGRA<0.35 IU/mL), 33 with LTBI (TST≥10mm and IGRA≥0.35 IU/mL) and 12 discordant (TST=0mm; IGRA≥1.0 IU/mL). There were no differences in concentrations of cytokines and chemokines in plasma between the different groups. Resisters had significantly lower concentrations of IFN-γ, IL-2, TNF-α, MIP-1α, MIP-1ß, ITAC, IL-13 and GM-CSF in supernatants compared with LTBI group. There were no significant differences in the concentrations in supernatants of IL-10, IL-1ß, IL-17A, IL-21, IL-23, MIP-3α, IL-4, IL-5, IL-6, IL-7, IL-8, Fractalkine and IL-12p70 between the groups. We observed that resisters had similar concentrations of total antibodies (IgG1, IgG2, IgG3, IgG4, IgA, and IgM) in plasma and supernatants compared to the LTBI and discordant groups. Conclusion: Resistance to Mtb infection despite sustained exposure is associated with lower Mtb-specific secretion of Th1-associated cytokines and chemokines. However, resisters showed secreted concentrations after Mtb stimulation of total antibodies and cytokines/chemokines associated with innate and Th17 immune responses similar to those with Mtb infection. This suggests an ability to mount non-IFN-γ immune responses to Mtb in apparent resisters.

Divergent metabolic programmes control two populations of MAIT cells that protect the lung.

Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127-Klrg1+ and CD127+Klrg1- antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127+ MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127+ MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1+ MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies.

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.

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.

The Missing Link in Correlates of Protective Tuberculosis Immunity: Recognizing the Infected Cell.

For most vaccination studies, the assessment of vaccine-induced CD4+ and CD8+ T cells has relied upon the measurement of antigen-specific polyfunctional cells, typically using recombinant antigen or peptide pools. However, this approach leaves open the question as to whether or not these cells are responsive to the Mtb-infected cell within the context of Mtb infection and hence leaves open the possibility that a key parameter of vaccine immunogenicity may be overlooked. In this review, we discuss the case that these measurements almost certainly over-estimate the capacity of both CD4+ and CD8+ T cells to recognize the Mtb-infected cell.

Cascade Immune Mechanisms of Protection against Mycobacterium tuberculosis (IMPAc-TB): study protocol for the Household Contact Study in the Western Cape, South Africa.

BACKGROUND: Natural immunity against Mycobacterium tuberculosis exists, and > 90% of those infected remain disease-free. Innate and adaptive immune responses required to mediate such protection against tuberculosis (TB) are, however, poorly understood. METHODS: This is an analytical study exploring protective and non-protective pathways of immunity against Mycobacterium tuberculosis. Adults without HIV infection are recruited at community healthcare clinics in high TB incidence areas of the Western Cape Province, South Africa. Data regarding participants' medical, social and medication usage will be collected, and clinical examinations and point-of-care tests documented. Reference tests for TB (chest radiographs and sputum tests for GeneXpert MTB/RIF Ultra®, Auramine smear and liquid cultures) and investigations to classify infection states [interferon-gamma release assay (IGRA) and SARS-CoV-2 polymerase chain reaction (PCR) nasopharyngeal swab and IgG], are done on all participants who meet the inclusion criteria. 18F-Fluorodeoxyglucose positron emission tomography combined with computerized tomography will be done on all close contacts (contacts) and healthy control (controls) participants. Participants are divided into 12 study groups representing a spectrum of TB clinical phenotypes and prior SARS-CoV-2 infection based on their TB status, exposure history, results of IGRA test at baseline and 3 months, SARS-CoV-2 serology, and PCR results, and for contacts and controls, PET-CT imaging findings indicative of sub-clinical TB lesions. Samples for experimental assays include whole blood for isolation of peripheral blood mononuclear cells and blood in PAXgene® tubes for RNA isolation. All SARS-CoV-2 PCR negative study participants undergo bronchoscopy for collecting bronchoalveolar lavage samples. DISCUSSION: The paired blood and BAL samples will be used for comprehensive analyses of the tissue-specific and systemic immunity that will include e.g., cytometry by time-of-flight analyses, RNA-sequencing, multiplex immunoassays, epigenetic analysis, and mechanistic studies of control of infection by Mycobacterium tuberculosis. Results will be integrated with those from mice and non-human primate studies to provide a comprehensive analysis of protective pathways in natural and vaccine-induced immunity against Mycobacterium tuberculosis.

Effects of BCG vaccination on donor unrestricted T cells in two prospective cohort studies.

BACKGROUND: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MR1-restricted MAIT cells, CD1b-restricted glucose monomycolate (GMM)-specific T cells, CD1d-restricted NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis. METHODS: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults. FINDINGS: BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CD1b-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CD1d-restricted NKT cells. By contrast, primary BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of CD26+CD161+TRAV1-2- IFN-γ-expressing CD4+ T cells in infants. INTERPRETATION: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies. FUNDING: Aeras, the National Institutes of Health, and the Bill and Melinda Gates Foundation.

Donor Unrestricted T Cells: Linking innate and adaptive immunity.

Donor Unrestricted T Cells (DURTs) are characterized by their use of antigen presentation molecules that are often invariant. As these cells recognize diverse mycobacterial antigens, often found in BCG, these cells have the potential to either serve as targets for vaccination, or as a means to enable the induction of traditional T and B cell immunity. Here, we will review specific DURT family members, and their relationship to BCG.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

INTRODUCTION: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments. METHODS: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions. Serum samples were collected from 289 participants enrolled in the Centers for Disease Control and Prevention TBTC Study 29 (NCT00694629) at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). RESULTS: After a peptide level proteome analysis utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS) and subsequent statistical analysis, a total of 183 core proteins demonstrated significant differences at both baseline and at week 8 timepoints between participants enrolled from African and non-African regions. The majority of the differentially expressed proteins were upregulated in participants from the African region, and included acute phase proteins, mediators of inflammation, as well as coagulation and complement pathways. Downregulated proteins in the African population were primarily linked to nutritional status and lipid metabolism pathways. CONCLUSIONS: We have identified differentially expressed nutrition and lipid pathway proteins by geographic region in TB patients undergoing treatment for pulmonary tuberculosis, which appear to be associated with differential treatment responses. Future TB clinical trials should collect expanded measures of nutritional status and further evaluate the relationship between nutrition and microbiologic treatment response.

MR1-Restricted MAIT Cells From The Human Lung Mucosal Surface Have Distinct Phenotypic, Functional, and Transcriptomic Features That Are Preserved in HIV Infection.

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αß T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.

Functional and Activation Profiles of Mucosal-Associated Invariant T Cells in Patients With Tuberculosis and HIV in a High Endemic Setting.

Background: MAIT cells are non-classically restricted T lymphocytes that recognize and rapidly respond to microbial metabolites or cytokines and have the capacity to kill bacteria-infected cells. Circulating MAIT cell numbers generally decrease in patients with active TB and HIV infection, but findings regarding functional changes differ. Methods: We conducted a cross-sectional study on the effect of HIV, TB, and HIV-associated TB (HIV-TB) on MAIT cell frequencies, activation and functional profile in a high TB endemic setting in South Africa. Blood was collected from (i) healthy controls (HC, n = 26), 24 of whom had LTBI, (ii) individuals with active TB (aTB, n = 36), (iii) individuals with HIV infection (HIV, n = 50), 37 of whom had LTBI, and (iv) individuals with HIV-associated TB (HIV-TB, n = 26). All TB participants were newly diagnosed and sampled before treatment, additional samples were also collected from 18 participants in the aTB group after 10 weeks of TB treatment. Peripheral blood mononuclear cells (PBMC) stimulated with BCG-expressing GFP (BCG-GFP) and heat-killed (HK) Mycobacterium tuberculosis (M.tb) were analyzed using flow cytometry. MAIT cells were defined as CD3+ CD161+ Vα7.2+ T cells. Results: Circulating MAIT cell frequencies were depleted in individuals with HIV infection (p = 0.009). MAIT cells showed reduced CD107a expression in aTB (p = 0.006), and reduced IFNγ expression in aTB (p < 0.001) and in HIV-TB (p < 0.001) in response to BCG-GFP stimulation. This functional impairment was coupled with a significant increase in activation (defined by HLA-DR expression) in resting MAIT cells from HIV (p < 0.001), aTB (p = 0.019), and HIV-TB (p = 0.005) patients, and higher HLA-DR expression in MAIT cells expressing IFNγ in aTB (p = 0.009) and HIV-TB (p = 0.002) after stimulation with BCG-GFP and HK-M.tb. After 10 weeks of TB treatment, there was reversion in the observed functional impairment in total MAIT cells, with increases in CD107a (p = 0.020) and IFNγ (p = 0.010) expression. Conclusions: Frequencies and functional profile of MAIT cells in response to mycobacterial stimulation are significantly decreased in HIV infected persons, active TB and HIV-associated TB, with a concomitant increase in MAIT cell activation. These alterations may reduce the capacity of MAIT cells to play a protective role in the immune response to these two pathogens.

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.

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.