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Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

A teenage girl presented with fever and altered mental status. MRI showed diffuse leptomeningeal enhancement of the brain and spine. She was diagnosed by a positive cerebrospinal fluid (CSF) culture with tuberculous (TB) meningitis and was started on anti-TB medications and corticosteroids. Her mental status improved, but she was noted to have proximal weakness of the lower extremities. In the course of tapering corticosteroids at week 11 of anti-TB therapy, she became acutely confused and febrile. MRI demonstrated interval development of tuberculomas in the brain and a mass lesion in the thoracic spine causing cord compression. Given the clinical picture was suggestive of a paradoxical reaction, the dose of corticosteroids was increased. Infliximab was added when repeat MRI revealed enlargement of the mass lesion in the spine with worsening cord compression. She was successfully tapered off of corticosteroids. Over several months, the patient's motor function recovered fully, and she returned to ambulating without assistance.

BackgroundInterleukin-17-producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined.MethodsWe performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context.ResultsWe identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3-cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17-producing CD4 T cells were preserved; we found that IL-17-producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies.ConclusionsWe found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Circular DNA offers benefits over linear DNA in diagnostic and field assays, but currently, circular DNA generation is lengthy, inefficient, highly dependent on the length and sequence of DNA, and can result in unwanted chimeras. We present streamlined methods for generating PCR-targeted circular DNA from a 700 bp amplicon of rv0678, the high GC content (65%) gene implicated in Mycobacterium tuberculosis bedaquiline resistance, and demonstrate that these methods work as desired. We employ self-circularization with and without splints, a Gibson cloning-based approach, and novel 2 novel methods for generating pseudocircular DNA. The circular DNA can be used as a template for rolling circle PCR followed by long-read sequencing, allowing for the error correction of sequence data, and improving the confidence in the drug resistance determination and strain identification; and, ultimately, improving patient treatment. IMPORTANCE Antimicrobial resistance is a global health threat, and drug resistant tuberculosis is a principal cause of antimicrobial resistance-related fatality. The long turnaround time and the need for high containment biological laboratories of phenotypic growth-based Mycobacterium tuberculosis drug susceptibility testing often commit patients to months of ineffective treatment, and there is a groundswell of effort in shifting from phenotypic to sequencing-based genotypic assays. Bedaquiline is a key component to newer, all oral, drug resistant, tuberculosis regimens. Thus, we focus our study on demonstrating the circularization of rv0678, the gene that underlies most M. tuberculosis bedaquiline resistance. We present 2 novel methods for generating pseudocircular DNA. These methods greatly reduce the complexity and time needed to generate circular DNA templates for rolling circle amplification and long-read sequencing, allowing for error correction of sequence data, and improving confidence in the drug resistance determination and strain identification.

Prospective, longitudinal clinical studies incorporating high-throughput, single-cell analyses could identify which bacterial antigens to include in TB vaccines — and which to avoid.

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the "FluoroDOT assay," which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.

In the initial stage of respiratory infection, Mycobacterium tuberculosis traverses from alveolar macrophages to phenotypically diverse monocyte-derived phagocytes and neutrophils in the lung parenchyma. Here, we compare the in vivo kinetics of early bacterial growth and cell-to-cell spread of two strains of M. tuberculosis: a lineage 2 strain, 4334, and the widely studied lineage 4 strain H37Rv. Using flow cytometry, live cell sorting of phenotypic subsets, and quantitation of bacteria in cells of the distinct subsets, we found that 4334 induces less leukocyte influx into the lungs but demonstrates earlier population expansion and cell-to-cell spread. The earlier spread of 4334 to recruited cells, including monocyte-derived dendritic cells, is accompanied by earlier and greater magnitude of CD4+ T cell activation. The results provide evidence that strain-specific differences in interactions with lung leukocytes can shape adaptive immune responses in vivo. IMPORTANCE Tuberculosis is a leading infectious disease killer worldwide and is caused by Mycobacterium tuberculosis. After exposure to M. tuberculosis, outcomes range from apparent elimination to active disease. Early innate immune responses may contribute to differences in outcomes, yet it is not known how bacterial strains alter the early dynamics of innate immune and T cell responses. We infected mice with distinct strains of M. tuberculosis and discovered striking differences in innate cellular recruitment, cell-to-cell spread of bacteria in the lungs, and kinetics of initiation of antigen-specific CD4 T cell responses. We also found that M. tuberculosis can spread beyond alveolar macrophages even before a large influx of inflammatory cells. These results provide evidence that distinct strains of M. tuberculosis can exhibit differential kinetics in cell-to-cell spread which is not directly linked to early recruitment of phagocytes but is subsequently linked to adaptive immune responses.

Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

Rapid and consistent protein identification across large clinical cohorts is an important goal for clinical proteomics. With the development of data-independent technologies (DIA/SWATH-MS), it is now possible to analyze hundreds of samples with great reproducibility and quantitative accuracy. However, this technology benefits from empirically derived spectral libraries that define the detectable set of peptides and proteins. Here, we apply a simple and accessible tip-based workflow for the generation of spectral libraries to provide a comprehensive overview on the plasma proteome in individuals with and without active tuberculosis (TB). To boost protein coverage, we utilized nonconventional proteases such as GluC and AspN together with the gold standard trypsin, identifying more than 30,000 peptides mapping to 3309 proteins. Application of this library to quantify plasma proteome differences in TB infection recovered more than 400 proteins in 50 min of MS acquisition, including diagnostic Mycobacterium tuberculosis (Mtb) proteins that have previously been detectable primarily by antibody-based assays and intracellular proteins not previously described to be in plasma.

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.

BackgroundHelminth infections can modulate immunity to Mycobacterium tuberculosis (Mtb). However, the effect of helminths, including Schistosoma mansoni (SM), on Mtb infection outcomes is less clear. Furthermore, HIV is a known risk factor for tuberculosis (TB) disease and has been implicated in SM pathogenesis. Therefore, it is important to evaluate whether HIV modifies the association between SM and Mtb infection.SettingHIV-infected and HIV-uninfected adults were enrolled in Kisumu County, Kenya, between 2014 and 2017 and categorized into 3 groups based on Mtb infection status: Mtb-uninfected healthy controls, latent TB infection (LTBI), and active TB disease. Participants were subsequently evaluated for infection with SM.MethodsWe used targeted minimum loss estimation and super learning to estimate a covariate-adjusted association between SM and Mtb infection outcomes, defined as the probability of being Mtb-uninfected healthy controls, LTBI, or TB. HIV status was evaluated as an effect modifier of this association.ResultsSM was not associated with differences in baseline demographic or clinical features of participants in this study, nor with additional parasitic infections. Covariate-adjusted analyses indicated that infection with SM was associated with a 4% higher estimated proportion of active TB cases in HIV-uninfected individuals and a 14% higher estimated proportion of active TB cases in HIV-infected individuals. There were no differences in estimated proportions of LTBI cases.ConclusionsWe provide evidence that SM infection is associated with a higher probability of active TB disease, particularly in HIV-infected individuals.

Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire provides a global view that is limited only in terms of theoretical sensitivity due to the depth of available sampling; however, the assignment of antigen specificities within TCR repertoires has become a bottleneck. This study combines antigen-driven expansion, deep TCR sequencing, and a novel analysis framework to show that homologous 'Clusters of Expanded TCRs (CETs)' can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse uncultured repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

There is limited understanding of the role of host metabolism in the pathophysiology of human tuberculosis (TB). Using high-resolution metabolomics with an unbiased approach to metabolic pathway analysis, we discovered that the tryptophan pathway is highly regulated throughout the spectrum of TB infection and disease. This regulation is characterized by increased catabolism of tryptophan to kynurenine, which was evident not only in active TB disease but also in latent TB infection (LTBI). Further, we found that tryptophan catabolism is reversed with effective treatment of both active TB disease and LTBI in a manner commensurate with bacterial clearance. Persons with active TB and LTBI also exhibited increased expression of indoleamine 2,3-dioxygenase-1 (IDO-1), suggesting IDO-1 mediates observed increases in tryptophan catabolism. Together, these data indicate IDO-1-mediated tryptophan catabolism is highly preserved in the human response to Mycobacterium tuberculosis and could be a target for biomarker development as well as host-directed therapies.

Background:Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method:We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results:Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion:Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients.

Secretion of specific proteins contributes to pathogenesis and immune responses in tuberculosis and other bacterial infections, yet the kinetics of protein secretion and fate of secreted proteins in vivo are poorly understood. We generated new monoclonal antibodies that recognize the Mycobacteriumtuberculosis secreted protein Ag85B and used them to establish and characterize a sensitive enzyme-linked immunosorbent assay (ELISA) to quantitate Ag85B in samples generated in vitro and in vivo We found that nutritional or culture conditions had little impact on the secretion of Ag85B and that there is considerable variation in Ag85B secretion by distinct strains in the M. tuberculosis complex: compared with the commonly used H37Rv strain (lineage 4), Mycobacteriumafricanum (lineage 6) secretes less Ag85B, and two strains from lineage 2 secrete more Ag85B. We also used the ELISA to determine that the rate of secretion of Ag85B is 10- to 100-fold lower than that of proteins secreted by Gram-negative and Gram-positive bacteria, respectively. ELISA quantitation of Ag85B in lung homogenates of M. tuberculosis H37Rv-infected mice revealed that although Ag85B accumulates in the lungs as the bacterial population expands, the amount of Ag85B per bacterium decreases nearly 10,000-fold at later stages of infection, coincident with the development of T cell responses and arrest of bacterial population growth. These results indicate that bacterial protein secretion in vivo is dynamic and regulated, and quantitation of secreted bacterial proteins can contribute to the understanding of pathogenesis and immunity in tuberculosis and other infections.IMPORTANCE Bacterial protein secretion contributes to host-pathogen interactions, yet the process and consequences of bacterial protein secretion during infection are poorly understood. We developed a sensitive ELISA to quantitate a protein (termed Ag85B) secreted by M. tuberculosis and used it to find that Ag85B secretion occurs with slower kinetics than for proteins secreted by Gram-positive and Gram-negative bacteria and that accumulation of Ag85B in the lungs is markedly regulated as a function of the bacterial population density. Our results demonstrate that quantitation of bacterial proteins during infection can reveal novel insights into host-pathogen interactions.

Mycobacterium tuberculosis causes chronic infection of mononuclear phagocytes, especially resident (alveolar) macrophages, recruited macrophages, and dendritic cells. Despite the importance of these cells in tuberculosis (TB) pathogenesis and immunity, little is known about the population dynamics of these cells at the sites of infection. We used a combination of congenic monocyte adoptive transfer, and pulse-chase labeling of DNA, to determine the kinetics and characteristics of trafficking, differentiation, and infection of mononuclear phagocytes during the chronic, adaptive immune phase of M. tuberculosis infection in mice. We found that Ly6Chi monocytes traffic rapidly to the lungs, where a subpopulation become Ly6Clo and remain in the lung vascular space, while the remainder migrate into the lung parenchyma and differentiate into Ly6Chi dendritic cells, CD11b+ dendritic cells, and recruited macrophages. As in humans with TB, M. tuberculosis-infected mice have increased numbers of blood monocytes; this is due to increased egress from the bone marrow, and not delayed egress from the blood. Pulse-chase labeling of dividing cells and flow cytometry analysis revealed a T1/2 of ~15 hrs for Ly6Chi monocytes, indicating that they differentiate rapidly upon entry to the parenchyma of infected lungs; in contrast, cells that differentiate from Ly6Chi monocytes turn over more slowly, but diminish in frequency in less than one week. New cells (identified by pulse-chase labeling) acquire bacteria within 1-3 days of appearance in the lungs, indicating that bacteria regularly encounter new cellular niches, even during the chronic stage of infection. Our findings that mononuclear phagocyte populations at the site of M. tuberculosis infection are highly dynamic provide support for specific approaches for host-directed therapies directed at monocytes, including trained immunity, as potential interventions in TB, by replacing cells with limited antimycobacterial capabilities with newly-recruited cells better able to restrict and kill M. tuberculosis.

BackgroundInfection with Mycobacterium tuberculosis is associated with inconsistent and incomplete elimination of the bacteria, despite development of antigen-specific T-cell responses. One mechanism used by M tuberculosis is to limit availability of antigen for activation of CD4 T cells.MethodsWe examined the utility of systemic administration of epitope peptides to activate pre-existing T cells in mice infected with M tuberculosis.ResultsWe found that systemic peptide administration (1) selectively activates T cells specific for the epitope peptide, (2) loads major histocompatibility complex class II on lung macrophages and dendritic cells, (3) activates CD4 T cells in the lung parenchyma, (4) and has little antimycobacterial activity.ConclusionsFurther studies revealed that CD4 T cells in lung lesions are distant from the infected cells, suggesting that, even if they can be activated, the positioning of CD4 T cells and their direct interactions with infected cells may be limiting determinants of immunity in tuberculosis.

Tuberculosis (TB) is a large global health problem, in part because of the long period of coevolution of the pathogen, Mycobacterium tuberculosis, and its human host. A major factor that sustains the global epidemic of TB is the lack of a sufficiently effective vaccine. While basic mechanisms of immunity that protect against TB have been identified, attempts to improve immunity to TB by vaccination have been disappointing. This Review discusses the mechanisms used by M. tuberculosis to evade innate and adaptive immunity and that likely limit the efficacy of vaccines developed to date. Despite multiple mechanisms of immune evasion, recent trials have indicated that effective TB vaccines remain an attainable goal. This Review discusses how knowledge from other systems can inform improvements on current vaccine approaches.

Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis-specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 M. tuberculosis Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of M. tuberculosis-unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis-specific T cell responses across different states of M. tuberculosis infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of M. tuberculosis-specific T cell responses associated with M. tuberculosis infection outcomes.

Two recent studies (Cambier et al., 2017; Madigan et al., 2017) reveal in vivo functions for specific phenolic glycolipids (PGLs) in the mycobacteria that cause tuberculosis or leprosy. M. tuberculosis (and M. marinum) PGL promotes bacterial spread to growth-permissive macrophages, while M. leprae PGL-1 induces macrophages to cause nerve demyelination characteristic of human leprosy.

Tuberculosis (TB) is the major cause of death from infectious diseases around the world, particularly in HIV infected individuals. TB vaccine design and development have been focused on improving Bacille Calmette-Guérin (BCG) and evaluating recombinant and viral vector expressed Mycobacterium tuberculosis (Mtb) proteins, for boosting BCG-primed immunity, but these approaches have not yet yielded significant improvements over the modest effects of BCG in protecting against infection or disease. On March 7-8, 2016, the National Institute of Allergy and Infectious Diseases (NIAID) convened a workshop on "The Impact of Mtb Immune Evasion on Protective Immunity: Implications for TB Vaccine Design" with the goal of defining immune mechanisms that could be targeted through novel research approaches, to inform vaccine design and immune therapeutic interventions for prevention of TB. The workshop addressed early infection events, the impact of Mtb evolution on the development and maintenance of an adaptive immune response, and the factors that influence protection against and progression to active disease. Scientific gaps and areas of study to revitalize and accelerate TB vaccine design were discussed and prioritized. These included a comprehensive evaluation of innate and Mtb-specific adaptive immune responses in the lung at different stages of disease; determining the role of B cells and antibodies (Abs) during Mtb infection; development of better assays to measure Mtb burden following exposure, infection, during latency and after treatment, and approaches to improving current animal models to study Mtb immunogenicity, TB disease and transmission.

One hundred healthy infants enrolled as controls in a tuberculosis vaccine study in Nyanza Province, Kenya provided anonymized samples for DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, -DRB1, and -DRB3/4/5 loci. The purpose of the study was to characterize allele frequencies in the local population, to support studies of T cell immunity against pathogens, including Mycobacterium tuberculosis. There are no detectable deviations from Hardy Weinberg proportions for the HLA-B, -C, -DRB1, -DPB1, -DQA1 and -DQB1 loci. A minor deviation was detected at the HLA-A locus due to an excess of HLA-A*02:02, 29:02, 30:02, and 68:02 homozygotes. The genotype data are available in the Allele Frequencies Net Database under identifier 3393.

Microbes that infect other organisms encounter host immune responses, and must overcome or evade innate and adaptive immune responses to successfully establish infection. Highly successful microbial pathogens, including M. tuberculosis, are able to evade adaptive immune responses (mediated by antibodies and/or T lymphocytes) and thereby establish long-term chronic infection. One mechanism that diverse pathogens use to evade adaptive immunity is antigenic variation, in which structural variants emerge that alter recognition by established immune responses and allow those pathogens to persist and/or to infect previously-immune hosts. Despite the wide use of antigenic variation by diverse pathogens, this mechanism appears to be infrequent in M. tuberculosis, as indicated by findings that known and predicted human T cell epitopes in this organism are highly conserved, although there are exceptions. These findings have implications for diagnostic tests that are based on measuring host immune responses, and for vaccine design and development.

Mycobacterium tuberculosis (Mtb) establishes a persistent infection, despite inducing antigen-specific T-cell responses. Although T cells arrive at the site of infection, they do not provide sterilizing immunity. The molecular basis of how Mtb impairs T-cell function is not clear. Mtb has been reported to block major histocompatibility complex class II (MHC-II) antigen presentation; however, no bacterial effector or host-cell target mediating this effect has been identified. We recently found that Mtb EsxH, which is secreted by the Esx-3 type VII secretion system, directly inhibits the endosomal sorting complex required for transport (ESCRT) machinery. Here, we showed that ESCRT is required for optimal antigen processing; correspondingly, overexpression and loss-of-function studies demonstrated that EsxH inhibited the ability of macrophages and dendritic cells to activate Mtb antigen-specific CD4+ T cells. Compared with the wild-type strain, the esxH-deficient strain induced fivefold more antigen-specific CD4+ T-cell proliferation in the mediastinal lymph nodes of mice. We also found that EsxH undermined the ability of effector CD4+ T cells to recognize infected macrophages and clear Mtb. These results provide a molecular explanation for how Mtb impairs the adaptive immune response.

Type I interferons (IFNs, including IFN-αβ) contribute to the pathogenesis of Mycobacterium tuberculosis strains that induce high IFN-αβ levels. In the current study we examined the role of IFN-αβ during infection with a Mycobacterium africanum strain that induces low IFN-β levels. We infected wild-type and IFN-αβ receptor knockout mice with M. africanum and monitored bacterial growth, lung disease, and survival over 292 days. We found reduced lung bacterial burdens and less severe histopathological findings in the absence of IFN-αβ signaling. We conclude that IFN-αβ is pathogenic during chronic M. africanum infection and that the pathogenic effects may be mediated through poorer control of bacterial growth.

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis.

Type I interferons (including IFNαβ) are innate cytokines that may contribute to pathogenesis during Mycobacterium tuberculosis (Mtb) infection. To induce IFNβ, Mtb must gain access to the host cytosol and trigger stimulator of interferon genes (STING) signaling. A recently proposed model suggests that Mtb triggers STING signaling through bacterial DNA binding cyclic GMP-AMP synthase (cGAS) in the cytosol. The aim of this study was to test the generalizability of this model using phylogenetically distinct strains of the Mtb complex (MTBC). We infected bone marrow derived macrophages with strains from MTBC Lineages 2, 4 and 6. We found that the Lineage 6 strain induced less IFNβ, and that the Lineage 2 strain induced more IFNβ, than the Lineage 4 strain. The strains did not differ in their access to the host cytosol and IFNβ induction by each strain required both STING and cGAS. We also found that the three strains shed similar amounts of bacterial DNA. Interestingly, we found that the Lineage 6 strain was associated with less mitochondrial stress and less mitochondrial DNA (mtDNA) in the cytosol compared with the Lineage 4 strain. Treating macrophages with a mitochondria-specific antioxidant reduced cytosolic mtDNA and inhibited IFNβ induction by the Lineage 2 and 4 strains. We also found that the Lineage 2 strain did not induce more mitochondrial stress than the Lineage 4 strain, suggesting that additional pathways contribute to higher IFNβ induction. These results indicate that the mechanism for IFNβ by Mtb is more complex than the established model suggests. We show that mitochondrial dynamics and mtDNA contribute to IFNβ induction by Mtb. Moreover, we show that the contribution of mtDNA to the IFNβ response varies by MTBC strain and that additional mechanisms exist for Mtb to induce IFNβ.

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

Persistence of Mycobacterium tuberculosis results from bacterial strategies that manipulate host adaptive immune responses. Infected dendritic cells (DCs) transport M. tuberculosis to local lymph nodes but activate CD4 T cells poorly, suggesting bacterial manipulation of antigen presentation. However, M. tuberculosis antigens are also exported from infected DCs and taken up and presented by uninfected DCs, possibly overcoming this blockade of antigen presentation by infected cells. Here we show that the first stage of this antigen transfer, antigen export, benefits M. tuberculosis by diverting bacterial proteins from the antigen presentation pathway. Kinesin-2 is required for antigen export and depletion of this microtubule-based motor increases activation of antigen-specific CD4 T cells by infected cells and improves control of intracellular infection. Thus, although antigen transfer enables presentation by bystander cells, it does not compensate for reduced antigen presentation by infected cells and represents a bacterial strategy for CD4 T cell evasion.

Mycobacterium tuberculosis commonly causes persistent or chronic infection, despite the development of Ag-specific CD4 T cell responses. We hypothesized that M. tuberculosis evades elimination by CD4 T cell responses by manipulating MHC class II Ag presentation and CD4 T cell activation and tested this hypothesis by comparing activation of Ag85B-specific CD4 T cell responses to M. tuberculosis and M. bovis bacillus Calmette-Guérin (BCG) Pasteur in vivo and in vitro. We found that, although M. tuberculosis persists in lungs of immunocompetent mice, M. bovis BCG is cleared, and clearance is T cell dependent. We further discovered that M. tuberculosis-infected macrophages and dendritic cells activate Ag85B-specific CD4 T cells less efficiently and less effectively than do BCG-infected cells, in vivo and in vitro, despite higher production and secretion of Ag85B by M. tuberculosis. During BCG infection, activation of Ag85B-specific CD4 T cells requires fewer infected dendritic cells and fewer Ag-producing bacteria than during M. tuberculosis infection. When dendritic cells containing equivalent numbers of M. tuberculosis or BCG were transferred to mice, BCG-infected cells activated proliferation of more Ag85B-specific CD4 T cells than did M. tuberculosis-infected cells. Differences in Ag85B-specific CD4 T cell activation were attributable to differential Ag presentation rather than differential expression of costimulatory or inhibitory molecules. These data indicate that suboptimal Ag presentation contributes to persistent infection and that limiting Ag presentation is a virulence property of M. tuberculosis.

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

[This corrects the article DOI: 10.1371/journal.ppat.1004376.].

Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.

Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise >1% of all Mycobacterium tuberculosis lipid. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and nuclear magnetic resonance, we established the structure of the previously unknown molecule, N(6)-tuberculosinyladenosine (N(6)-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N(6)-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M. tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria, and vaccine strains. With no substantially similar known molecules in nature, the discovery and in vivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.

Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB), is an intracellular pathogen of mononuclear phagocytes. Although M. tuberculosis has traditionally been thought to survive and replicate in macrophages, recent work in our laboratory and others has revealed that M. tuberculosis infects multiple subsets of mononuclear phagocytes in vivo and in vitro. In experimental animals, M. tuberculosis infects no fewer than five distinct cell subsets in the lungs, including resident alveolar macrophages and 4 types of cells that recruited to the lungs in response to inflammatory signals: neutrophils, monocytes, interstitial macrophages, and dendritic cells. A characteristic of the adaptive immune response in TB is that it is delayed for several weeks following infection, and we have determined that this delay is due to prolonged residence of the bacteria in lung phagocytes prior to acquisition of the bacteria by dendritic cells. Among the mechanisms used by M. tuberculosis to delay acquisition by dendritic cells is to inhibit apoptosis of alveolar macrophages and neutrophils, which sequester the bacteria and prevent their acquisition by dendritic cells in the early stages of infection. We hypothesize that each infected cell subset makes a distinct contribution to the overall biology of M. tuberculosis and allows the bacteria to evade elimination by T-cell responses and to avoid rapid killing by antimycobacterial drugs.

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.

During Mycobacterium tuberculosis and other respiratory infections, optimal T cell activation requires pathogen transport from the lung to a local draining lymph node (LN). However, the infected inflammatory monocyte-derived dendritic cells (DCs) that transport M. tuberculosis to the local lymph node are relatively inefficient at activating CD4 T cells, possibly due to bacterial inhibition of antigen presentation. We found that infected migratory DCs release M. tuberculosis antigens as soluble, unprocessed proteins for uptake and presentation by uninfected resident lymph node DCs. This transfer of bacterial proteins from migratory to local DCs results in optimal priming of antigen-specific CD4 T cells, which are essential in controlling tuberculosis. Additionally, this mechanism does not involve transfer of the whole bacterium and is distinct from apoptosis or exosome shedding. These findings reveal a mechanism that bypasses pathogen inhibition of antigen presentation by infected cells and generates CD4 T cell responses that control the infection.

UnlabelledThe Mycobacterium tuberculosis genome includes the large family of pe_pgrs genes, whose functions are unknown. Because of precedents in other pathogens in which gene families showing high sequence variation are involved in antigenic variation, a similar role has been proposed for the pe_pgrs genes. However, the impact of immune selection on pe_pgrs genes has not been examined. Here, we sequenced 27 pe_pgrs genes in 94 clinical strains from five phylogenetic lineages of the M. tuberculosis complex (MTBC). We found that pe_pgrs genes were overall more diverse than the remainder of the MTBC genome, but individual members of the family varied widely in their nucleotide diversity and insertion/deletion (indel) content: some were more, and others were much less, diverse than the genome average. Individual pe_pgrs genes also differed in the ratio of nonsynonymous to synonymous mutations, suggesting that different selection pressures act on individual pe_pgrs genes. Using bioinformatic methods, we tested whether sequence diversity in pe_pgrs genes might be selected by human T cell recognition, the major mechanism of adaptive immunity to MTBC. We found that the large majority of predicted human T cell epitopes were confined to the conserved PE domain and experimentally confirmed the antigenicity of this domain in tuberculosis patients. In contrast, despite being genetically diverse, the PGRS domains harbored few predicted T cell epitopes. These results indicate that human T cell recognition is not a significant force driving sequence diversity in pe_pgrs genes, which is consistent with the previously reported conservation of human T cell epitopes in the MTBC.ImportanceRecognition of Mycobacterium tuberculosis antigens by T lymphocytes is known to be important for immune protection against tuberculosis, but it is unclear whether human T cell recognition drives antigenic variation in M. tuberculosis. We previously discovered that the known human T cell epitopes in the M. tuberculosis complex are highly conserved, but we hypothesized that undiscovered epitopes with naturally occurring sequence variants might exist. To test this hypothesis, we examined the pe_pgrs genes, a large family of genes that has been proposed to function in immune evasion by M. tuberculosis. We found that the pe_pgrs genes exhibit considerable sequence variation, but the regions containing T cell epitopes and the regions of variation are distinct. These findings confirm that the majority of human T cell epitopes of M. tuberculosis are highly conserved and indicate that selection forces other than T cell recognition drive sequence variation in the pe_pgrs genes.

Effector T cells control intracellular infection by secreting cytokines and through contact-dependent cytolysis. Because cytokines can diffuse and act at a distance, we determined whether cytokine diffusion is sufficient to control Mycobacterium tuberculosis or whether direct recognition of infected cells by CD4 T cells is required. Using MHC class II (MHC II) mixed bone marrow chimeras, we compared the bacterial burdens in lung myeloid cells that were capable (MHC II(+/+)) or not (MHC II(-/-)) of being recognized by CD4 T cells. MHC II(+/+) cells had lower bacterial burdens than did MHC II(-/-) cells. CD4 T cell depletion increased the number of bacteria associated with MHC II(+/+)cells but not MHC II(-/-) cells, indicating that direct recognition of infected cells by CD4 T cells is required for control of intracellular M. tuberculosis. These results show that the effector mechanisms required for CD4 T cell control of distinct intracellular pathogens differ and that long-range cytokine diffusion does not contribute to control of M. tuberculosis.

In a recent Science paper, Teles et al. (2013) show that type I and II interferons (IFNs) are reciprocally expressed in the polar immune forms of leprosy, with type I IFNs inducing IL-10 that interferes with the antimycobacterial effects of type II IFNs (IFNγ) at the site of infection.