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Mycobacterium tuberculosis (Mtb) infection, including active tuberculosis (TB) and latent Mtb infection (LTBI), leads to diverse outcomes owing to different host immune responses. However, the immune mechanisms that govern the progression from LTBI to TB remain poorly defined in humans. Here, we profiled the lung immune cell populations within the bronchoalveolar lavage fluid (BALF) from patients with LTBI or TB using single-cell RNA sequencing (scRNA-seq). We found that Mtb infection substantially changed the immune cell compartments in the BALF, especially for the three subsets of macrophages, monocyte macrophage (MM)-CCL23, MM-FCN1, and MM-SPP1, which were found to be associated with the disease status of TB infection. Notably, MM-CCL23 cells derived from monocytes after stimulation with Mtb were characterized by high levels of chemokine ( CCL23 and CXCL5 ) production and might serve as a marker for Mtb infection. The MM-CCL23 population mainly recruited CD8-CCR6 T cells through CCL20/CCR6, which was a prominent feature associated with protection immunity in LTBI. This study improves our understanding of the lung immune landscape during Mtb infection, which may inform future vaccine design for protective immunity.

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required.

The hallmark of tuberculosis (TB) is the formation of immune cell-enriched aggregates called granulomas. While granulomas are pathologically diverse, their tissue-wide heterogeneity has not been spatially resolved at the single-cell level in human tissues. By spatially mapping individual immune cells in every lesion across entire tissue sections, we report that in addition to necrotizing granulomas, the human TB lung contains abundant non-necrotizing leukocyte aggregates surrounding areas of necrotizing tissue. These cellular lesions were more diverse in composition than necrotizing lesions and could be stratified into four general classes based on cellular composition and spatial distribution of B cells and macrophages. The cellular composition of non-necrotizing structures also correlates with their proximity to necrotizing lesions, indicating these are foci of distinct immune reactions adjacent to necrotizing granulomas. Together, we show that during TB, diseased lung tissue develops a histopathological superstructure comprising at least four different types of non-necrotizing cellular aggregates organized as satellites of necrotizing granulomas., (© 2023 Sawyer et al.)

Mycobacterium tuberculosis (M.tb) infects a quarter of the world's population and may progress to active tuberculosis (ATB). There is no gold standard for diagnosing latent tuberculosis infection (LTBI). Some immunodiagnostic tests are recommended to detect LTBI but can not distinguish ATB from LTBI. The breath test is useful for diagnosing ATB compared to healthy subjects but was never studied for LTBI. This proof-of-concept study (Chinese Clinical Trials Registry number: ChiCTR2200058346) was the first to explore a novel, rapid, and simple LTBI detection method via breath test on high-pressure photon ionization time-of-flight mass spectrometry (HPPI-TOFMS). The case group of LTBI subjects (n = 185) and the control group (n = 250), which included ATB subgroup (n = 121) and healthy control (HC) subgroup (n = 129), were enrolled. The LTBI detection model indicated that a breath test via HPPI-TOFMS could distinguish LTBI from the control with a sensitivity of 80.0% (95% CI: 67.6%, 92.4%) and a specificity of 80.8% (95% CI: 71.8%, 89.9%). Nevertheless, further intensive studies with a larger sample size are required for clinical application.

Orchestration of an effective T lymphocyte response at infection sites is critical for protection against Mycobacterium tuberculosis (Mtb) infection. However, the local T cell immunity landscape in human tuberculosis is poorly defined. Tuberculous pleural effusion (TPE), caused by Mtb, is characterized by an influx of leukocytes to the pleural space, providing a platform suitable for delineating complex tissue responses to Mtb infection. Using single-cell transcriptomics and T cell receptor sequencing, we analyzed mononuclear cell populations in paired pleural fluid and peripheral blood of TPE patients. While all major cell clusters were present in both tissues, their relative proportions varied significantly by anatomic location. Lineage tracking analysis revealed subsets of CD8 and CD4 T cell populations with distinct effector functions specifically expanded at pleural sites. Granzyme K-expressing CD8 T cells were preferentially enriched and clonally expanded in pleural fluid from TPE, suggesting that they are involved in the pathogenesis of the disease. The findings collectively reveal the landscape of local T cell immunity in tuberculosis., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2022 Cai et al.)

Background: Biomarker-based tests for diagnosing TB currently rely on detecting Mycobacterium tuberculosis (Mtb) antigen-specific cellular responses. While this approach can detect Mtb infection, it is not efficient in diagnosing TB, especially for patients who lack aetiological evidence of the disease., Methods: We prospectively enrolled three cohorts for our study for a total of 630 subjects, including 160 individuals to screen protein biomarkers of TB, 368 individuals to establish and test the predictive model and 102 individuals for biomarker validation. Whole blood cultures were stimulated with pooled Mtb-peptides or mitogen, and 640 proteins within the culture supernatant were analysed simultaneously using an antibody-based array. Sixteen candidate biomarkers of TB identified during screening were then developed into a custom multiplexed antibody array for biomarker validation., Results: A two-round screening strategy identified eight-protein biomarkers of TB: I-TAC, I-309, MIG, Granulysin, FAP, MEP1B, Furin and LYVE-1. The sensitivity and specificity of the eight-protein biosignature in diagnosing TB were determined for the training (n=276), test (n=92) and prediction (n=102) cohorts. The training cohort had a 100% specificity (95% CI 98% to 100%) and 100% sensitivity (95% CI 96% to 100%) using a random forest algorithm approach by cross-validation. In the test cohort, the specificity and sensitivity were 83% (95% CI 71% to 91%) and 76% (95% CI 56% to 90%), respectively. In the prediction cohort, the specificity was 84% (95% CI 74% to 92%) and the sensitivity was 75% (95% CI 57% to 89%)., Conclusions: An eight-protein biosignature to diagnose TB in a high-burden TB clinical setting was identified., Competing Interests: Competing interests: HZ, YY and R-PH are employees of RayBiotech Life, a company producing commercial antibody arrays, including the antibody array targeting 640 human proteins that was used in this study. The custom antibody array targeting 16 proteins was also produced by RayBiotech., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Recruitment of monocytes to the infection site is critical for host resistance against Mycobacterium tuberculosis CD157 has a crucial role in neutrophil and monocyte transendothelial migration and adhesion, but its role in tuberculosis (TB) is unclear. Here, we show that both mRNA and protein levels of Cd157 are significantly increased during M. tuberculosis infection. Deficiency of Cd157 impaired host response to M. tuberculosis infection by increasing bacterial burden and inflammation in the lung in the murine TB model. In vitro experiments show that the bactericidal ability was compromised in Cd157 knockout (KO) macrophages, which was due to impaired M. tuberculosis -induced reactive oxygen species (ROS) production. We further reveal that CD157 interacts with TLR2 and PKCzeta and facilitates M. tuberculosis -induced ROS production in Cd157 KO macrophages, which resulted in enhanced M. tuberculosis killing. For the clinic aspect, we observe that the expression of CD157 decreases after effective anti-TB chemotherapy. CD157 is specifically increased in pleural fluid in tuberculous pleurisy patients compared to pneumonia and lung cancer patients. Interestingly, the levels of soluble CD157 (sCD157) correlate with human peripheral monocyte-derived macrophage bactericidal activity. Exogenous application of sCD157 could compensate for macrophage bactericidal ability and restore ROS production. In conclusion, we have identified a novel protective immune function of CD157 during M. tuberculosis infection via TLR2-dependent ROS production. Application of sCD157 might be an effective strategy for host-directed therapy against TB in those with insufficient CD157 production. IMPORTANCE Tuberculosis, a chronic bacterial disease caused by Mycobacterium tuberculosis , remains a major global health problem. CD157, a dual-function receptor and β-NAD + -metabolizing ectoenzyme, promotes cell polarization, regulates chemotaxis induced through the high-affinity fMLP receptor, and controls transendothelial migration. The role of CD157 in TB pathogenesis remains unknown. In this study, we find that both mRNA and protein levels of CD157 are significantly increased in TB. Deficiency of CD157 impaired host defense against M. tuberculosis infection both in vivo and in vitro , which is mediated by an interaction among CD157, TLR2, and PKCzeta. This interaction facilitates M. tuberculosis -induced macrophagic ROS production, which enhances macrophage bactericidal activity. Interestingly, the sCD157 level in plasma is reversibly associated with MDM M. tuberculosis killing activity. By uncovering the role of CD157 in pathogenesis of TB for the first time, our work demonstrated that application of soluble CD157 might be an effective strategy for host-directed therapy against TB., (Copyright © 2019 Yang et al.)

Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF -/- ) are highly susceptible to infection with Mycobacterium tuberculosis , and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4 + T cells as the infection progresses. M. tuberculosis -specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune response to IMPORTANCE Mycobacterium tuberculosis is the bacterium that causes tuberculosis, the leading cause of death by any infection worldwide. T cells are critical components of the immune response to Mycobacterium tuberculosis While gamma interferon (IFN-γ) is a key effector function of T cells during infection, a failed phase IIb clinical trial and other studies have revealed that IFN-γ production alone is not sufficient to control M. tuberculosis In this study, we demonstrate that CD4 + , CD8 + , and nonconventional T cells produce GM-CSF during Mycobacterium tuberculosis infection in mice and in the peripheral blood of infected humans. Under conditions where other sources of GM-CSF are absent, T cell production of GM-CSF is protective and is required for control of infection. GM-CSF activation of macrophages to limit bacterial growth requires host expression of the transcription factor PPARγ. The identification of GM-CSF production as a T cell effector function may inform future host-directed therapy or vaccine designs., (Copyright © 2017 Rothchild et al.)

Gamma interferon (IFN-gamma) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-gamma enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-gamma responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-gamma ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure (r = -0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-gamma ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the primary causes of death worldwide. Rapid and accurate diagnosis of TB is one of the most direct means to reduce the incidence of TB. In this study, urinary proteomic profiling of TB patients and non‐TB individual controls (HCs) was performed, and differentially expressed urinary proteins between TB and HCs were compared and exclusively expressed proteins in TB patients were selected to establish a clinically useful disease marker panel. In total, these top 11 targeted proteins with 265 peptides were scheduled for multiple reaction monitoring validation analysis by using urine samples from 52 TB patients and 52 HCs. The result demonstrated that a three‐protein combination out of the five‐protein panel (namely P22352, Q9P121, P15151, Q13291, and Q8NDA2) exhibited sensitivity rate of 82.7% in the diagnosis of TB. Furthermore, the three‐protein combination could differentiate TB from the latent tuberculosis (LTB) effectively, which exhibited specificity rate of 92.3% for the diagnosis of TB from the LTB category. Although more numbers of clinical samples are required for further verification, the results provided preliminary evidence that this "three‐protein combination" out of the five‐protein panel could probably be a novel TB diagnostic biomarker in clinical application. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus-negative (Bn) TB laboratory diagnosis, are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultraperformance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N -glycans of IgG in TB patients, including smear-negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG–Gal ratio G0/(G1 + G2 × 2) is 90.48% for bacillus-positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%–98.39%. Similar results were observed in Mycobacterium tuberculosis (M. tb) infection mouse models. The decrease in galactosylation of IgG in TB patients was also qualitatively confirmed using specific lectin blot assay. Using the above techniques, we can discriminate the content of IgG G0 with terminal N -acetylglucosamine and IgG–Gal ratio G0/(G1 + G2 × 2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG–Gal ratio G0/(G1 + G2 × 2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate. [ABSTRACT FROM AUTHOR]

Background: Biomarker-based tests for diagnosing TB currently rely on detecting Mycobacterium tuberculosis (Mtb) antigen-specific cellular responses. While this approach can detect Mtb infection, it is not efficient in diagnosing TB, especially for patients who lack aetiological evidence of the disease.Methods: We prospectively enrolled three cohorts for our study for a total of 630 subjects, including 160 individuals to screen protein biomarkers of TB, 368 individuals to establish and test the predictive model and 102 individuals for biomarker validation. Whole blood cultures were stimulated with pooled Mtb-peptides or mitogen, and 640 proteins within the culture supernatant were analysed simultaneously using an antibody-based array. Sixteen candidate biomarkers of TB identified during screening were then developed into a custom multiplexed antibody array for biomarker validation.Results: A two-round screening strategy identified eight-protein biomarkers of TB: I-TAC, I-309, MIG, Granulysin, FAP, MEP1B, Furin and LYVE-1. The sensitivity and specificity of the eight-protein biosignature in diagnosing TB were determined for the training (n=276), test (n=92) and prediction (n=102) cohorts. The training cohort had a 100% specificity (95% CI 98% to 100%) and 100% sensitivity (95% CI 96% to 100%) using a random forest algorithm approach by cross-validation. In the test cohort, the specificity and sensitivity were 83% (95% CI 71% to 91%) and 76% (95% CI 56% to 90%), respectively. In the prediction cohort, the specificity was 84% (95% CI 74% to 92%) and the sensitivity was 75% (95% CI 57% to 89%).Conclusions: An eight-protein biosignature to diagnose TB in a high-burden TB clinical setting was identified. [ABSTRACT FROM AUTHOR]

Abstract: Although it has been recognized that ectopic follicle-like B cell aggregate formation is common in the lungs of patients with tuberculosis, the role of infiltrated B cells in human tuberculosis remains to be elucidated. In the present study, we showed that ectopic B cell aggregate formation was associated with containment of Mycobacterium tuberculosis. The area ratio of ectopic B cell aggregates was correlated with localized IL-17 mRNA expression and peripheral TGF-β and IL-6 mRNA expression. Depletion of B cells from pleural fluid mononuclear cells resulted in significantly diminished M. tuberculosis antigen-specific IL-17 and IL-22 production, but not in IFN-γ secretion. Therefore, ectopic lung B cell formation is important for containment of M. tuberculosis, and up-regulation of IL-17 and IL-22 responses may be an important mechanism underlying the protective role B cells in human tuberculosis. [Copyright &y& Elsevier]