Mycobacterium tuberculosis–Induced Bronchoalveolar Lavage Gene Expression Signature in Latent Tuberculosis Infection Is Dominated by Pleiotropic Effects of CD4+ T Cell–Dependent IFN-γ Production despite the Presence of Polyfunctional T Cells within the Airways

BackgroundRapid and effective discrimination between active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains a challenge. There is an urgent need for developing practical and affordable approaches targeting this issue.MethodsParticipants with ATB and LTBI were recruited at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort) based on positive T-SPOT results from June 2020 to January 2021. The expression of activation markers including HLA-DR, CD38, CD69, and CD25 was examined on Mycobacterium tuberculosis (MTB)-specific CD4+ T cells defined by IFN-γ, TNF-α, and IL-2 expression upon MTB antigen stimulation.ResultsA total of 90 (40 ATB and 50 LTBI) and another 64 (29 ATB and 35 LTBI) subjects were recruited from the Qiaokou cohort and Caidian cohort, respectively. The expression patterns of Th1 cytokines including IFN-γ, TNF-α, and IL-2 upon MTB antigen stimulation could not differentiate ATB patients from LTBI individuals well. However, both HLA-DR and CD38 on MTB-specific cells showed discriminatory value in distinguishing between ATB patients and LTBI individuals. As for developing a single candidate biomarker, HLA-DR had the advantage over CD38. Moreover, HLA-DR on TNF-α+ or IL-2+ cells had superiority over that on IFN-γ+ cells in differentiating ATB patients from LTBI individuals. Besides, HLA-DR on MTB-specific cells defined by multiple cytokine co-expression had a higher ability to discriminate patients with ATB from LTBI individuals than that of MTB-specific cells defined by one kind of cytokine expression. Specially, HLA-DR on TNF-α+IL-2+ cells produced an AUC of 0.901 (95% CI, 0.833–0.969), with a sensitivity of 93.75% (95% CI, 79.85–98.27%) and specificity of 72.97% (95% CI, 57.02–84.60%) as a threshold of 44% was used. Furthermore, the performance of HLA-DR on TNF-α+IL-2+ cells for differential diagnosis was obtained with validation cohort data: 90.91% (95% CI, 72.19–97.47%) sensitivity and 68.97% (95% CI, 50.77–82.73%) specificity.ConclusionsWe demonstrated that HLA-DR on MTB-specific cells was a potentially useful biomarker for accurate discrimination between ATB and LTBI.

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1405. The Accuracy of Mycobacterium tuberculosis Specific IFN-γ/IL-2/TNF-α- FluoroSpot in Differential Diagnosis of Active Tuberculosis and Latent Tuberculosis Infection: A Case-Control Study

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.1597 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S786-S787

Author(s):

Lifan Zhang ◽

Huimin Ma ◽

Qiping Ge ◽

Yueqiu Zhang ◽

Xiaochun Shi ◽

...

Keyword(s):

T Cells ◽

Differential Diagnosis ◽

Mycobacterium Tuberculosis ◽

Latent Tuberculosis Infection ◽

Specific Antigen ◽

Latent Tuberculosis ◽

Active Tuberculosis ◽

Tnf Α ◽

Ifn Γ ◽

Stimulation Of

Abstract Background To establish the Mycobacterium tuberculosis (MTB) specific IFN-γ/IL-2/TNF-α-FluoroSpot assay, and preliminarily evaluate its accuracy of differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI). Methods Patients with pathologically confirmed and clinically diagnosed ATB in Peking Union Medical College Hospital and Beijing Chest Hospital from April 2020 to May 2021 were enrolled as case group, while patients with LTBI in the same period were enrolled as control group. The FluoroSpot assay was used to simultaneously detect the secretion of IFN-γ, IL-2 and TNF-α in T cells stimulated by the MTB specific antigens ESAT-6 and CFP-10 at the single-cell level. A binary logistic regression model was used to fit the combined diagnostic parameters, and the sensitivity, specificity, predictive value and likelihood ratio of the differential diagnosis of ATB and LTBI were calculated. Figure 1. Schematic diagram of FluoroSpot (IFN-γ/IL-2/TNF-α) detecting cytokine-secreting specific T cells after stimulation with MTB specific antigen. A. The green spots are the total IFN-γ-secreting T cells; B. The red spots are the total IL-2-secreting T cells; C. The blue spots are the total TNF-α-secreting T cells; D. The green spots are the single IFN-γ-secreting T cells; the red spots are the single IL-2-secreting T cells; the blue spots are the single TNF-α-secreting T cells; the yellow spots are the dual IFN-γ/IL-2-secreting T cells; the cyan spots are the dual IFN-γ/TNF-α-secreting T cells; the purple spots are the dual IL-2/TNF-α-secreting T cells; the white spots are the triple IFN-γ/IL-2/TNF-α-secreting T cells. Results 62 patients with ATB (37 pathogen-confirmed ATB, 25 clinical diagnosed ATB), 87 patients with LTBI were included. There was significant correlation of the frequencies of total IFN-γ-secreting T cells detected by IFN-γ/IL-2/TNF-α-FluoroSpot assay compared with T-SPOT.TB after stimulation of MTB-specific antigen (r=0.829 for ESAT-6, P< 0.001, r=0.804 for CFP-10, P< 0.001). ROC curve was drawn for both T-SPOT.TB and Fluorospot. For T-SPOT.TB, the AUROC was 0.669 (95%CI 0.574-0.765), the sensitivity and specificity of differentiating ATB from LTBI were 70.97% (95%CI 58.05%-81.80%) and 56.32% (95CI 45.26%-66.94%) respectively. While for Fluorospot, the AUROC was 0.906 (95 CI 0.856-0.957), the sensitivity and specificity of differentiating ATB from LTBI were 80.65% (95%CI 68.63% - 89.58%) and 88.51% (95%CI 79.88% - 94.35%) respectively. Figure 2. Correlation between the frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay and those of T-SPOT.TB. (A) Stimulated by EAST-6. (B) Stimulated by CFP-10. Figure 3. ROC curves and the corresponding AUROC for measurement of frequencies of specific T cells in differentiating ATB and LTBI under stimulation of ESAT-6 or CFP-10. The blue line is drawn with the frequency of IFN-γ-secreting T cells detected by T-SPOT.TB, and the AUC is 0.669 (95%CI, 0.574-0.765). The red line is drawn with combination of the frequencies and proportion of single IFN-γ-'single IL-2-'single TNF-α-'dual IFN-γ/IL-2-'dual IFN-γ/ /TNF-α-'dual IL-2/TNF-α-secreting T cells detected by FluoroSpot, and the AUC is 0.906 (95% CI, 0.856-0.957). Table 1. Diagnostic value of T-SPOT.TB and FluoroSpot for differentiating ATB from LTBI Conclusion Compared with T-SPOT.TB, the IFN-γ/IL-2/TNF-α-Fluorospot assay may be helpful to distinguish ATB from LTBI, and the results need to be verified by large sample prospective cohort study. Disclosures All Authors: No reported disclosures

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Treatment of latent tuberculosis infection induces changes in multifunctional Mycobacterium tuberculosis-specific CD4+ T cells

Medical Microbiology and Immunology ◽

10.1007/s00430-015-0424-z ◽

2015 ◽

Vol 205 (1) ◽

pp. 37-45 ◽

Cited By ~ 2

Author(s):

Ilaria Sauzullo ◽

Fabio Mengoni ◽

Claudia Mascia ◽

Raffaella Rossi ◽

Miriam Lichtner ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

Cd4 T Cells ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

Tuberculosis Infection

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Utility of interferon gamma/tumor necrosis factor alpha FluoroSpot assay in differentiation between active tuberculosis and latent tuberculosis infection: a pilot study

BMC Infectious Diseases ◽

10.1186/s12879-021-06351-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Lifan Zhang ◽

Shijun Wan ◽

Ziyue Zhou ◽

Yueqiu Zhang ◽

Xiaoqing Liu

Keyword(s):

T Cells ◽

Pilot Study ◽

Diagnostic Accuracy ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

Active Tuberculosis ◽

Tuberculosis Infection ◽

Tnf Α ◽

Ifn Γ ◽

Sensitivity Specificity

Abstract Background The differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) remains challenging in clinical practice. We aimed to evaluate the diagnostic accuracy of the IFN-γ/TNF-α FluoroSpot assay for differentiating ATB from LTBI. Methods We conducted a pilot study of case-control design, using the FluoroSpot assay to simultaneously detect IFN-γ and TNF-α secretion at the single-cell level. The frequencies of antigen-specific single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells were detected. The optimal cutoffs value of frequencies for differentiating ATB from LTBI were determined according to receiver operating characteristic curve analysis. The sensitivity, specificity, predictive values (PV) and likelihood ratios (LR) of the FluoroSpot assay were calculated. Results Thirty patients diagnosed microbiologically with ATB, 36 healthcare workers with LTBI and 36 healthy controls were enrolled. After stimulated by ESAT-6 or CFP-10 peptides, the median frequencies of single TNF-α-, total TNF-α-, single IFN-γ-, total IFN-γ- and dual IFN-γ/TNF-α-secreting T cells in ATB patients were all significantly higher than those in LTBI and HC groups (P < 0.01). The frequencies of total IFN-γ-secreting T cells detected by FluoroSpot assay correlated significantly with those of T-SPOT.TB (r = 0.910 for ESAT-6, P < 0.001, r = 0.845 for CFP-10, P < 0.001). After stimulated by ESAT-6 peptides, with total TNF-α-secreting T cells frequencies at a cut off value of 21 iSFCs/250,000 PBMCs, the sensitivity, specificity, PLR, NLR, PPV, NPV of IFN-γ/TNF-α FluoroSpot assay in differentiating ATB from LTBI were 96.7% (95%CI, 82.8–99.9%), 94.3% (95%CI, 80.8–99.3%), 16.92 (95%CI, 4.40–65.08), 0.04 (95%CI, 0.01–0.24), 93.6% (95%CI,78.6–99.2%) and 97.1% (95%CI, 84.7–99.9%), respectively. With the frequencies of total TNF-α- and total IFN-γ-secreting T cells stimulated by ESAT-6 peptides combined, the specificity was increased to 97.1%, and the positive likelihood ratio to 31.5. The combination with CFP-10 might not improve the diagnostic accuracy of the ESAT-6 for differentiating ATB from LTBI. Conclusions IFN-γ/TNF-α FluoroSpot assay might have potential to help differentiate ATB from LTBI, but the findings need to be further verified by cross-sectional or prospective cohort studies.

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Characterization of Mycobacterium tuberculosis-specific Th22 cells and the effect of tuberculosis disease and HIV co-infection

10.1101/2020.10.22.20217521 ◽

2020 ◽

Author(s):

Mohau S. Makatsa ◽

F. Millicent A. Omondi ◽

Rubina Bunjun ◽

Robert J. Wilkinson ◽

Catherine Riou ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Mycobacterium Tuberculosis ◽

Cd4 T Cells ◽

Th17 Cells ◽

Latent Tuberculosis ◽

Th22 Cells ◽

Distinct Subset ◽

Increased Risk ◽

Ifn Γ

ABSTRACTThe development of a highly effective tuberculosis (TB) vaccine is likely dependent on our understanding of what constitutes a protective immune response to TB. Accumulating evidence suggests that CD4+ T cells producing IL-22, a distinct subset termed ‘Th22’ cells, may contribute to protective immunity to TB. Thus, we characterized Mycobacterium tuberculosis (Mtb)-specific Th22 (and Th1 and Th17) cells in 72 individuals with latent tuberculosis infection (LTBI) or TB disease, with and without human immunodeficiency virus (HIV)-1 infection. We investigated the functional properties (IFN-γ, IL-22 and IL-17 production), memory differentiation (CD45RA, CD27 and CCR7) and activation profile (HLA-DR) of Mtb-specific CD4+ T cells. In HIV-uninfected individuals with LTBI, we detected abundant IFN-γ producing CD4+ T cells (median: 0.93%) and IL-22-producing CD4+ T cells (median: 0.46%) in response to Mtb. The frequency of IL-17 producing CD4+ T cells was much lower, at a median of 0.06%. Consistent with previous studies, IL-22 was produced by a distinct subset of CD4+ T cells and not co-expressed with IL-17. Mtb-specific IL-22 responses were markedly reduced (median: 0.08%) in individuals with TB disease and HIV co-infection compared to IFN-γ responses. Mtb-specific Th22 cells exhibited a distinct memory and activation phenotype compared to Th1 and Th17 cells. Furthermore, Mtb-specific IL-22 was produced by conventional CD4+ T cells that required T cell receptor (TCR) engagement. In conclusion, we confirm that Th22 cells contribute substantially to the immune response to TB. Depletion of Mtb-specific Th22 cells during HIV co-infection may contribute to increased risk of TB disease.

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Immune profiling of Mycobacterium tuberculosis-specific T cells in recent and remote infection

10.1101/2020.11.13.20230946 ◽

2020 ◽

Author(s):

Cheleka A.M. Mpande ◽

Virginie Rozot ◽

Boitumelo Mosito ◽

Munyaradzi Musvosvi ◽

One B Dintwe ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Differentially Expressed ◽

Cd4 T Cell ◽

Remote Infection ◽

Ifn Γ

AbstractBackgroundRecent Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of progression to tuberculosis disease, compared to persistent infection after remote exposure. However, current immunodiagnostic tools fail to distinguish between recent and remote infection. We aimed to characterise the immunobiology associated with acquisition of M.tb infection and identify a biomarker that can distinguish recent from remote infection.MethodsHealthy South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 months) and persistent (QuantiFERON-TB+ for >1.5 year) infection. We characterized M.tb-specific CD4 T cell functional (IFN-γ, TNF, IL-2, CD107, CD154), memory (CD45RA, CCR7, CD27, KLRG-1) and activation (HLA-DR) profiles by flow cytometry after CFP-10/ESAT-6 peptide pool or M.tb lysate stimulation. We then assessed the diagnostic performance of immune profiles that were differentially expressed between individuals with recent or persistent QuantiFERON-TB+.FindingsCFP-10/ESAT-6-specific CD4 T cell activation but not functional or memory phenotypes distinguished between individuals with recent and persistent QuantiFERON-TB+. In response to M.tb lysate, recent QuantiFERON-TB+ individuals had lower proportions of highly differentiated IFN-γ+TNF+ CD4 T cells expressing a KLRG-1+ effector phenotype and higher proportions of early differentiated IFN-γ-TNF+IL-2+ and activated CD4 T cells compared to persistent QuantiFERON-TB+ individuals. Among all differentially expressed T cell features CFP-10/ESAT-6-specific CD4 T cell activation was the best performing diagnostic biomarker of recent infection.InterpretationRecent M.tb infection is associated with highly activated and moderately differentiated functional M.tb-specific T cell subsets, that can be used as biomarkers to distinguish between recent and remote infection.

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Depletion of Cd4+ T Cells Causes Reactivation of Murine Persistent Tuberculosis despite Continued Expression of Interferon γ and Nitric Oxide Synthase 2

Journal of Experimental Medicine ◽

10.1084/jem.192.3.347 ◽

2000 ◽

Vol 192 (3) ◽

pp. 347-358 ◽

Cited By ~ 221

Author(s):

Charles A. Scanga ◽

V.P. Mohan ◽

Keming Yu ◽

Heather Joseph ◽

Kathryn Tanaka ◽

...

Keyword(s):

Nitric Oxide ◽

T Cells ◽

T Cell ◽

Persistent Infection ◽

Cd4 T Cells ◽

Macrophage Activation ◽

Latent Tuberculosis ◽

Cd4 T Cell ◽

Ifn Γ ◽

Oxide Synthase

Tuberculosis is a major cause of death in much of the world. Current estimates are that one-third of the world's population is infected with Mycobacterium tuberculosis. Most infected persons control the infection but in many cases may not eliminate the organism. Reactivation of this clinically latent infection is responsible for a large proportion of active tuberculosis cases. A major risk factor for reactivation of latent tuberculosis is HIV infection, suggesting a role for the CD4+ T cell subset in maintaining the latent persistent infection. In this study, we tested the requirement for CD4+ T cells in preventing reactivation in a murine model of latent tuberculosis. Antibody-mediated depletion of CD4+ T cells resulted in rapid reactivation of a persistent infection, with dramatically increased bacterial numbers in the organs, increased pathology in the lungs, and decreased survival. Although CD4+ T cells are believed to be a major source of interferon (IFN)-γ, expression of the gene for IFN-γ in the lungs of CD4+ T cell–depleted mice was similar to that in control mice. In addition, inducible nitric oxide synthase production and activity was unimpaired after CD4+ T cell depletion, indicating that macrophage activation was present even during CD4+ T cell deficiency. These data indicate that CD4+ T cells are necessary to prevent reactivation but may have roles in addition to IFN-γ production and macrophage activation in controlling a persistent tuberculous infection.

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Gene expression tryptophan aspartate coat protein in determining latent tuberculosis infection using immunocytochemistry and real time polimerase chain reaction

Infectious Disease Reports ◽

10.4081/idr.2020.8733 ◽

2020 ◽

Author(s):

Rebekah J. Setiabudi ◽

Ni Made Mertaniasih ◽

Muhammad Amin ◽

Wayan Tunas Artama

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Coat Protein ◽

Real Time ◽

Latent Tuberculosis Infection ◽

Latent Tuberculosis ◽

Peripheral Blood Monocyte ◽

Tuberculosis Infection ◽

Chain Reaction ◽

The Difference

Background: Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide. Problem of Latent Tuberculosis Infection (LTBI) is increasing in number especially in countries with high TB incidence rate, such as Indonesia. Although not every LTBI will become active TB, if untreated and not handled appropriately it can still be a source of transmission and may increase the rate of resistance to the first-line TB drugs. Mycobacterium tuberculosis as a cause of tuberculosis disease is an intracellular pathogens that survives within the phagosome of host macrophages. Several host factors are involved in this process, including the Tryptophan Aspartate-containing Coat Protein (TACO). TACO is a protein recruited and retained by viable Mycobacterium tuberculosis on the surface of the phagosome membrane to maintain its survival in phagosome, because the presence of TACO plays an important role in inhibiting the fusion of phagosomes and lysosomes. Objective: the aim of this studyis to assess the difference of gene expression TACO protein in Latent Tuberculosis Infection (LTBI) and healthy people. Method: A preliminary studyof mRNA examination of TACO protein using Immunocytochemistry (ICC) and Real Time-Polimerase Chain Reaction (RT-PCR) method by a PCR Light Cycler 2.0 machine (Roche) in LTBI and healthy groups. Results: 18 samples of peripheral blood monocyte cells (PBMCs) were collected and divided into 2 groups. We found that there was a significantly difference between the 2 groups of samples. Conclusion: Further research is required to consider that the measurement of TACO expression using RT-PCRcan used as one of the other method to determine LTBI.

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HIV Infection Functionally Impairs Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses

Journal of Virology ◽

10.1128/jvi.01728-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 4

Author(s):

Patrizia Amelio ◽

Damien Portevin ◽

Jerry Hella ◽

Klaus Reither ◽

Lujeko Kamwela ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Hiv Infection ◽

Cd4 T Cells ◽

Latent Tuberculosis ◽

Cd4 T Cell ◽

Content Type ◽

Cell Responses ◽

Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus (HIV) infection is the major risk factor predisposing for Mycobacterium tuberculosis progression from latent tuberculosis infection (LTBI) to tuberculosis disease (TB). Since long-term-treated aviremic HIV-infected individuals remained at higher risk of developing TB than HIV-uninfected individuals, we hypothesized that progression from LTBI to pulmonary TB (PTB) might be due not only to CD4 T-cell depletion but also to M. tuberculosis-specific CD4 T-cell functional impairment. To test this hypothesis, M. tuberculosis-specific T-cell frequencies and cytokine profiles were investigated in untreated Tanzanian individuals suffering from LTBI (n = 20) or PTB (n = 67) and compared to those of untreated M. tuberculosis/HIV-coinfected individuals suffering from LTBI (n = 15) or PTB (n = 10). We showed that HIV infection significantly reduced the proportion of Th2 (interleukin 4 [IL-4]/IL-5/IL-13) producing M. tuberculosis-specific CD4 T cells and IL-2-producing M. tuberculosis-specific CD4 and CD8 T cells in individuals with LTBI or PTB (P < 0.05). Interestingly, the loss of IL-2 production was associated with a significant increase of PD-1 expression on M. tuberculosis-specific CD4 and CD8 T cells (P < 0.05), while the loss of Th2 cytokine production was associated with a significant reduction of Gata-3 expression in memory CD4 T cells (P < 0.05). Finally, we showed that the serum levels of IL-1α, IL-6, C-reactive protein (CRP), IL-23, and IP-10 were significantly reduced in M. tuberculosis/HIV-coinfected individuals with PTB compared to those in HIV-negative individuals with PTB (P < 0.05), suggesting that HIV infection significantly suppresses M. tuberculosis-induced systemic proinflammatory cytokine responses. Taken together, this study suggests that in addition to depleting M. tuberculosis-specific CD4 T cells, HIV infection significantly impairs functionally favorable M. tuberculosis-specific CD4 T-cell responses in Tanzanian individuals with LTBI or PTB. IMPORTANCE Mycobacterium tuberculosis and human immunodeficiency virus (HIV) infections are coendemic in several regions of the world, and M. tuberculosis/HIV-coinfected individuals are more susceptible to progression to tuberculosis disease. We therefore hypothesized that HIV infection would potentially impair M. tuberculosis-specific protective immunity in individuals suffering from latent tuberculosis infection (LTBI) or active pulmonary tuberculosis (PTB). In this study, we demonstrated that M. tuberculosis/HIV-coinfected individuals have fewer circulating M. tuberculosis-specific CD4 T cells and that those that remained were functionally impaired in both LTBI and PTB settings. In addition, we showed that HIV infection significantly interferes with M. tuberculosis-induced systemic proinflammatory cytokine/chemokine responses. Taken together, these data suggest that HIV infection impairs functionally favorable M. tuberculosis-specific immunity.

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