Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis

doi:10.1016/j.jinf.2015.07.009

Journal of Infection

Volume 71, Issue 5, November 2015, Pages 526-533

https://doi.org/10.1016/j.jinf.2015.07.009 Get rights and content

Highlights

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Active TB associates with Mycobacterium tuberculosis-specific CD45RA⁻ CD27⁻ CD4⁺ IFNγ⁺ T-cells.
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Active TB associates with M. tuberculosis-specific CD27⁻ CD4⁺ IFN-γ⁺ T-cells.
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Active TB associates with a high CD27 MFI RATIO on Mtb-specific CD4⁺ T-cells.
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Several cytometric methods support the use of CD27 as a biomarker for active TB.
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The above immune-approaches have been studied among the IGRA-responsive subjects.

Summary

There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4⁺ T-cells has been suggested as a tool to diagnose different TB stages.

Objectives

To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4⁺ T-cells to differentiate TB stages.

Methods

55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27^−/+ within the IFN-γ⁺ CD4⁺ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4⁺ T-cells over the CD27 MFI of IFN-γ⁺ CD4⁺ T-cells was evaluated.

Results

The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27⁺ CD45RA⁻ cells within the IFN-γ⁺ CD4⁺ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator.

Conclusions

the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages.

Introduction

The global tuberculosis (TB) epidemic is still not under control.¹ Diagnosis of pulmonary active TB relies on the evaluation of clinical symptoms, radiological images and detection of Mycobacterium tuberculosis (Mtb) in patient respiratory samples, such as sputum. Microscopic detection of Mtb in sputum smears is the most commonly used approach for diagnosing pulmonary TB and monitoring response to treatment.2, 3, 4 However, sputum smears have poor sensitivity, and a high proportion (20%–66%) of tuberculosis (TB) cases is smear-negative.² Nucleic acid amplification–based tests are more sensitive for diagnosing ATB,² but do not differentiate between live and dead Mtb, thus are not useful for monitoring treatment-mediated clearance of Mtb. Although it takes a long time to obtain microbiological isolation and culture of Mtb from sputum, it remains the TB diagnostic gold standard.² Blood-based host biomarkers for diagnosing TB are attractive alternatives to tests that rely on detecting mycobacteria.

Interferon (IFN)-γ release assays (IGRAs) are new tools for latent TB infection (LTBI) diagnosis. These tests are based on the IFN-γ response to Mtb antigens as ESAT-6 and CFP-10.3, 4They represent a breakthrough, however, they do not discriminate between active TB disease and LTBI.1, 3, 4, 5, 6, 7 IGRA accuracy for LTBI diagnosis may be enhanced using other Mtb-specific antigens8, 9 or peptides selected from ESAT-6/CFP-10,10, 11, 12 evaluating the response at the site of TB disease13, 14 or investigating host biomarkers other than IFN-γ in whole blood or peripheral blood mononuclear cells (PBMC).10, 14, 15, 16, 17, 18, 19, 20, 21

Cytometry has been proposed as a potential tool to improve TB diagnosis by phenotypical and functional characterization of antigen-specific T-cells. The cytokine profile of Mtb-specific T-cells has been studied in depth with the aim of finding a correlation with TB status.20, 22, 23, 24, 25, 26, 27, 28, 29 However, existing data are currently inconclusive due to contrasting findings on the distribution of the various cytokine-producing CD4⁺ and CD8⁺ T-cell subsets.

T-cell expression of surface molecules such as CD45RA, CD27 and CCR7 identifies different T-cell subsets that reflect different stages of cell differentiation.30, 31, 32, 33, 34 Interestingly, the effector T-cells are expanded during active Mtb replication, whereas the memory cells associate with control and eradication of Mtb infection.14, 35, 36, 37 Moreover, several reports demonstrate that the decrease of CD27 surface expression on circulating Mtb-specific CD4 T-cells associates with the status of active TB disease.22, 38, 39, 40, 41, 42, 43

Studies in mice have demonstrated that the lack of CD27 expression on T lymphocytes identifies the functionally mature highly differentiated effector T-cells.⁴⁴ In vivo studies show that in the lungs of Mtb-infected mice, the effector CD4⁺ T-cells with a low CD27 expression differentiate from effector CD4⁺ T-cell precursors with a high CD27 expression.⁴⁵ Moreover, it is shown that the Mtb infection leads to the accumulation of effector CD4⁺ T lymphocytes with low CD27 expression in the lungs, blood, and other organs.44, 45 However no consequences on the clinical outcome have been observed in CD27 KO mice infected with Mtb.⁴⁶

Interestingly, CD4⁺ T-cells with a low CD27 surface expression have also been described as a marker of lung tissue destruction during pulmonary TB, suggesting its use as an immune assay to monitor the efficacy of TB therapy.³⁸ The studies on CD27 expression on Mtb-specific T-cells show similar and reproducible results, despite the differences of the experimental settings and clinical characteristics of the enrolled patients.22, 38, 39, 40, 41, 42, 43, 47 Therefore, the phenotype data of the CD4⁺ T-cells are more consistent compared to the cytokine profile studies, suggesting that CD27 evaluation on Mtb specific-CD4⁺ T-cells may be a useful tool for diagnosing TB stages. Recently, a new assay, the T-cell activation marker of TB (TAM-TB), has been proposed.³⁹ Different from the other approaches, it evaluates the ratio of the median fluorescence intensity (MFI) of CD27 within the CD4⁺ T-cells over the MFI of CD27 in the Mtb-specific CD4 T-cells.³⁹ The TAM-TB assay differentiates active TB from LTBI in children.³⁹

In the present study, we used several concomitant approaches to evaluate whether detection of the CD27 surface expression on Mtb specific-CD4⁺ T-cells is a useful marker to discriminate active disease from LTBI in adult individuals from a low TB-endemic country such as Italy.

Section snippets

Study population and sample collection

This study was approved by the Ethical Committee of the L. Spallanzani National Institute of Infectious Diseases (INMI), approval number 02/2007. Informed written consent was required to participate in the study that was conducted at INMI.

Active TB was defined based on Mtb isolation from sputum culture. Mtb was drug-sensitive to the first line of TB drugs; patients were enrolled within 7 days of starting the specific treatment. Cured TB was defined as microbiological pulmonary TB after a

Characteristics of the patient population

We enrolled 55 subjects, 13 active TB, 12 cured TB and 30 LTBI, selected to score positive to the QFT-IT to have better chances of obtaining a positive cytometric response after RD1-specific stimulation. The majority was male and BCG-unvaccinated and 50.9% were from Western Europe. Significant differences were found for origin (p < 0.0001) and BCG vaccination (p = 0.001) (Table 1).

Active TB associates with down modulation of CD27 expression

CD4⁺ T-cells can be phenotypically divided into at least four different populations, based on the surface

Discussion

In this study, among the IGRA-responders, we show that: (i) active TB significantly associates with an increase of the CD27⁻ subset of Mtb-specific CD4 T-cells in whole blood; (ii) this result is Mtb-specific, as shown by the absence of CD27 modulation among the groups studied after in vitro CMV stimulation; (iii) the most accurate results for TB diagnosis are reached by evaluating the CD27 surface expression in the CD4⁺ IFN-γ⁺ T-cell subset (within the total IFN-γ⁺ or IFN-γ⁺ CD45RA⁻ T-cells),

Conflicts of interest

I declare that there is no conflict of interest.

Acknowledgments

The authors are grateful to all the patients, nurses and physicians who helped to perform this study. We are deeply grateful to Ms Andrea Baker (INMI, Rome, Italy) for the editing. The study was supported by grants from the Italian Ministry of Health: “Ricerca Corrente” and a grant from the European Union: HEALTH-F3-2009-241642. The funders had no role in the decision to publish the study, in analyzing the data or drafting the manuscript.

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Immune profiling of Mycobacterium tuberculosis-specific T cells in recent and remote infection
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TST and IGRAs have a low predictive value for tuberculosis [26]. Other functional assays that measure M.tb-specific T cell properties such as Th1 cytokine co-expression profiles [27,28], T cell differentiation (CD27, [29,30]) and T cell activation [17–22] have been proposed as new potential immunodiagnostic concepts that can distinguish between M.tb infection and disease. In line with this principle, potential biomarkers of recent M.tb infection based on proportions of TNF-only (IFN-γ-IL-2-) TE (CD45RA-CCR7-CD127-) CD4 T cells or T cell proliferation, as well as a blood transcriptomic signature, have been described [31–33].
Recent 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.
Healthy 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 characterised 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+.
CFP-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.
Recent 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.
US National Institutes of Health (NIH), Bill and Melinda Gates Foundation, South African National Research Foundation, South African Medical Research Council, and Aeras.
Performance of a simple flow cytometric assay in diagnosing active tuberculosis
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A recently developed flow cytometry-based assay for detection of antigen-specific CD4 T cell recall responses measures the upregulation of co-expression of surface markers of CD25 (subunit of IL-2 receptor) and CD134 (OX40, a member of TNF receptor superfamily) after antigen exposure. There are several data showing potential promising work of these cellular assays to be suitable for routine diagnostic use [8–11]. A recent data from Sauzullo, I. et al. showed that the diagnostic performance of QFT-Plus assay and co-expression of CD25/CD134 in response to active TB was good [12].
A flow cytometric assay measuring Mycobacterium tuberculosis-specific CD4 T-cell responses using co-expression of CD25/CD134 (OX40 assay) was explored as a diagnostic tool for active tuberculosis (TB) in a Thai population with and without HIV infection. Peripheral blood mononuclear cells (PBMC) obtained from 133 participants at TB diagnosis were cryopreserved. Seventy-six participants had a clinical diagnosis of TB which were confirmed by a positive culture. CD4 T-cell responses were measured after stimulation with a pool of overlapping peptides covering RD-1 antigens: CFP-10 + ESAT-6. The performance of the assay was also compared to the Xpert MTB/RIF assay. The overall sensitivity of the OX40 assay was 94.7% (95%CI 87.1–98.5); its specificity was 71.9% (95%CI, 58.5–83). The sensitivity of the OX40 assay among HIV-infected participants was 100% (95%CI, 88.8–100) with a specificity of 92.9% (95%CI, 66.1–99.8). OX40 assay performed particularly well in those with active TB and HIV infection.
Loss of T cells expressing CD27 at the site of active tuberculosis – A prospective diagnostic study
2020, Tuberculosis
The lack of a rapid and reliable diagnostic test for active tuberculosis is still a burden to the control of the infection. The accumulation of Mycobacterium tuberculosis (MTB)-specific CD4⁺ T cells at the site of infection and the increase of MTB-specific CD27⁻ cells seem to be characteristic for active tuberculosis. We evaluated CD27 expression of non-stimulated T cells at the site of infection compared to peripheral blood of seventy-two patients (n = 72) presenting with symptoms of active MTB-infection. Twenty patients (n = 20, 27.8%) were actually confirmed to have active tuberculosis. Overall, a significant increase of terminally differentiated CD27⁻ CD4⁺ T cells at the site of disease was noted when compared to peripheral blood (<0.001). However, the loss of CD27 at the site of disease was not restricted to active tuberculosis (p = 0.253). The CD27 expression profile of tuberculosis patients was only discriminative to patients with malignancy.
Multi-parameter flow cytometry immunophenotyping distinguishes different stages of tuberculosis infection
2020, Journal of Infection
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Although in our study the CD27 marker was used in order to identify the different memory subsets, modulation of the surface expression of CD27 was also assessed by means of its MFI values. Inspired by recent works assessing the MFI CD27 ratio between CD4+ T-cells and CD4+IFNγ+ T-cells,19,39 we analysed the ratio between MFI of CD27 in CD4+ cells over MFI CD27 in CD4+CD154+ T cells after Mtb-stimulation. In our hands, active TB patients and LTBI contacts showed a greater MFI CD27 ratio than NoTBI contacts, although no significant differences were found between groups (Figure S.4).
To identify new potential host biomarkers in blood to discriminate between active TB patients, uninfected (NoTBI) and latently infected contacts (LTBI).
A blood cell count was performed to study parent leukocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated and a multi-parameter flow cytometry assay was conducted to study the distribution of basal and Mycobacterium tuberculosis (Mtb)-stimulated lymphocytes. Differences between groups and the area under the ROC curve (AUC) were investigated to assess the diagnostic accuracy.
Active TB patients presented higher Monocyte-to-lymphocyte and Neutrophil-to-lymphocyte ratios than LTBI and NoTBI contacts (p<0.0001; AUC>0.8). Lymphocyte subsets with differences (p >0.05; AUC >0.7) between active TB and both contact groups include the basal distribution of Th1/Th2 ratio, Th1-Th17, CD4⁺ Central Memory (T_CM) or MAIT cells. Expression of CD154 is increased in Mtb-activated CD4⁺ T_CM and Effector Memory T cells in active TB and LTBI compared to NoTBI. In CD4⁺ T cells, expression of CD154 showed a higher accuracy than IFNγ to discriminate Mtb-specific activation.
We identified different cell subsets with potential use in tuberculosis diagnosis. Among them, distribution of CD4 T_CM cells and their expression of CD154 after Mtb-activation are the most promising candidates.
Combined IFN-γ and TNF-α release assay for differentiating active tuberculosis from latent tuberculosis infection
2018, Journal of Infection
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The results of the IGRA against ESAT-6 and CFP-10 were not significantly different in active TB versus not active TB with LTBI, whereas there were significantly more spot-forming cells (SFC) in the TARA in active TB than in not active TB with LTBI (Fig. 3). To distinguish active TB from not active TB with LTBI, many studies have analyzed specific T cell phenotypes by flow cytometry or measured levels of multiple cytokines using microbead-based immunoassays.12,13,17,24 Prabhavathi et al. suggested using the ratio of IFN-γ/TNF-α levels against the TB specific antigen, Rv2626c, for diagnosis of LTBI.14
The IFN-γ-release assay (IGRA) cannot differentiate active tuberculosis (TB) from latent TB infection (LTBI). We hypothesized that the TNF-α-release assay (TARA) combined with IGRA might discriminate active TB from not active TB without LTBI.
Adult patients with suspected TB, and with unrelated diseases such as herpes zoster as controls, were enrolled in an intermediate TB-burden country. Patients with confirmed or probable TB were regarded as active TB, and patients with not active TB were further classified as those having not active TB with and without LTBI based on IGRA results. The IGRA and TARA by using ELISPOT assays were performed on peripheral mononuclear cells.
Thirty six patients with active TB and 53 patients including 18 not active TB with LTBI and 35 not active TB without LTBI were finally included. The sensitivity and specificity of the IGRA for those patients found to have active TB were 94% (CI, 80-99) and 66% (CI 52-78), respectively. Combining the IGRA and the TARA substantially increased the specificity for active TB (93%, CI, 82-98; P = 0.001) compared with the IGRA only, without compromising sensitivity (89%, CI, 73-96; P = 0.67).
Combining the IGRA and TARA appears to be useful for diagnosing active TB.
Diagnosis and therapeutic approach of latent tuberculosis infection
2018, Enfermedades Infecciosas y Microbiologia Clinica
La detección y tratamiento de la infección tuberculosa latente (ITBL) constituye una medida esencial para el control de la tuberculosis (TB) en países de baja incidencia. Esta estrategia está limitada, sin embargo, por la falta de capacidad predictiva de las pruebas diagnósticas para el desarrollo de TB activa entre las personas infectadas y la larga duración y toxicidad de las pautas de tratamiento. Las técnicas in vitro de liberación de interferón-gamma son más específicas y sensibles que la prueba de la tuberculina, y permiten seleccionar mejor los casos que requieren tratamiento. Aun así, su capacidad para predecir el desarrollo de TB sigue siendo pobre. Además, las pautas de tratamiento de ITBL son largas, y las tasas de cumplimiento, bajas. Esta revisión discute el uso de las técnicas diagnósticas disponibles y las nuevas aproximaciones al diagnóstico de la ITBL y su abordaje terapéutico en diferentes escenarios clínicos.
Detection and treatment of latent tuberculosis infection (LTBI) is an essential measure for tuberculosis (TB) control in low-incidence countries. However, such strategy is limited by the low predictive ability of the diagnostic tests for the development of active TB among infected people and the long-term and toxic treatment regimens. The in vitro interferon-gamma release assays are more specific and sensitive than the tuberculin skin test (TST), and enable a better selection of cases requiring treatment. Nonetheless, their capacity to predict development of TB is still poor. In addition, treatment regimens for LTBI are long, and compliance rates are low. This review discusses the use of the available diagnostic tests and the new approaches to the diagnosis of LTBI, as well as its management in different clinical scenarios.

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Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis

Highlights

Summary

Objectives

Methods

Results

Conclusions

Introduction

Section snippets

Study population and sample collection

Characteristics of the patient population

Active TB associates with down modulation of CD27 expression

Discussion

Conflicts of interest

Acknowledgments

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