Mycobacteriology
Antigen-specific CD4- and CD8-positive signatures in different phases of Mycobacterium tuberculosis infection

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Abstract

Current diagnostic standards for Mycobacterium tuberculosis (MTB) infection do not distinguish between active and latent tuberculosis (TB). To identify specific biomarkers characterizing the different forms of TB infection, we investigated in parallel with the QuantiFERON -TB Gold In-Tube (QFT-IT) the use of flow cytometry measuring CD4 and CD8 MTB-specific immune response in 17 active-TB patients, 21 health care workers (HCW), 14 recent contacts of TB patients (RC-TB), and 10 bacille Calmette Guerin (BCG)–vaccinated healthy controls (BCG-HC).

A correlation (r = 0.4526, P = 0.0002) was found only between the amount of IFN-γ measured by QFT-IT and the frequency of CD4+/CD69+/IFN-γ+ T cells. The frequency of CD4+/CD69+/IFNγ+ responding T cells was higher in active-TB patients (0.254 ± 0.336%, P < 0.01) compared to the other groups. The response of QFT-IT antigen-specific CD8+/CD69+/IFNγ+ T cells was significantly higher in RC-TB (0.245 ± 0.305%, P < 0.05) compared to the other study groups.

Introduction

Tuberculosis (TB) still remains a public health problem of paramount importance (WHO, 2006). It has been estimated that nearly 2 billion people, one third of the world's population, are infected by Mycobacterium tuberculosis (MTB), the agent causing the disease, resulting in about 9 million new cases every year together with 2 million deaths (WHO, 2006). The control of TB requires a multifaceted approach by integrating public health interventions with the use of efficient new diagnostic tools, vaccines, and drugs. In particular, the rapid and exact discrimination between subjects with active and latent tuberculosis infection (LTBI) represents one of the main measures of the efficient control of disease diffusion (WHO, 2007).

Different approaches have been described recently in the literature to help in discriminating between LTBI and active-TB status. On one hand, the use of immune response against proteins of MTB associated with different phases of infection, such as dormancy-related proteins (DosR) (Leyten et al., 2006) as well as resuscitating protein factors (Commandeur et al., 2011), gives some insight without a clear-cut solution to the problem. On the other hand, a better discrimination between the different TB stages could be envisaged by a more accurate definition of the immune response to the MTB antigens (Billeskov et al., 2007, Ryan et al., 2009). However, all these approaches are still confined to research laboratories without an immediate transfer to the laboratory of clinical pathology and with no impact on patient management.

Upon infection, MTB stimulates both CD4+ and CD8+ T cells as well other cells of the immune system, eliciting a strong type 1 response dominated by interferon (IFN)-γ secretion (Leyten et al., 2006). The overall response is at the basis of the so-called delayed-type hypersensitivity (DTH) caused by MTB antigens. This phenomenon has been used for more than 1 century to identify MTB-infected subjects by the tuberculin skin test (TST), i.e., the intradermal reaction to tuberculin or PPD (Kaufmann et al., 2005).

In the last decade, extensive studies on subtractive DNA hybridization of pathogenic M. bovis and bacille Calmette–Guerin (BCG) together with a comparative genome-wide DNA microarray analysis of MTB H37Rv and BCG allowed the identification of antigens for diagnostic and vaccine development, as the immunodominant early secreted antigenic target (ESAT-6) and culture filtrate protein (CFP-10) (Mostowy and Behr 2002). Based on these studies, one of the most significant developments in the diagnostic armamentarium for TB in the last hundred years seems to be the assays based on IFN-γ determination (IGRAs), tests that adapted the T-cell response measure to the need of simplification, and the possibility of automation for clinical laboratories. The evaluation in different clinical settings of the IGRA assays using the MTB RD1 antigens has shown many advantages over the tuberculin skin test. Although their sensitivity has not been consistent across the different tests and populations, IGRAs have excellent specificity unaffected by BCG vaccination (Pai et al., 2008). However, IGRAs did not distinguish between LTBI and active TB either (Pai et al., 2008), and contradictory results have been obtained with recently exposed subjects (Mack et al., 2009).

Flow cytometry analysis by intracellular cytokine staining (ICS) following appropriate in vitro stimulation adds to the diagnostic advantages of IGRAs, as it allows the phenotypic differentiation between antigen-specific lymphocyte subsets (Sargentini et al., 2009, Sutherland et al., 2009).

In this study, with the aim of identifying specific biomarkers characterizing the different forms of TB infection, we investigated in parallel with the IGRA test QuantiFERON-TB Gold In-Tube (QFT-IT; Cellestis Ltd., Victoria, Australia) the use of single-cell ICS by flow cytometry for the identification of CD4+- and CD8+-specific immune responses elicited against the same antigens present in the QFT-IT test, by using the cells that are discarded from the QFT-IT test after antigen stimulation and recovery of the plasma supernatant for measuring the IFN-γ.

Section snippets

Study population

This cross-sectional study was performed from February to December 2009 at the Regional Dispensary for Tuberculosis and Lung Diseases, Sofia (Bulgaria). Table 1 summarizes the demographic and principal clinical characteristics for all the study groups.

Specifically, 17 MTB culture–positive confirmed pulmonary active-TB patients were evaluated before the start of the chemotherapy treatment.

A group of 14 recent family contacts of newly diagnosed acid fast bacilli (AFB)–positive active-TB patients

Results

In this subject series, the QFT-IT assay was positive in 13/17 (76.4%) active-TB patients, 7/14 (50%) RC-TB subjects, 10/21 (47.6%) HCW working in wards with TB patients, and 2/10 (20%) BCG-HC subjects (Table 1).

In order to assess the role of different antigen-specific T cells in determining the IFN-γ release in the QFT-IT assay, we first evaluated by ICS the correlation of the CD4+ and CD8+/CD69+/IFN-γ+ T-cell response against the same pool of antigens. A positive correlation (r = 0.4526, P =

Discussion

The IGRA tests, the current standard for the diagnosis of MTB infection, are not capable of discriminating between active and latent TB infection, while the efficient control of TB requires the use of tools for the identification of the 2 TB forms. In fact, even if the immunodominant antigens of the RD1 region used in the IGRAs have been capable of circumventing a number of problems related to the use of the old TST (Richeldi, 2006), there is still the need for specific biomarkers

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    The study has been supported in part by grant PRIN2007 to MA and by the grants L-1503/05 and BIn-6/06 of the NSC, Ministry of Education, Youth and Science of Republic of Bulgaria, to RM.

    1

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