Elsevier

Journal of Infection

Volume 66, Issue 6, June 2013, Pages 475-486
Journal of Infection

IFNγ/TNFα specific-cells and effector memory phenotype associate with active tuberculosis

https://doi.org/10.1016/j.jinf.2013.02.004Get rights and content

Summary

Objectives

Controversial results were reported on the role of polyfunctional T-cells in tuberculosis (TB). Our aim was to simultaneously characterize the Mycobacterium tuberculosis (Mtb)-specific immune response as cytokine production and memory phenotype by flow cytometry after in vitro stimulation with region of difference 1 (“RD1”) recombinant proteins (ESAT-6 and CFP-10) in patients at different TB stage in a low TB endemic country. To assess the specificity of these findings, we evaluated the response to cytomegalovirus (CMV), an unrelated antigen.

Methods

We enrolled subjects with active TB, cured TB, latent TB infection (LTBI). Cytokine and phenotype profiles of T-cells from whole blood stimulated with “RD1” proteins and CMV were characterized by multi-parametric flow cytometry.

Results

Bifunctional IFNγ+ TNFα+ CD4+ T-cells and effector memory phenotype significantly associated with active TB compared to the LTBI group (p = 0.008, at least p ≤ 0.009 respectively) whereas “RD1”-T-cell response in cured TB and LTBI was characterized by a central memory phenotype (at least p ≤ 0.013 and p ≤ 0.004 respectively vs active TB). In contrast, response to CMV antigen was not associated with a TB-specific status.

Conclusion

We identified qualitative associations between Mtb-specific T-cell and TB status in terms of functional capacity and memory status. These immune correlates may be helpful to trace natural history of TB.

Introduction

Tuberculosis (TB) kills approximately 1.4 million individuals annually.1 Over 2 billion people are latently infected with Mycobacterium tuberculosis, as estimated by tuberculin skin testing (TST), representing a huge reservoir from which most new TB cases arise. Between 3% and 10% of all latently infected individuals will develop TB during their lifetime. The control and elimination process of the global TB epidemic could be enhanced by identifying and treating individuals with latent TB infection (LTBI).

CD4+ T helper 1 (Th1) cells play an essential role in protecting against M. tuberculosis producing interferon gamma (IFNγ) and tumor necrosis factor alfa (TNFα) which synergize to activate microbicidal effector mechanisms in human macrophages.2 IFNγ-deficient mice and humans with IL12- or IFNγ-receptor abnormalities demonstrate increased susceptibility to mycobacterial diseases.3, 4, 5 The importance of TNFα is suggested by observing LTBI reactivation,6 when patients with autoimmune disorders are treated with neutralizing anti-TNFα monoclonal antibodies (mAbs).7

The ability of antigen-specific T-cells to simultaneously produce a range of cytokines (i.e. polyfunctional T-cells) has been associated with superior functional capacity8 and has been correlated with the control of human chronic viral infections such as HIV8, 9, 10 and hepatitis C.11 However, while the role of polyfunctional T-cells in protective immunity against other intracellular pathogens as Leishmania major12 has been convincingly proven, the existing data in human TB are currently inconclusive. Previous studies investigating polyfunctional T-cells in human TB have focused solely on IFNγ and IL2 producing T-cells.13, 14, 15, 16, 17 Recent papers have focused predominantly on triple-positive T-cells producing IFNγ IL2 TNFα in human TB disease and infection.18, 19, 20, 21, 22, 23, 24 However, contrasting findings regarding distribution of the various cytokine-producing CD4+ T-cell subsets in individuals with LTBI and TB have been reported.

Based on the expression of surface markers, CD4+ T-cells can be divided into at least four different populations: naïve (N) referred to as CD45RA+ CD27+, central memory (CM) as CD4RA CD27+, effector memory (EM) as CD45RA CD27, and terminally differentiated effector memory (E) T-cells as CD4RA+ CD27.25 EM T-cells rapidly respond to re-infection, whereas CM T-cells are long-lived T-cells with self-renewal and a proliferative capacity that might contribute to EM pool maintenance.26 In several infection models including TB, it has been shown that effector T-cells are expanded during active replication and memory cells are detectable after control and eradication.25, 27

It has been demonstrated that individuals with active TB show a higher proportion of antigen-specific EM T-cells28 and a reduced frequency of CM CD4+ T-cells than LTBI individuals.29

The aim of this study was to simultaneously characterize the M. tuberculosis-specific immune response in terms of cytokine production and memory phenotype by flow cytometry after in vitro whole blood stimulation with region of difference 1 (“RD1”) recombinant proteins (ESAT-6 and CFP-10) in patients at different TB stages in a low TB endemic country as Italy.1 To evaluate the specificity of the findings in terms of TB response, we also evaluated the response to cytomegalovirus (CMV), an unrelated antigen.

Section snippets

Study population and sample collection

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

Enrolled subjects were selected to be positive to Quantiferon-Gold in Tube (QFT-IT) (Cellestis Limited, Carnegie, Victoria, Australia). Active TB patients, diagnosed with drug-susceptible pulmonary TB (positive culture for M. tuberculosis from sputa),

Characteristics of the population

Seventy-two subjects at different TB stages were enrolled. They were selected to be QFT-IT positive. The majority of the subjects enrolled was male and BCG-unvaccinated, and almost 60% were from Western Europe. Significant differences among the TB groups were found for origin (p = 0.002) and BCG vaccination (p < 0.0001) (Table 1).

Cytokine frequency of “RD1”-response is higher in subjects with active TB than those with LTBI

The cytokine response to “RD1” proteins, considering the production of any cytokines (IFNγ, IL2 or TNFα), was higher in the active TB group than the LTBI subjects,

Discussion

In this study, performed in a low TB endemic country, we characterized (by flow cytometry in a cross-sectional setting) the functional and memory/effector status of M. tuberculosis-specific CD4+ T-cells in subjects with active TB disease, cured TB and LTBI.

We found that bifunctional “RD1”-specific CD4+ T-cells and EM phenotype are associated with active TB disease. Conversely in cured TB and LTBI subjects the “RD1”-specific CD4+ T-cells are associated with a CM phenotype. The response to an

Acknowledgments

The authors are grateful to all the patients, nurses (in particular Gilda Cuzzi, Cristina Copertino and Immacolata Mauceri) and physicians (Alessia Rianda, Giampiero D’Offizi, Nicola Petrosillo, Susanna Grisetti, Andrea Antinori, Angela Corpolongo, Marco Gentile, Nazario Bevilacqua, Daniele Biagioli, Roberto Tonnarini, Francesco Nicola Lauria) who helped to perform this study. We also thank Teresa Chiacchio, Rita Casetti, Alexandre Harari and Giuseppe Pantaleo for their support and scientific

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