Characterization of regulatory T cells identified as CD4⁺CD25^highCD39⁺ in patients with active tuberculosis

Abstract

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T_reg), but it is also present on activated T cells, thus hindering correct T_reg identification. Using classical markers for T_reg recognition, discordant results were found in terms of T_reg expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T_reg detection. The objectives of this study were: (i) to identify T_reg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T_reg are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T_reg function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T_reg were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39⁺ cells within the CD4⁺CD25^high cells have T_reg properties (absence of interferon-γ production and transforming growth factor-β1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T_reg by classical or novel markers. In contrast, a significantly higher percentage of T_reg was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39⁺ T_reg increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T_reg identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

Introduction

Tuberculosis (TB) remains one of the world's leading causes of mortality because of a single infectious agent, with approximately 1·5 million deaths and 9·2 million new cases per year as estimated in 2006 [1]. It is estimated that worldwide one-third of the human population is infected with the causative agent Mycobacterium tuberculosis and is therefore at risk of developing the disease.

Regulatory T cells (T_reg) play an important role in immune regulation to prevent autoimmunity diseases and to control the immune responses by down-regulating the function of effector CD4⁺ or CD8⁺ T cells [2]. It is believed that T_reg down-modulate immune responses after pathogen eradication to avoid exacerbated pathology. Although this mechanism is generally to the benefit of the host in acute infections, it poses problems in chronic infections, notably when pathogen persistence is sustained in the face of an active immune response. T_reg comprise multiple subsets: naturally occurring thymus-derived CD4⁺CD25^high T cells, that have the ability of cell-contact-dependent suppression of immune response, and different subsets of adaptive T_reg, such as Tr1 cells secreting high levels of interleukin (IL)-10, and T helper type 3 cells which produce high levels of transforming growth factor (TGF)-β1 [3,4]. It has been shown that several factors such IL-2, IL-10 and TGF-β1 are involved in the generation and maintenance of T_reg[5].

Natural T_reg express constitutively CD25 (α chain of IL-2 receptor); they are identified as CD4 T cells with high expression of CD25 and they have regulatory properties. This fact introduced certain difficulties and confusion in distinguishing T_reg from conventional non-regulatory activated CD4⁺CD25⁺ T cells. At present, the intracellular forkhead winged-helix family transcriptional repressor p3 (FoxP3) is described as the most specific marker of T_reg. FoxP3 expression correlates well with regulatory activity; it is expressed in CD4⁺CD25⁺ T_reg and is considered a key player for their development and function [6]. FoxP3 represses IL-2, IL-4 and interferon (IFN)-γ gene expression and interacts with nuclear transcription factors of activated T cells (nuclear factor-κB, nuclear factor of activated cells), resulting in poor cytokine production and impaired proliferation [7]. However, the FoxP3 marker is also present in activated T cells [8]. Moreover, recent studies have shown that a lack or low expression of CD127 (the α chain of the IL-7 receptor) is linked with T_reg identification similar to CD4⁺CD25⁺FoxP3⁺ T cells [9]. Recently CD39, an ectoenzyme that degrades adenosine triphosphate (ATP) to adenosine 5′-monophosphate, has been shown to be associated specifically to T_reg, as described in inflammatory autoimmune diseases [10].

The T_reg have been implicated in infectious diseases, particularly in chronic or persistent infections [3,11]. Discordant results were found ex vivo in terms of T_reg expansion during active TB disease, some authors reporting an increase of CD4⁺CD25⁺FoxP3⁺ T cells, whereas others reported absence of modulation of this T cell subset [2,12–15]. Thus, the objectives of this study were to: (i) identify T_reg expressing CD39 in patients with TB and compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T_reg can be expanded in vitro by exogenous IL-2 or by antigen-specific stimulation with region of difference (RD)-1 proteins; and (iii) to characterize T_reg functions in the modulation of antigen-specific responses.

Materials and methods

Patient population

Thirteen patients with newly diagnosed pulmonary TB and 12 healthy control subjects were recruited at the National Institute for Infectious Diseases ‘Lazzaro Spallanzani’. The study was approved by the Ethics Committee of the Institute, and all enrolled individuals provided written informed consent. All the patients included in the study were sputum culture positive for M. tuberculosis, tested negative for human immunodeficiency virus infection and did not receive immunosuppressive drugs. Healthy subjects were chosen if: (i) no exposure to M. tuberculosis was reported; (ii) negative response to the QuantiFERON-TB Gold (Cellestis Limited, South Melbourne, Australia) was shown; and (iii) negative tuberculin skin test (TST) scoring. The characteristics of all participants are shown in Table 1.

Table 1.

Epidemiological and demographic characteristics of the subjects enrolled.

	Active TB n 13 (%)	Healthy subjects n 12 (%)
Median age in years (range)	33(21–65)	34(27–45)
Female (positive over total)	4(30·7)	7(58·3)
Origin (positive over total)
West Europe	1(7·7)	12(100)
East Europe	6(46·1)	0
Asia	3(23·1)	0
Africa	3(23·1)	0
TST (positive over total)	9(69·2)	0
BCG-vaccinated (positive over total)	13(100)	0
QuantiFERON-TB Gold In-Tube (positive over total)	11(84·6)	0

BCG, bacillus Calmette–Guérin; TB, tuberculosis; TST, tuberculin skin test.

Patients with active TB were studied within 7 days of admission and before they started anti-TB therapy. For each enrolled participant, a blood sample was drawn into tubes containing heparin.

The TST was administered by the Mantoux procedure using 5 IU of purified protein derivative (Chiron, Siena, Italy). Results were read after 72 h. Induration of at least 10 mm was considered a positive response [16].

QuantiFERON-TB Gold In-Tube (Cellestis Limited, Carnegie, Victoria, Australia) was performed and its results were scored as indicated by the manufacturer (cut-off value for a positive test was 0·35 IU/ml).

In a group of patients we also evaluated cytomegalovirus (CMV) serology by VIDAS CMV immunoglobulin (Ig)G and IgM (Biomerieux, Marcy l'Etoile, France).

Isolation of peripheral blood mononuclear cells and cultures

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using Ficoll density gradient centrifugation. After washing, PBMC at a concentration of 1 × 10⁶ cells/ml were cultured for 6 days at 37°C and 5% CO₂ in a complete medium (RPMI-1640, 10% heat-inactivated human serum, 10 mM HEPES, 2 mM L-glutamine and 10 U/ml penicillin–streptomycin, all from Euroclone Ltd, Weatherby, UK). Cultures were prepared in 48-well tissue-culture plates (Costar, Corning Inc., NY, USA) in 1 ml/well. PBMC were stimulated with M. tuberculosis-specific antigens identified as early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (RD1) intact proteins (Lionex, Braunschweig, Germany) at 4 µg/ml in the presence or absence of IL-2 (Chiron) at 5 U/ml.

Depletion of CD4⁺CD25⁺ T cells

In some experiments, CD4⁺ T cells were isolated from PBMC by negative selection (Miltenyi Biotec, Bergisch Gladbach, Germany), then CD4⁺CD25⁺ T cells were selected positively from CD4⁺ T cells using a human CD4⁺CD25⁺ T_reg isolation kit, according to the manufacturer's instructions (Miltenyi Biotec). Approximately 90% of T_reg were eliminated after depletion, as determined by flow cytometry analysis. The cell fractions CD4^– and CD4⁺CD25^– were pooled and used as T_reg-depleted PBMC.

Cytokine detection

From PBMC different cell subsets were sorted (MoFlo, Beckman Coulter, Fullerton, CA, USA) based on the expression of CD39 on CD25^high/lowCD4⁺ T cells, and were used to determine TGF-β1 production; B cells (CD19⁺ cells) and monocytes (CD14⁺CD3^– cells) were used as controls. The different cell subsets were in vitro stimulated for 1 day with anti-CD3 and anti-CD28 antibodies (Becton Dickinson, San José, CA, USA). TGF-β1 and IL-10 release was evaluated by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA).

The IFN-γ in the cell cultures' supernatants was evaluated by ELISA according to the manufacturer's instructions (QuantiFERON-CMI; Cellestis Limited). Cytokines results were expressed as pg/ml.

Antigen-specific response evaluation

Freshly isolated T_reg-undepleted PBMC and T_reg-depleted PBMC from TB patients were cultured in 96-well plates at a concentration of 2×10⁵ cells per well in 250 µl of complete medium. The cells were stimulated with M. tuberculosis-specific RD1 intact proteins (Lionex), phytohaemagglutinin at 5 µg/ml (Sigma Aldrich, St Louis, MO, USA) and CMV lysate (StrainAD 169; ABI Inc., Columbia, MD, USA) at 2 µg/ml and cultured for 3 days.

Phenotypic analysis

Phenotypic analysis of PBMC (1 × 10⁶ cells/ml) was performed by flow cytometry either ex vivo or after 1 and 6 days of culture in complete medium in the presence or absence of the stimuli as indicated above. Expression of different markers was assessed by staining with appropriate combinations of monoclonal antibodies (mAb) conjugated directly to fluorochromes: fluorescein isothiocyanate-conjugated anti-CD39 (Ancell, Bayport, MN, USA), phycoerythrin-conjugated anti-CD25 (Becton Dickinson), peridinin chlorophyll–protein complex-conjugated anti-CD4 (Becton Dickinson) and Alexa Fluor 647-conjugated anti-FoxP3 (Becton Dickinson).

To detect intracellular expression of IFN-γ, 10 µg/ml of brefeldin A (Sigma Aldrich) was used, as described previously [17]. Phorbol-12-myristate-13-acetate (PMA) plus ionomycin (Sigma Aldrich) were used as positive controls at 3 nM and 1·5 µM respectively. Briefly, production of IFN-γ was assessed by staining with appropriate combinations of mAb conjugated directly to fluorochromes. Data acquisition and analysis were performed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQUEST software (version 3.1; Becton Dickinson). For all staining procedures, an isotype-matched negative control was processed in parallel.

Statistical analysis

Statistical significance of results was determined with the statistics programme included in the GraphPad Prism software (GraphPad). Statistical analysis was performed using Student's t-test to assess differences between the different study groups. The Wilcoxon matched-pairs t-test was used to analyse cytokines production by T_reg. Differences were considered significant when the P value was less than 0·05.

Results

Epidemiological and demographic characteristics of the subjects enrolled

Demographic and clinical characteristics of the 13 patients with active TB and 12 healthy control subjects are summarized in Table 1. Among the 13 patients with active TB, nine (69%) had TST-positive results. All TB subjects were bacillus Calmette–Guérin (BCG)-vaccinated. Among the 12 control subjects, all were TST-negative, BCG-unvaccinated and none was infected with M. tuberculosis (negative result to QuantiFERON-TB Gold).

Phenotypic analysis of T_reg

To compare the classical markers for T_reg (CD25, FoxP3 and CD127) with the recently identified CD39 marker, we stained PBMC ex vivo with the appropriate combination of mAbs.

As shown in Fig. 1a, the CD25 marker identified three distinct populations of CD4⁺ T cells (high, low and negative) that also express FoxP3 (Fig. 1b–d), CD127 (Fig. 1e–g) and CD39 (Fig. 1h–j) differently. Interestingly, the CD39 marker is expressed almost exclusively by the CD4⁺CD25^high population (median 40 ± 20% Fig. 1h), similar to FoxP3 (median 86 ± 14% Fig. 1b). Conversely, CD127 is expressed at very low levels by CD4⁺CD25^high compared with the CD4⁺CD25^low and CD4⁺CD25^neg cells.

Fig. 1.

Phenotypic analysis of T regulatory cells (T_reg). Classical markers for CD4 T_reg[CD25, forkhead box P3 (FoxP3) and CD127] were compared with the innovative CD39 marker. Peripheral blood mononuclear cells (PBMC) from healthy control subjects were stained with appropriate combinations of monoclonal antibodies and fluorescence activated cell sorter (FACS) analysis was performed, as described in the Materials and methods section. The expression of FoxP3 (b–d), CD127 (e–g) or CD39 (h–j) was evaluated among CD4⁺CD25^high, CD4⁺CD25^low and CD4⁺CD25^neg T cells (a). Representative flow cytometric panels from one of five donors are shown.

Characterization of CD39⁺ T cells

To evaluate whether CD39⁺ T cells have the functional characteristics of T_reg, intracellular IFN-γ cytokine expression was evaluated. As shown previously, the CD4 marker is down-modulated after PMA stimulation [18,19], therefore we analysed IFN-γ cytokine production by CD8^–CD3⁺CD39⁺ T cells. Among the gated CD3⁺CD8^– T cells (Fig. 2a), we found that CD39 expression correlates with the inability to produce IFN-γ (Fig. 2b), consistent with a regulatory phenotype. Conversely, IFN-γ was produced significantly by CD8^–CD3⁺CD39^– cells. TGF-β1 production characterizes T_reg function further; however, it cannot be evaluated by intracellular staining. Therefore its release was evaluated by immunoenzymatic methods in different sorted-cell subsets according to the expression of CD39 on CD25^high/lowCD4⁺ Tcells; B cells (CD19⁺ cells) and monocytes (CD14⁺ CD3^– cells) were used as controls (Fig. 2c and d). The different cell subsets were stimulated in vitro with anti-CD3 and anti-CD28 antibodies, with the exception of B cells and monocytes. As shown in Fig. 2c, IFN-γ production was significantly lower in CD4⁺CD25^highCD39⁺ compared with the other cell subsets. Conversely, TGF-β1 release (Fig. 2d) was significantly higher in CD4⁺CD25^highCD39⁺ compared with the other cell subsets, substantiating the notion that these cells have T_reg characteristics.

Fig. 2.

Characterization of CD39⁺ T cells. Peripheral blood mononuclear cells (PBMC) from a healthy control subjects of five tested were stained with CD8 and CD3 monoclonal antibodies (mAbs), as shown in (a). Interferon (IFN)-γ cytokine expression on CD39 T cells was evaluated among CD3⁺CD8^– gated T cells by flow cytometer (b). IFN-γ (c) and transforming growth factor (TGF)-β1 (d) production was evaluated in different cell subsets that were sorted within the CD4⁺ T cells based on the expression of CD39 on CD25^high/low. Moreover, B cells (CD19⁺ cells) and monocytes were used as controls. Cell subsets, with the exception of monocytes and B cells, were stimulated in vitro for 1 day with anti-CD3 and anti-CD28 mAbs. IFN-γ and TGF-β1 release was evaluated by enzyme-linked immunosorbent assay. Results are shown as IFN-γ and TGF-β1 production from a representative healthy subject of two tested (c–d). Fluorescence activated cell sorter analysis and sorting were performed as described in the Materials and methods section.

Ex vivo and in vitro evaluation of CD4 T_reg by different markers in healthy donors and TB patients

We then measured the frequency of CD25^highFoxP3⁺, CD25^highCD39⁺ and CD25^highCD127^– on gated CD4⁺ T cells in ex vivo PBMC of subjects with or without active TB (Fig. 3a). The percentage of CD4⁺CD25^highCD39⁺ and CD4⁺CD25^highCD127^– cells was higher in TB patients than in healthy donors, although the difference was not statistically significant. The percentage of CD4⁺CD25^highFoxP3⁺ cells was comparable in TB versus healthy donors. Thus, no increase of T_reg was found in the ex vivo analysis in patients with active TB disease compared with controls.

Fig 3.

Ex vivo and in vitro evaluation of CD4 T regulatory cells (T_reg) by different markers in healthy donors and patients with tuberculosis (TB). Ex vivo evaluation of T_reg in patients with or without active TB. Ex vivo expression of CD25^high in combination with forkhead box P3 (FoxP3) (white bar), CD39 (grey bar) or CD127 (black bar) on gated CD4⁺ T lymphocytes, from healthy donors and TB patients, was evaluated by flow cytometry (a). Overtime evaluation of CD25 and FoxP3 expression on gated CD4 T cells after RD1-specific stimulation. In vitro culture of peripheral blood mononuclear cells (PBMC) stimulated for 1 or 6 days by RD1 proteins, in the presence or absence of interleukin (IL)-2, was performed. CD4⁺CD25^highFoxP3⁺ cells were significantly higher in TB patients at days 1 (P = 0·01) and 6 (P = 0·01) in response to RD1 proteins compared with healthy donors. Exogenous IL-2 increased significantly the expansion of T_reg in RD1 protein-stimulated PBMC at days 1 (P = 0·04) and 6 (P = 0·04) (b). Overtime evaluation of CD25 and CD39 expression on gated CD4 T cells after RD1-specific stimulation. CD4⁺CD25^highCD39⁺ T cells were increased significantly at days 1 (P = 0·0009) and 6 in TB patients (P = 0·03) after RD1 protein-specific stimulation which was increased further by exogenous IL-2 at days 1 (P = 0·0058) and 6 (P = 0·04) (c). Data are shown as the mean of six independent experiments in which 12 healthy donors and 13 patients with active TB were studied. Standard deviations are reported. HD, healthy donors; TB, active pulmonary TB disease patients; RD, region of difference.

Further, we evaluated if in vitro antigen-specific stimulation could expand T_reg in TB patients. Concerning the CD4⁺CD25^highFoxP3⁺ T cells, a significant difference was found in TB patients at days 1 (P = 0·01) and 6 (P = 0·01) stimulated with RD1 proteins compared with healthy donors. Exogenous IL-2 increased the expansion of T_reg significantly in RD1 protein-stimulated PBMC at days 1 (P = 0·04) and 6 (P = 0·04) (Fig. 3b).

Similarly, using CD39 as a T_reg marker, a significantly higher increase of this cell population was found at days 1 (P = 0·0009) and 6 (P = 0·03) in TB patients compared with healthy donors after RD1 protein-specific stimulation, which was increased further by the addition of exogenous IL-2 at days 1 (P = 0·005) and 6 (P = 0·04) (Fig. 3c). Thus, RD1-specific stimulation induces a significant expansion of T_reg in patients with active TB.

Cytokine analysis after RD1-specific stimulation

Among patients with active TB we evaluated whether IL-10 and TGF-β1 were produced upon stimulation with RD1 proteins in the presence or absence of exogenous IL-2. The results indicate that, at day 6, IL-10 production was increased in response to RD1 proteins in TB patients (median 298·7 pg/ml; range: 10·88–1058·26 pg/ml) compared with healthy donors (median: 262·5 pg/ml; range: 48·21–493·6 pg/ml), although it was not statistically significant. On the other hand, TGF-β1 production was not modulated by M. tuberculosis-specific stimulation [TB patients (median 8189 pg/ml, range: 2080–18328 pg/ml) versus healthy donors (median 8460 pg/ml, range: 2378–14607 pg/ml)] (data not shown).

Depletion of CD4⁺CD25⁺CD39⁺ T cells from TB patients PBMC increases responses to recall antigens

To investigate whether T_reg may hinder an anti-TB immune response, CD4⁺CD25^highCD39⁺ T cells were depleted from TB patients' PBMC (Fig. 4).

Fig. 4.

Flow cytometric analysis before and after depletion of T regulatory cells (T_reg) from tuberculosis (TB) patients' peripheral blood mononuclear cells (PBMC). TB patients' PBMC were stained with CD4 and CD25 before (a) and after (c) depletion of T_reg. CD39 expression was evaluated on gated CD4⁺CD25^high T cells (b). Fluorescence activated cell sorter (FACS) analysis was performed as described in the Materials and methods section. Representative panels from one patient are shown. The percentage of T_reg evaluated as CD4⁺CD25^high in T_reg-undepleted PBMC is 2% (a). Among them 88% are CD39⁺ (b). After T_reg depletion the percentage of T_reg is 0·2% (c).

The T_reg (Fig. 4a) were depleted from PBMC; after this depletion, the cells were characterized by fluorocitometric analysis by being almost all CD39⁺ (88% positivity) (Fig. 4b). The T_reg-undepleted (Fig. 4a) and T_reg-depleted (Fig. 4c) cell populations were used to perform functional experiments.

In those with detectable serum IgG antibodies to CMV, we used specific stimulation with CMV as control to evaluate the response to a recall antigen. In the four patients analysed, CD4⁺CD25⁺CD39⁺-depleted T cells showed a significantly enhanced RD1 protein- and CMV-mediated production of IFN-γ (P = 0·001) compared with the T_reg-undepleted PBMC fraction (Fig. 5a) after 3 days of antigen-specific stimulation. Data are also shown as fold of increase of IFN-γ responses to antigens in T_reg-depleted PBMC/T_reg-undepleted PBMC (Fig. 5b).

Fig. 5.

Depletion of CD4⁺CD25⁺CD39⁺ T cells from tuberculosis (TB) patients' peripheral blood mononuclear cells (PBMC) increases interferon (IFN)-γ production in response to recall antigens. PBMC from TB patients were cultured in the presence or absence of RD1-specific proteins and cytomegalovirus (CMV). After 3 days of culture IFN-γ production was evaluated in the supernatants by enzyme-linked immunosorbent assay in T regulatory cells (T_reg)-undepleted PBMC (grey bar) and T_reg-depleted PBMC (black bar). IFN-γ production was increased in response to RD1 proteins and CMV in T_reg-depleted PBMC. Results are shown as IFN-γ production from a representative TB patient (a) and as fold of increase of IFN-γ responses to antigens in T_reg-depleted PBMC/T_reg-undepleted PBMC from four TB patients (b). RD, region of difference; Pt, patient.

Discussion

In this study we evaluated whether T_reg were expanded in active TB disease using different T_reg markers using ex vivo and in vitro cell culture systems assessment. We demonstrated that CD39 is a useful marker to detect T_reg because, within CD4⁺CD25^high cells, it identifies a cell subset characterized by high production of TGF-β1 and the absence of IFN-γ expression. Moreover, we show that ex vivo evaluation of CD4 T_reg, identified by either the expression of CD25^highFoxP3⁺CD127^– molecules or by the CD39 marker, did not show an increase in the peripheral blood of active TB patients compared with healthy donors. Conversely, in active TB patients, RD1 protein-specific stimulation in vitro expanded T_reg, evaluated by the classical and the new marker, which was increased further by IL-2. This T_reg expansion was associated with an increase of endogenous IL-10, although it was not statistically significant. Depletion of CD4⁺CD25^highCD39⁺ increased M. tuberculosis-specific responses, as well as other recall antigens responses. In conclusion, for the first time, to our knowledge, we demonstrated that CD39 is a good marker for T_reg identification in an infectious disease such as TB. This information can be useful for future studies to monitor M. tuberculosis-specific response during TB.

To validate CD39 as an appropriate marker for T cells endowed with suppressive abilities, we measured the capability of CD39⁺ T cells to produce IFN-γ and TGF-β. Interestingly, we found that CD39⁺ T cells do not produce IFN-γ and those within the CD4⁺CD25^high subset do produce TGF-β. These data, together, indicate that the CD4⁺CD25^highCD39⁺ present T_reg functions.

Discordant results were found ex vivo in terms of T_reg expansion in those with TB disease, with some authors reporting an increase of CD4⁺CD25⁺FoxP3⁺ T cells [2,12–14] whereas others showed the absence of modulation of this cell subset [15]. In this study we did not observe any statistical difference between healthy and diseased subjects analysing three different combinations of markers identifying T_reg. Conversely, an increase of T_reg after in vitro specific-stimulation in patients with active TB was found. The antigen-specific increase of T_reg was amplified by exogenous IL-2, which is a well-known factor for T_reg generation [5]. T_reg increase was demonstrated by the expression of classical (FoxP3, CD25 and CD127) and innovative (CD39) markers.

The mechanisms by which T_reg control immune responses are understood incompletely, but there is evidence for a central role of the inhibitory cytokines IL-10 and TGF-β1. When these cytokines were evaluated in TB patients, we found an increase of IL-10 after specific stimulation, although not statistically significant. On the other hand, no modulation of TGF-β1 was found. These data are in line with those found by Roberts et al. [15], who did not find any statistical difference between TB patients and latent TB infection (LTBI) controls in terms of mRNA expression for IL-10 and TGF-β1 after in vitro BCG vaccination.

Depletion of CD4⁺CD25^highCD39⁺ increased M. tuberculosis-specific responses, as well as other recall antigens responses, indicating that T_reg broadly modulate antigen-specific immunity. This result is consistent with the findings by other authors [2,12,13], who reported an increase of IFN-γ response, specific either to BCG or RD1- or heparin-binding haemagglutinin adhesin-proteins after the depletion of CD4⁺CD25⁺ T cells.

This study provides the first evidence for a role of T_reg identified as CD4⁺CD25^highCD39⁺ in TB. The added value of this finding is that CD39 may allow an easier detection of T_reg compared with FoxP3, which is an intracellular marker. Moreover, at least in humans, FoxP3 is up-regulated during activation, and a fraction of FoxP3⁺ cells produces IFN-γ (unpublished observations). On the contrary, CD39⁺ cells do not produce IFN-γ and their role in the control of unwanted immune reactions (such as those that occur in autoimmune diseases) has been suggested recently. Indeed, in patients affected by multiple sclerosis, an autoimmune disease of the central nervous system, CD39⁺ T cells are greatly reduced compared with healthy subjects. Our results are consistent with the hypothesis that T_reg help to control the critical balance between immune-mediated suppression of M. tuberculosis and immunopathology in patients with TB.

More work is required to delineate the role of these T_reg in TB disease and infection. It will be interesting to analyse prospectively whether dynamic changes in T_reg frequencies are associated with the paradoxical worsening of symptoms and tissue inflammation observed in a proportion of patients with TB during treatment. It is also important to ascertain whether or not T_reg are expanded in latent TB infection, where pathogen and antigen loads are low and immunopathology is absent.

Disclosure

The authors have no conflicting financial interests.