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Published ahead of print on December 9, 2005, doi:10.1164/rccm.200508-1294OC
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American Journal of Respiratory and Critical Care Medicine Vol 173. pp. 803-810, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200508-1294OC

Original Article

Regulatory T Cells Are Expanded in Blood and Disease Sites in Patients with Tuberculosis

Valerie Guyot-Revol, John A. Innes, Sarah Hackforth, Tim Hinks and Ajit Lalvani

Tuberculosis Immunology Group, Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and Department of Infection and Tropical Medicine, Birmingham Heartlands Hospital, Birmingham, United Kingdom

Correspondence and requests for reprints should be addressed to A. Lalvani, D.M., Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Level 7, Oxford OX3 9DU, UK. E-mail: ajit.lalvani{at}ndm.ox.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: T-cell responses during tuberculosis (TB) help contain Mycobacterium tuberculosis in vivo but also cause collateral damage to host tissues. Immune regulatory mechanisms may limit this immunopathology, and suppressed cellular immune responses in patients with TB suggest the presence of regulatory activity. CD4+CD25high regulatory T cells mediate suppressed cellular immunity in several chronic infections but have not been described in TB.

Objective: To determine whether regulatory T cells are increased in patients with TB and whether they suppress cellular immune responses.

Methods: We compared the frequency of circulating regulatory T cells in 27 untreated patients with TB and 23 healthy control subjects using two specific markers: cell-surface CD25 expression and FoxP3 mRNA expression in peripheral blood mononuclear cells.

Measurements and Main Results: We detected a threefold increase in the frequency of CD4+CD25high T cells (p < 0.001) and a 2.2-fold increase in FoxP3 expression (p = 0.006) in patients with TB, and there was a positive correlation between these markers (r = 0.58, p < 0.001). Increased expression of interleukin-10 and transforming growth factor-beta1 mRNA was also detected in patients with TB but did not correlate with regulatory T-cell markers. Ex vivo depletion of CD4+CD25high cells from peripheral blood mononuclear cells resulted in increased numbers of M. tuberculosis antigen–specific IFN-{gamma}–producing T cells in seven of eight patients with TB (p = 0.005). Finally, FoxP3 expression was increased 2.3-fold in patients with extrapulmonary TB compared with patients with purely pulmonary TB (p = 0.01) and was amplified 2.6-fold at disease sites relative to blood (p = 0.043).

Conclusions: Regulatory T cells are expanded in patients with TB and may contribute to suppression of Th1-type immune responses.

Key Words: CD4+ • CD25+ • regulatory T cells • FoxP3 • immunopathology • Mycobacterium tuberculosis

Tuberculosis (TB) is associated with chronic, persistent antigen stimulation in vivo that maintains a sustained immune response that suppresses, but generally fails to eradicate, Mycobacterium tuberculosis (MTB). The host response involves many limbs of the cellular immune system but consists predominantly of MTB-specific Th1-type IFN-{gamma}–secreting CD4 and CD8 effector T cells (1–3). Although this response helps to limit bacterial replication and dissemination in vivo, it also causes significant immunopathology. Indeed, the clinical manifestations and natural history of TB disease are to a large extent determined by the balance between the protective and immunopathologic effects of the Th1-type cellular immune response.

The immune system has regulatory mechanisms for suppressing the effector response to persistent antigens, and these are believed to limit immune-mediated tissue pathology (4). The impaired responsiveness of peripheral blood T cells in patients with TB suggests that T-cell responses to MTB may be subject to regulatory mechanisms. Patients with active TB have reduced MTB antigen–specific IFN-{gamma} production and lymphoproliferative responses in vitro compared with healthy control subjects (5, 6). Impaired cellular immunity in vivo manifests in about 15% of patients as absence of a delayed-type hypersensitivity reaction to intradermal injection with tuberculin purified protein derivative; this cutaneous anergy is associated with the absence of granuloma formation and poor clinical outcomes (7). The regulatory cytokines interleukin 10 (IL-10) and transforming growth factor beta1 (TGF-beta1) have been implicated in suppression of in vitro antigen-specific cellular immune responses in TB. IL-10 is associated with cutaneous anergy and is secreted by Tr1 T cells, whereas TGF-beta1 is secreted by MTB-infected macrophages (7–9) and Th3 cells.

Naturally occurring CD4+CD25+ regulatory T cells, or natural Tregs, are a subset of CD4+ T cells with suppressive properties inhibiting effector functions of CD4+ and CD8+ T cells (10, 11). Arising from the thymus, they enter peripheral tissues where they suppress the activation of other self-antigen–reactive T cells (12, 13). Natural Tregs maintain self-tolerance and prevent autoimmune diseases (14, 15) but also regulate the host response to infection. In murine models, they suppress T-cell responses to several intracellular pathogens, and their depletion in vivo leads to increased immune-mediated tissue pathology. More recently, Tregs have been implicated in the immunologic hyporesponsiveness associated with certain chronic infections in humans (16, 17), including cytomegalovirus (18), hepatitis B virus (HBV) (19), hepatitis C virus (HCV) (20–23), HIV (18, 24), and Helicobacter pylori (25, 26), but no role for Tregs has been described in TB.

CD4+CD25+ cells represent 5 to 10% of human circulating CD4+ T cells, but only the subset expressing high levels of CD25, the IL-2 receptor {alpha}-chain, exhibit strong regulatory function (27). These CD4+CD25high T cells constitute 1 to 2% of the CD4+ T-cell population. However, because CD25 is also a marker of activation of T cells, its specificity as a marker of Tregs is limited. FoxP3 is a master gene governing the development and function of Tregs (28, 29). Expression of FoxP3 in transgenic mice and ectopic expression of FoxP3 in human cells has been shown to genetically reprogram T cells to a regulatory phenotype (30–32), and FoxP3 expression distinguishes Tregs from activated effector cells within the CD25+ T-cell population (33). Thus, relative FoxP3 mRNA expression is considered to be the most accurate marker of Treg activity.

Given the critical balance between immune-mediated suppression of mycobacteria and immune-mediated tissue pathology in TB, it is important to determine whether Tregs play a role in this disease. We hypothesized that patients with active, untreated TB would have higher frequencies of Tregs compared with healthy control subjects and that Treg populations would be expanded at sites of active disease where inflammation and immune-mediated pathology most require containment.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
See the online supplement for an extended version of METHODS.

Subjects and Samples
Twenty-three healthy laboratory personnel with no known TB exposure were invited to take part in the study (Table 1). All had negative IFN-{gamma} enzyme-linked immunospot (ELISPOT) assay results using overlapping peptides spanning the length of the highly MTB-specific antigens, early secretory antigenic target-6 (ESAT-6), and culture filtrate protein-10 (CFP-10) (34). Thus, although not subjected to tuberculin skin testing, all healthy control subjects were deemed to be uninfected (American Thoracic Society [ATS] classification: category 0).


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  		TABLE 1. DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF PATIENTS WITH TUBERCULOSIS AND HEALTHY CONTROL SUBJECTS

 
Twenty-seven patients with active TB disease (Table 1) were prospectively recruited at Birmingham Heartlands Hospital, United Kingdom. Protocols were approved by the Birmingham Heartlands Hospital Institutional Review Board, and written, informed consent was obtained from all participants. All patients diagnosed with active TB had positive cultures for MTB from one or more clinical specimens or clinical and radiologic features highly suggestive of TB together with a good response to anti-TB treatment over 6 mo (ATS classification: category 3; Table 1). None had started treatment at the time of enrolment. Patients coinfected with HIV, HBV, or HCV were excluded from the study. For five additional patients with TB whose disease was anatomically localized at clinically accessible sites, a sample from the disease site was obtained simultaneously with a blood sample.

Purification of CD4+CD25+ T Cells
CD4+CD25+ and CD4+CD25– T cells were enriched from peripheral blood mononuclear cells (PBMCs) by using the Dynal CD4+CD25+ Treg kit according to the manufacturer's instructions (Dynal Biotech, Wirral, UK). Briefly, CD4+ T cells were negatively isolated using a cocktail of antibodies to CD14, CD56, CD19, CD8, CD235{alpha}, and Depletion Dynabeads. Purified CD4+ T cells were then incubated with Dynabeads CD25. CD4+CD25– cells were obtained by negative selection, and CD4+CD25+ cells were positively selected and detached from the beads. The purity of the enriched T cells was verified by flow-cytometric analysis and ranged from 80 to 90% for CD4+CD25+ T cells and 90 to 97% for CD4+CD25– T cells.

CD25high T-Cell Depletion
PBMCs were depleted of CD25high T cells using CD25+ Dynabeads according to the manufacturer's instructions (Dynal). This resulted in an average 89% depletion of the CD25high cells in the depleted PBMCs (average percentage of CD25high cells before depletion, 1.13%; average percentage after depletion, 0.13%). After this maneuver, the absolute percentage of CD25+ cells was reduced from 5.27% (predepletion) to 4.22% (postdepletion; average 20% depletion).

Real-Time Quantitative Reverse Transcriptase–Polymerase Chain Reaction
Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. RNA samples were treated with RNase-free DNase I to eliminate contaminating genomic DNA. RNA was reverse transcribed using Sensiscript reverse transcriptase and 0.01 µg/µl random primers (Invitrogen, Paisley, UK) in a final volume of 20 µl. Amplification was performed in a total volume of 25 µl containing qPCR Master Mix (Eurogentec, Romsey, UK), primers, and internal fluorescent TaqMan probe (Applied Biosystems, Warrington, UK). Polymerase chain reaction (PCR) cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min using the ABI7000 Thermocycler (Applied Biosystems, Foster City, CA). All samples were run in duplicate. Foxp3, TGF-beta1, and IL-10 were normalized to glyceraldehyde-3-phosphate dehydrogenase.

Statistical Analysis
All comparisons were tested using two-tailed t tests on natural logarithmically transformed data to account for positive skew in distribu- tion, except where mentioned otherwise. Tests were two-tailed with a significance level of p <= 0.05. Results are expressed as median ± interquartile range (IQR). Analyses were performed using SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Characteristics of Patients with TB and Healthy Control Subjects
Demographic and clinical characteristics of the 23 healthy control subjects and 27 patients with active TB are summarized in Table 1. Patients with TB represented a broad clinical spectrum, and all were untreated at the time of study. Of the 27 patients with TB, 24 had positive TST results (see Table 1 and online supplement).

Patients with TB Have Increased Frequencies of Circulating CD4+CD25high Treg and FoxP3 Expression
We first compared by flow cytometry the percentage of CD4+CD25high T cells (Figure 1A; see online supplement) present in PBMCs of 24 active patients with TB and 21 uninfected healthy control subjects. Patients with active TB had a threefold higher percentage of CD25high T cells within the CD4+ T-cell population in peripheral blood compared with healthy control subjects (median, 3.51% [interquartile range (IQR), 2.18–4.92%] vs. median, 1.17% [IQR, 1.03–1.96%]; p < 0.001; Figure 1B).


Figure 1
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  		Figure 1. Increased percentage of circulating regulatory T cells in patients with active tuberculosis (TB) compared with healthy donors. (A) Representative flow cytometric analysis of CD4 and CD25 on peripheral blood mononuclear cells (PBMCs) of a healthy control subject and a patient with TB. CD4+CD25high T cells are shown gated (green box). Numbers represent percentage of events within the CD4+CD25high T-cell gate in the total CD4+ population. (B) Percentage of CD4+CD25high T cells from PBMC of 21 healthy control subjects and 24 patients with active TB. The median and individual frequencies for every patient are shown. (C) FoxP3 mRNA relative expression in PBMC of 22 healthy control subjects and 26 patients with active TB. The median and individual relative expressions for every patient are shown. (D) Correlation between FoxP3 mRNA relative expression and the percentage of CD4+CD25high cells in PBMC from patients with active TB and healthy control subjects (n = 43). The linear regression line is shown.

 
Because CD25 is a marker of T-cell activation and Tregs, we used the transcription factor FoxP3 as a definitive marker for Tregs. The relative level of FoxP3 mRNA was assessed by real-time reverse transcriptase–PCR (RT-PCR) in PBMCs of patients with active TB (n = 26) and healthy control subjects (n = 22). Consistent with the increased percentage of CD4+CD25high T cells, there was a significant 2.2-fold increase in FoxP3 mRNA expression in patients with TB compared with healthy control subjects (0.041 [IQR, 0.028–0.086] vs. 0.019 [IQR, 0.012–0.035], p = 0.006; Figure 1C).

To determine whether the increased FoxP3 expression in PBMC in patients with TB was due to an increased level of FoxP3 expression per cell with a constant number of Tregs or an increased number of Tregs with a constant level of FoxP3 expression per cell, we compared FoxP3 expression in freshly isolated CD4+CD25+ T cells from patients with TB (n = 5) and healthy control subjects (n = 6). There was no quantitative difference between the two groups (p = 0.855, Mann Whitney test), suggesting that the increased level of FoxP3 expression in patients with TB is due to increased frequencies of Tregs.

We compared the level of expression of FoxP3 and the percentage of CD4+CD25high cells in PBMC from 43 donors (patients with TB and healthy control subjects) and found a significant positive correlation between these two markers (Spearman's r = 0.58; p < 0.001; Figure 1D).

To determine whether the relative FoxP3 expression was specific for CD4+CD25+ Tregs, we compared FoxP3 expression in CD4+CD25+ versus CD4+CD25– T cells. Freshly isolated CD4+CD25+ but not CD4+CD25– T cells from TB patients (n = 5) and healthy control subjects (n = 6) expressed high levels of FoxP3 mRNA (Table 2 and Figures 2A–2D). No expression of FoxP3 mRNA was detected in the non-CD4+ T-cell fraction (data not shown).


Figure 2
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  		Figure 2. FoxP3 is specifically expressed in CD4+CD25+ Treg cells but not in CD4+CD25– T cells nor in in vitro–generated CD4+CD25+ activated T cells. Flow cytometric analysis of CD4 and CD25 on (A) PBMCs, (B) freshly isolated CD4+CD25+ T cells, (C) freshly isolated CD4+CD25– T cells, and (D) real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) for FoxP3 on each of these cell populations (PBMCs, isolated CD4+CD25– cells, and CD4+CD25+ cells). Flow cytometric analysis of CD4 and CD25 on (E) isolated CD25– and (F) CD25+ T-cell populations generated after 3 d of in vitro anti-CD3 and anti-CD28 stimulation of purified CD4+ CD25– T cells (see online supplement). (G) Real-time quantitative RT-PCR for FoxP3 on cells derived from 3 d in vitro–stimulated CD4+CD25– cells. Results with freshly isolated CD4+CD25+ cells (green bars) are shown for reference in the same graph as the leftward most column. Values above the cytometry graphs represent the percentage of cells in each quadrant. Results shown are from one experiment performed on PBMCs from a patient with active TB and are representative of four independent experiments from four patients with TB. Similar results were obtained with PBMC from three healthy control subjects.

 

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  		TABLE 2. RELATIVE EXPRESSION OF FOXP3, IL-10, AND TGF-beta1 mRNA IN FRESHLY ISOLATED CD4+CD25+ AND CD4+CD25– T-CELL SUBSETS IN 11 DONORS (FIVE PATIENTS WITH TUBERCULOSIS AND SIX HEALTHY CONTROL SUBJECTS)

 
The patients with TB and healthy control subjects were not matched for ethnicity. To test whether the different ethnic composition of the two groups might be responsible for the differences in Treg markers, we compared the percentage of CD4+CD25high cells and FoxP3 expression between the largest ethnic groups in the patients with TB and control subjects. There were no significant differences in either Treg marker in whites versus the other ethnic groups combined or in Indians versus the other ethnic groups combined (p > 0.09 for every comparison). Therefore, the difference in Treg markers between the patients with TB and the uninfected control subjects is not due to the different ethnic composition of the two groups.

FoxP3 Expression Is Specific for CD4+CD25+ Tregs and Is Not Up-regulated in Activated Effector CD4+CD25+ T Cells
Although in mice FoxP3 is not induced upon in vitro activation of CD4+CD25– T cells (30–32), regulation of FoxP3 expression in human T-cell subsets has not been extensively investigated. We therefore wanted to confirm that activated effector T cells that have recently up-regulated CD25 cell-surface expression do not express FoxP3 mRNA. Freshly isolated CD4+CD25– T cells from healthy control subjects (n = 3) and patients with active TB (n = 4) were stimulated with plate-bound anti-CD3 and soluble anti-CD28 antibodies in vitro for 3 d. Activated CD25– and CD25+ T cells were purified, and FoxP3 expression was analyzed; neither subset of T cells derived after 3 d culture expressed high levels of FoxP3 mRNA (Figure 2E–2G; see online supplement). Longer activation periods (10 or 17 d) with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2 (5 ng/ml) did not reveal induction of FoxP3 mRNA in activated T cells (n = 3; data not shown).

Correlation of FoxP3 Expression with Clinical Subtypes of TB
To determine whether Tregs correlated with disease subtype, we compared relative expression of FoxP3 mRNA and frequency of CD4+CD25high T cells in PBMC from patients who had purely pulmonary disease with patients who had extrapulmonary disease. Patients with extrapulmonary TB (extrapulmonary, disseminated and lymphatic, n = 17) had a significant 2.3-fold higher FoxP3 mRNA expression than patients with purely pulmonary TB (n = 10) (0.064 [IQR, 0.035–0.094] vs. 0.028 [IQR, 0.012–0.466], respectively, p = 0.010). Because lymphatic TB is localized and paucibacillary, we repeated the comparison after excluding lymphatic patients from the extrapulmonary group. Patients with extrapulmonary TB (extrapulmonary and disseminated only, n = 12) had a significant 1.7-fold higher FoxP3 expression (0.046 [IQR, 0.028–0.089], p = 0.046) than the patients with purely pulmonary TB. The percentage of CD4+CD25high T cells in patients with extrapulmonary TB (n = 17) was not significantly higher than in patients with pulmonary TB (n = 10) (3.69% [IQR, 2.43–5.45] vs. 3.08% [IQR, 1.49–4.94], p = 0.29).

CD4+CD25high Treg Cells Suppress IFN{gamma}-producing MTB Antigen–specific T Cells Ex Vivo
To determine whether circulating Tregs from patients with TB were functionally able to suppress MTB antigen–specific Th1-type T-cell responses, we performed CD25high T-cell depletions (n = 8) and assessed the number of IFN-{gamma}–producing cells specific for ESAT-6 and CFP-10–derived peptides by ex vivo ELISPOT assay (35, 36; see online supplement).

The number of ESAT-6/CFP-10–specific IFN-{gamma} spot-forming cells (SFCs) from the eight donors as a group was significantly higher postdepletion (median, 249 SFCs per million PBMCs) than in the undepleted PBMC (median, 168 SFCs per million PBMCs; p = 0.005, Wilcoxon signed-rank test). The mean percentage change in the number of ESAT-6/CFP-10–specific IFN-{gamma} SFCs in the CD25high-depleted PBMCs compared with the undepleted PBMCs was calculated and plotted for each patient (Figure 3). Two patients responded to ESAT-6 only, two responded to CFP-10 only, and four responded to both ESAT-6 and CFP-10. In all but one patient, an increase was seen after CD25high depletion compared with undepleted PBMC (Figure 3).


Figure 3
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  		Figure 3. CD4+ CD25high Treg cells suppress IFN-{gamma} production by Mycobacterium tuberculosis (MTB)–specific T cells ex vivo. Freshly isolated PBMCs and CD25high-depleted PBMCs from eight patients with TB were stimulated for 16 h with ESAT-6 and CFP-10–derived peptides in the ex vivo IFN-{gamma} ELISPOT assay. Data are expressed for each patient as the average of the normalized percentage change in total ESAT-6/CFP-10–specific summated IFN-{gamma} spot-forming cells (SFCs) from CD25high-depleted PBMC (gray bars) compared with undepleted PBMC (white bar and red line).

 
Patients with TB Have Increased IL-10 and TGF-beta1 mRNA Expression in PBMC, but These Cytokines Are Not Preferentially Expressed in CD4+CD25+ Tregs
We measured the constitutive level of expression ex vivo without in vitro stimulation of these two cytokines by real-time RT-PCR in PBMCs from patients with TB and healthy control subjects. Significant increases of 2.2-fold and 1.5-fold in expression of, respectively, IL-10 (22 patients with TB: median, 1.52 x 10–3 [IQR, 0.76 x 10–3 to 2.90 x 10–3] vs. 18 healthy control subjects: median, 0.69 x 10–3 [IQR, 0.50 x 10–3 to 1.41 x 10–3]; p = 0.005) and TGF-beta1 expression (25 patients with TB: median, 0.70 [0.62–1.12] vs. 23 healthy control subjects: median, 0.46 [0.31–0.64]; p < 0.001) were detected in patients with TB compared with healthy control subjects (Figure 4). Neither FoxP3 mRNA expression in PBMC nor the percentage of CD4+CD25high T cells correlated significantly with IL-10 or TGF-beta1 mRNA expression in the patients with TB or in the healthy control subjects (r < 0.20 and p > 0.10 for all comparisons).


Figure 4
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  		Figure 4. Increased IL-10 and TGF-beta1 mRNA relative expression in PBMC from patients with active TB compared with healthy donors. Real-time quantitative RT-PCR for (A) IL-10 in 18 healthy control subjects and 23 patients with active TB and (B) TGF-beta1 in 22 healthy control subjects and 25 patients with active TB. The median and individual relative expressions for every patient are shown.

 
We analyzed freshly isolated populations of CD4+CD25– T cells and CD4+CD25+ T cells in five patients with active TB and six healthy control subjects to determine whether these cytokines were preferentially expressed in CD4+CD25+ Treg cells. In contrast with FoxP3, significant preferential expression of IL-10 was not observed in CD4+CD25+ T cells, although there was a weak, statistically nonsignificant trend (Table 2). TGF-beta1 seemed to be preferentially expressed in CD4+CD25– T cells (Table 2). No quantitative differences between the patients and control subjects were observed for IL-10 (p = 1.000 within CD4+CD25– cells and p = 0.855 within CD4+CD25+ cells, Mann-Whitney test) and TGF-beta1 (p = 0.144 within CD4+CD25– cells and p = 0.584 within CD4+CD25+ cells, Mann-Whitney test).

Increased Frequencies of Tregs at Sites of Active TB Disease Relative to Peripheral Blood
In five patients, we analyzed matched samples from sites of active TB disease (pleural, ascitic, and pericardial fluids) and peripheral blood obtained at the same time. We measured the frequency of total ESAT-6/CFP-10 peptide-specific IFN-{gamma}–secreting T cells, the frequency of CD4+ CD25high T cells, and FoxP3 mRNA expression at the site of disease and in corresponding time-matched blood samples. We observed enrichment of ESAT-6/CFP-10 peptide-specific IFN-{gamma}–secreting T cells at disease sites compared with PBMCs (Figure 5A) in four out of four patients, but this increased frequency did not reach statistical significance (median, 1,186 SFC per million cells [IQR, 727–2,031] at disease sites vs. 228 SFCs per million cells [IQR, 57–487.5] in blood; p = 0.068, Wilcoxon signed-rank test), probably because of the small number of patients. We also observed an increase in the percentage of CD4+CD25high T cells in the pleural, ascitic, or pericardial fluids in four out of four patients compared with the percentage of these cells circulating simultaneously in peripheral blood (Figure 5B). The detected increase did not reach statistical significance (median, 3.87% [IQR, 2.54–12.53] vs. 2.96% [IQR, 0.94–10.08]; p = 0.068, Wilcoxon signed-rank test). Relative mRNA expression of FoxP3 by real-time RT-PCR (Figure 5C) was significantly increased 2.6-fold at the sites of disease compared with peripheral blood (median, 0.051 [IQR, 0.032–0.35] vs. 0.020 [IQR, 0.007–0.034]; p = 0.043, Wilcoxon signed-rank test).


Figure 5
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  		Figure 5. Increased percentage of CD4+CD25high Treg cells and FoxP3 expression at sites of active disease compared with peripheral blood. (A) Summated frequencies of IFN-{gamma}–secreting ESAT-6/CFP-10 peptide-specific T cells in PBMCs and at disease sites (n = 4), as enumerated by ex vivo ELISPOT assay. (B) Percentage of CD4+CD25high T cells in PBMCs and disease sites (n = 4) determined by flow cytometric analysis. (C) FoxP3 mRNA relative expression in PBMCs and disease sites (n = 5) determined by real-time RT-PCR and normalized to GAPDH. Patients 1, 3, and 5 had tuberculous pleural effusions, Patient 2 had a tuberculous pericardial effusion, and Patient 4 had tuberculous ascites. The results of individual pairs of time-matched samples from the same donor are linked by lines.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Definitive characterization of Tregs has until recently been difficult because of the lack of accurate and specific markers for this T-cell population. We therefore used the two most accurate available markers for Tregs: high levels of cell-surface CD25 expression and FoxP3 mRNA expression. We found a significantly higher percentage of circulating CD4+CD25high T cells and an increased level of FoxP3 mRNA expression in PBMC from patients with TB compared with uninfected healthy control subjects. Moreover, there was a positive correlation between these two markers, and we confirmed that FoxP3 was strongly and almost exclusively expressed by CD4+CD25high Tregs. This represents, to the best of our knowledge, the first demonstration of a role for Tregs in human TB. Our results do not directly demonstrate augmented FoxP3 expression by individual CD4+CD25high T cells; therefore, we do not know what proportion of CD4+CD25high T cells expresses FoxP3. However, it has recently been shown using newly available anti-FoxP3 monoclonal antibodies that FoxP3 is expressed by the majority of CD4+CD25high T cells in peripheral blood of healthy donors (37).

The functional hallmark of Tregs is their ability to suppress cellular immune responses, and specific depletion of CD4+CD25high Tregs should therefore lead to an increase in T-cell responses. We assessed the impact of Treg depletion on MTB antigen–specific, IFN-{gamma}–secreting T cells. The significant increase in the number of IFN-{gamma}–secreting T cells detected after specific depletion of CD25high cells from PBMCs confirmed that Tregs from patients with TB have demonstrable regulatory activity and are capable of controlling a crucial component of the host immune response to MTB. To confirm that Tregs play a functional role in vivo in humans is challenging because, in contrast to murine models, in vivo depletion of CD25high cells is not possible. However, the fact that ex vivo depletion of CD25high cells from freshly isolated PBMCs resulted in increased numbers of detectable IFN-{gamma}–secreting T cells in the short incubation overnight ELISPOT assay suggests that this suppressive activity is likely to be present in the blood of patients with TB. We did not assess the effect of CD4+CD25+ Treg depletion on Th1-type responses to non-MTB antigens. Therefore, we do not know whether the suppressive effect we observed is specific for T-cell responses to MTB. In chronic viral infections, the regulatory effect of CD4+CD25+ Tregs has been found to be antigen specific by some investigators and non–antigen specific by others (19, 21, 38).

Our results suggest that Tregs contribute to the impaired immune response in TB and may therefore play a role in limiting immune-mediated pathology. If this were the case, one would expect to find particularly high concentrations of Tregs at sites of active inflammation and tissue pathology. This is exactly what we observed; in all patients for whom clinical samples from disease sites were available, we observed an increase in FoxP3 expression and the percentage of CD4+CD25high T cells at the disease site compared with time-matched blood samples. This local amplification of the Treg population occurred irrespective of the anatomic location of disease. Increased (39) and unchanged (40) expression of IL-2R{alpha} (CD25) in bronchoalveolar lavage cells from sites of active pulmonary TB has previously been observed in patients. In the light of our findings, this observation may have been due to locally increased concentrations of Tregs. We also found increased frequencies of MTB antigen–specific, IFN-{gamma}–secreting T cells at disease sites, as we have previously observed (41). These findings are consistent with a widely accepted model of Treg homeostasis in infection, which proposes that in the presence of a pathogen, Tregs may proliferate in parallel with pathogen-specific effector T cells (42). Based on our results, we cannot distinguish whether the high concentrations of Tregs at disease sites are a result of preferential homing to inflamed tissue or local proliferation and retention. Although the magnitude of the suppressive effect of CD4+CD25high T cells we observed in PBMC was modest, the expansion of Tregs at disease sites suggests that suppression of effector responses may be amplified in inflamed tissues. However, the fact that tissue inflammation was manifest at disease sites despite the high local concentration of Tregs suggests that the increased Treg activity we observed is not sufficient to prevent immunopathology in the patients with TB whom we studied.

If Tregs play a significant role in TB, a correlation between clinical phenotype of disease and Treg markers might be expected. We observed a significantly higher level of FoxP3 expression in PBMCs from patients with extrapulmonary TB compared with patients who had purely pulmonary disease. Although a similar association of extrapulmonary TB with CD4+CD25high T cells was not seen, FoxP3 mRNA expression is considered to be the most accurate marker of Treg activity, suggesting that the observed correlation of Treg activity with clinical phenotype is likely to be real. Extrapulmonary dissemination can represent a failure to maintain localized disease within the organ of primary infection and is associated with suppressed Th1-type immunity, as in HIV infection. It is not clear whether the increased Tregs in extrapulmonary TB predispose to bacillary dissemination or whether Tregs expand as a result of more widespread pathology in these patients. It is also conceivable that increased Treg activity may reflect the chronicity of active TB. Although IL-10–secreting Tr1 T cells are specifically associated with cutaneous anergy in TB (7, 8), the Tregs described here are associated more generally with active TB per se because only 2 of 27 patients with TB had anergic tuberculin skin test responses.

The mechanisms that mediate the suppressive functions of natural Tregs are poorly understood. Some studies have shown that direct cell–cell contact is required, whereas others have suggested a role for IL-10 (43). The mechanism of action of Tregs was not directly addressed in this study. However, given that IL-10 and TGF-beta1 have been implicated in mediating the impaired cellular immune responses associated with active TB (7–9), we investigated whether the activity of the Tregs we identified might be mediated by these cytokines. We first confirmed that PBMCs from patients with active TB do express significantly higher levels of IL-10 and TGF-beta1 than do PBMCs from healthy control subjects. However, neither FoxP3 expression nor the percentage of CD4+CD25high T cells correlated with IL-10 or TGF-beta1 expression in the patients with TB or the healthy control subjects. We then confirmed that there is no significant preferential expression of these cytokines in CD4+CD25high T cells, suggesting that these cells are not IL-10–producing Tr1 nor TGF-beta1–producing Th3 cells. Definitive elucidation of the mechanism of action requires demonstration that anti–IL-10 and anti– TGF-beta1 blocking antibodies do not abrogate the suppressive activity of Tregs, and transwell experiments are needed to determine whether direct cell–cell contact is required. Even if these cytokines do not mediate the activity of natural Tregs, it is conceivable that they may induce or enhance the development of Tregs in vivo, as has been observed previously in vitro (44, 45).

This study provides the first evidence for a role for Tregs in TB. Our results are consistent with the hypothesis that Tregs help to control the critical balance between immune-mediated suppression of MTB and immunopathology in patients with TB. More work is required to delineate the role of these cells in TB disease and infection. It will be interesting to analyze prospectively whether Treg frequencies decline after curative treatment for TB and whether dynamic changes in Treg frequencies are associated with the paradoxic 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 Tregs are expanded in latent TB infection, where pathogen and antigen load are low and immunopathology is absent.


   Acknowledgments
 
The authors thank all participants of this study. They thank Ben Marshall and Jeremy McNally for collection of site of disease samples; Fiona Powrie, Paul Klenerman, and Helen Fletcher for helpful discussions; and Jonathan Deeks for statistical advice.


   FOOTNOTES
 
Supported by the Wellcome Trust. A.L. is a Wellcome Senior Research Fellow in Clinical Science.

Originally Published in Press as DOI: 10.1164/rccm.200508-1294OC on December 9, 2005

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Received in original form August 20, 2005; accepted in final form December 7, 2005


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, Williamson R, Levin M. A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996;335:1941–1949.[Abstract/Free Full Text]
  2. Ottenhoff TH, Kumararatne D, Casanova JL. Novel human immunodeficiencies reveal the essential role of type-I cytokines in immunity to intracellular bacteria. Immunol Today 1998;19:491–494.[CrossRef][Medline]
  3. Stenger S, Modlin RL. T cell mediated immunity to Mycobacterium tuberculosis. Curr Opin Microbiol 1999;2:89–93.[CrossRef][Medline]
  4. Belkaid Y, Piccirillo CA, Mendez S, Shevach EM, Sacks DL. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 2002;420:502–507.[CrossRef][Medline]
  5. Hirsch CS, Toossi Z, Othieno C, Johnson JL, Schwander SK, Robertson S, Wallis RS, Edmonds K, Okwera A, Mugerwa R, et al. Depressed T-cell interferon-gamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. J Infect Dis 1999;180:2069–2073.[CrossRef][Medline]
  6. Zhang M, Lin Y, Iyer DV, Gong J, Abrams JS, Barnes PF. T-cell cytokine responses in human infection with Mycobacterium tuberculosis. Infect Immun 1995;63:3231–3234.[Abstract]
  7. Boussiotis VA, Tsai EY, Yunis EJ, Thim S, Delgado JC, Dascher CC, Berezovskaya A, Rousset D, Reynes JM, Goldfeld AE. IL-10-producing T cells suppress immune responses in anergic tuberculosis patients. J Clin Invest 2000;105:1317–1325.[Medline]
  8. Delgado JC, Tsai EY, Thim S, Baena A, Boussiotis VA, Reynes JM, Sath S, Grosjean P, Yunis EJ, Goldfeld AE. Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia. Proc Natl Acad Sci USA 2002;99:7576–7581.[Abstract/Free Full Text]
  9. Othieno C, Hirsch CS, Hamilton BD, Wilkinson K, Ellner JJ, Toossi Z. Interaction of Mycobacterium tuberculosis-induced transforming growth factor beta1 and interleukin-10. Infect Immun 1999;67:5730–5735.[Abstract/Free Full Text]
  10. Murakami M, Sakamoto A, Bender J, Kappler J, Marrack P. CD25+CD4+ T cells contribute to the control of memory CD8+ T cells. Proc Natl Acad Sci USA 2002;99:8832–8837.[Abstract/Free Full Text]
  11. Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998;188:287–296.[Abstract/Free Full Text]
  12. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat Rev Immunol 2003;3:253–257.[CrossRef][Medline]
  13. Cozzo C, Larkin J III, Caton AJ. Cutting edge: self-peptides drive the peripheral expansion of CD4+CD25+ regulatory T cells. J Immunol 2003;171:5678–5682.[Abstract/Free Full Text]
  14. Gavin M, Rudensky A. Control of immune homeostasis by naturally arising regulatory CD4+ T cells. Curr Opin Immunol 2003;15:690–696.[CrossRef][Medline]
  15. Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance. Nat Rev Immunol 2003;3:199–210.[CrossRef][Medline]
  16. Belkaid Y, Rouse BT. Natural regulatory T cells in infectious disease. Nat Immunol 2005;6:353–360.[CrossRef][Medline]
  17. Mills KH. Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 2004;4:841–855.[CrossRef][Medline]
  18. Aandahl EM, Michaelsson J, Moretto WJ, Hecht FM, Nixon DF. Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens. J Virol 2004;78:2454–2459.[CrossRef][Medline]
  19. Stoop JN, van der Molen RG, Baan CC, van der Laan LJ, Kuipers EJ, Kusters JG, Janssen HL. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection. Hepatology 2005;41:771–778.[CrossRef][Medline]
  20. Cabrera R, Tu Z, Xu Y, Firpi RJ, Rosen HR, Liu C, Nelson DR. An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection. Hepatology 2004;40:1062–1071.[CrossRef][Medline]
  21. Rushbrook SM, Ward SM, Unitt E, Vowler SL, Lucas M, Klenerman P, Alexander GJ. Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection. J Virol 2005;79:7852–7859.[Abstract/Free Full Text]
  22. Sugimoto K, Ikeda F, Stadanlick J, Nunes FA, Alter HJ, Chang KM. Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection. Hepatology 2003;38:1437–1448.[Medline]
  23. Boettler T, Spangenberg HC, Neumann-Haefelin C, Panther E, Urbani S, Ferrari C, Blum HE, von Weizsacker F, Thimme R. T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J Virol 2005;79:7860–7867.[Abstract/Free Full Text]
  24. Kinter AL, Hennessey M, Bell A, Kern S, Lin Y, Daucher M, Planta M, McGlaughlin M, Jackson R, Ziegler SF, et al. CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status. J Exp Med 2004;200:331–343.[Abstract/Free Full Text]
  25. Kullberg MC, Jankovic D, Gorelick PL, Caspar P, Letterio JJ, Cheever AW, Sher A. Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis. J Exp Med 2002;196:505–515.[Abstract/Free Full Text]
  26. Lundgren A, Suri-Payer E, Enarsson K, Svennerholm AM, Lundin BS. Helicobacter pylori-specific CD4+ CD25high regulatory T cells suppress memory T-cell responses to H. pylori in infected individuals. Infect Immun 2003;71:1755–1762.[Abstract/Free Full Text]
  27. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001;167:1245–1253.[Abstract/Free Full Text]
  28. Ochs HD, Ziegler SF, Torgerson TR. FOXP3 acts as a rheostat of the immune response. Immunol Rev 2005;203:156–164.[CrossRef][Medline]
  29. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, Wilkinson JE, Galas D, Ziegler SF, Ramsdell F. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 2001;27:68–73.[Medline]
  30. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330–336.[CrossRef][Medline]
  31. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–1061.[Abstract/Free Full Text]
  32. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol 2003;4:337–342.[CrossRef][Medline]
  33. Fontenot JD, Rasmussen JP, Williams LM, Dooley JL, Farr AG, Rudensky AY. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 2005;22:329–341.[CrossRef][Medline]
  34. Lalvani A, Pathan AA, Durkan H, Wilkinson KA, Whelan A, Deeks JJ, Reece WH, Latif M, Pasvol G, Hill AV. Enhanced contact tracing and spatial tracking of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Lancet 2001;357:2017–2021.[CrossRef][Medline]
  35. Lalvani A, Brookes R, Hambleton S, Britton WJ, Hill AV, McMichael AJ. Rapid effector function in CD8+ memory T cells. J Exp Med 1997;186:859–865.[Abstract/Free Full Text]
  36. Liu XQ, Dosanjh D, Varia H, Ewer K, Cockle P, Pasvol G, Lalvani A. Evaluation of T-cell responses to novel RD1- and RD2-encoded Mycobacterium tuberculosis gene products for specific detection of human tuberculosis infection. Infect Immun 2004;72:2574–2581.[Abstract/Free Full Text]
  37. Roncador G, Brown PJ, Maestre L, Hue S, Martinez-Torrecuadrada JL, Ling K-L, Pratap S, Toms C, Fox BC, Cerundolo V, et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol 2005;35:1681–1691.[CrossRef][Medline]
  38. McGuirk P, McCann C, Mills KH. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J Exp Med 2002;195:221–231.[Abstract/Free Full Text]
  39. Tchou-Wong K-M, Tanabe O, Chi C, Yie T-A, Rom WN. Activation of NF-{kappa}B in Mycobacterium tuberculosis-induced interleukin-2 receptor expression in mononuclear phagocytes. Am J Respir Crit Care Med 1999;159:1323–1329.[Abstract/Free Full Text]
  40. Schwander SK, Sada E, Torres M, Escobedo D, Sierra JG, Alt S, Rich EA. T lymphocytic and immature macrophage alveolitis in active pulmonary tuberculosis. J Infect Dis 1996;173:1267–1272.[Medline]
  41. Wilkinson KA, Wilkinson RJ, Pathan A, Ewer K, Prakash M, Klenerman P, Maskell N, Davies R, Pasvol G, Lalvani A. Ex vivo characterization of early secretory antigenic target 6-specific T cells at sites of active disease in pleural tuberculosis. Clin Infect Dis 2005;40:184–187.[CrossRef][Medline]
  42. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat Immunol 2001;2:816–822.[CrossRef][Medline]
  43. Jonuleit H, Schmitt E, Kakirman H, Stassen M, Knop J, Enk AH. Infectious tolerance: human CD25(+) regulatory T cells convey suppressor activity to conventional CD4(+) T helper cells. J Exp Med 2002;196:255–260.[Abstract/Free Full Text]
  44. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:737–742.[CrossRef][Medline]
  45. Yamagiwa S, Gray JD, Hashimoto S, Horwitz DA. A role for TGF-beta in the generation and expansion of CD4+ CD25+ regulatory T cells from human peripheral blood. J Immunol 2001;166:7282–7289.[Abstract/Free Full Text]



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