Regulatory T Cells Depress Immune Responses to Protective Antigens in Active Tuberculosis

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1 Regulatory T Cells Depress Immune Responses to Protective Antigens in Active Tuberculosis Jean-Michel Hougardy 1, Sammy Place 1, Marc Hildebrand 2, Annie Drowart 3, Anne-Sophie Debrie 4,5, Camille Locht 4,5, and Françoise Mascart 1,6 1 Laboratory of Vaccinology and Mucosal Immunity, Hôpital Erasme, Brussels, Belgium; 2 Infectious Disease Department, Hôpital Saint-Pierre, Université Libre de Bruxelles, Brussels, Belgium; 3 Chest Department, Hôpital Brugmann, Université Libre de Bruxelles, Brussels, Belgium; 4 Institut Pasteur de Lille, Lille, France; 5 INSERM, U629, Lille, France; and 6 Immunobiology Clinic, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium Rationale: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN- responses to the protective antigen heparinbinding hemagglutinin (HBHA), whereas patients with active TB do not. Objectives: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. Methods: The impact of anti transforming growth factor (TGF)- and anti IL-10 antibodies and of Treg cell depletion on the HBHAinduced IFN- secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. Measurements and Main Results: Although the addition of anti TGF- or anti IL-10 antibodies had no effect on the HBHA-induced IFN- secretion in patients with active TB, depletion of CD4 CD25 high FOXP3 T lymphocytes resulted in the induction by HBHA of IFN- concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target 6 or candidin T-cell responses. Conclusions: Specific CD4 CD25 high FOXP3 T cells depress the T-cell mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans. Keywords: tuberculosis; regulatory T cells; heparin-binding hemagglutinin Tuberculosis (TB) remains one of the world s leading causes of mortality due to a single infectious agent, with approximately 2 million deaths and 8 million new cases per year (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 to develop the disease. Most infected individuals will stay healthy throughout their lifetime and develop a latent infection with no sign of disease. They can be regarded as being protected against the disease by the immune response induced through natural infection. However, in 5 to 10% of the cases, infected subjects will eventually progress to active TB, suggesting that their immune response is not protective against active disease. A better understanding of the immunologic differences between subjects with latent M. tuberculosis (Received in original form January 16, 2007; accepted in final form May 30, 2007 ) Supported by a grant from the Fond de la Recherche Scientifique Médicale and by the European Commission, within the 6th Framework Program, contract no. LSHP-CT J.M.H. was supported by a fellowship from the Fond pour la Formation à la Recherche dans l Industrie et dans l Agriculture. S.P. was supported by a fellowship from the Fondation Erasme. Correspondence and requests for reprints should be addressed to Professor Françoise Mascart, Hôpital Erasme, Immunobiology Clinic, 808 Route de Lennik, B-1070 Brussels, Belgium. fmascart@ulb.ac.be Am J Respir Crit Care Med Vol 176. pp , 2007 Originally Published in Press as DOI: /rccm OC on May 31, 2007 Internet address: AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Elevated levels of regulatory T cells with the CD4 CD25 FOXP3 phenotype have been detected in patients with tuberculosis. What This Study Adds to the Field We show that regulatory T cells suppress IFN- responses preferably to protective antigens, such as the novel heparinbinding hemagglutinin, suggesting that the induction of these cells constitutes an important escape mechanism of Mycobacterium tuberculosis. infection (LTBI) and patients with active TB may ultimately lead to the definition of correlates for protection, to differential immunodiagnosis of LTBI and active TB, and to the development of new vaccines against the disease. CD4 and CD8 T-cell mediated immunity is essential for protection against TB (2). We have previously reported that subjects with LTBI mount strong T-cell responses to the protective M. tuberculosis heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not (3, 4). This impairment is reversible because patients with active TB may gain their capacity to produce HBHA-specific IFN- upon successful treatment (3). These observations indicate that patients with active TB do not have an intrinsic defect in their ability to mount a T-cell response to HBHA; rather, this response is likely to be suppressed during active TB, suggesting that regulatory mechanisms play a role in immune evasion by M. tuberculosis. This article provides evidence that a subset of the circulating CD4 CD25 high T cells characterized by the intracellular expression of FOXP3 suppress the HBHA T-cell responses in patients with active TB but have no effect on the T-cell responses to other antigens, such as the early-secreted antigen target (ESAT) 6 or candidin. Some of the results of these studies have been previously reported in the form of abstracts (5 7). METHODS Subjects Blood was obtained from 58 patients with active, newly diagnosed, and untreated TB (mean age, yr; range, yr; 33 Caucasian, 21 patients from Africa, 2 from Pakistan, 1 from Latin America, and 1 from Asia), from 22 LTBI subjects (mean age, yr; range, yr; 15 Caucasians, six subjects from Africa and one from Latin America), and from 12 uninfected subjects (mean age, yr; range, yr; all Caucasian). The uninfected subjects were tuberculin

2 410 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL skin test negative, had not been vaccinated, and had no known exposure to M. tuberculosis. The subjects with LTBI were selected according to the recommended criteria from the national foundation against respiratory affections (8): a risk-stratified induration 72 hours after intradermal injection of 2 U tuberculin (PPD-RT23 SST; Statens Serum Institute, Copenhagen), normal chest radiographs, and no clinical sign of active TB. All 58 patients with TB had pulmonary TB. For 42 of these patients, diagnosis was based on smear positivity, M. tuberculosis culture, and/ or polymerase chain reaction positivity. For four patients, diagnosis was based on the presence of granulomas, and for 12 patients, diagnosis was based on clinical and radiologic responses to therapy. All patients were recruited from Belgian hospitals linked to the Université Libre de Bruxelles hospital network. All individuals were living in Belgium at the time of enrollment and were HIV seronegative. None of the patients presented immunodepressive illness or received immunosuppressive treatment. All had normal IFN- secretion in response to mitogen ( 10 ng/ml IFN- upon stimulation with phytohemagglutinin). The study was approved by the local ethics committee of the hospital where the patients were enrolled, and informed consent was given by all individuals. Antigens and Antigen-specific IFN- Determinations Native and recombinant HBHA were purified as described (3, 9, 10). Peripheral blood mononuclear cells (PBMCs) were purified from fresh blood samples and in vitro stimulated as described (3, 4) with 2 g/ml HBHA, 4 g/ml purified protein derivative (PPD) (batch RT49; Statens Serum Institute, Copenhagen, Denmark), 5 g/ml ESAT-6 (kindly provided by M. Doherty, Statens Serum Institute), 5 g/ml antigen 85 (kindly provided by K. Huygen, Institut Pasteur de Bruxelles, Brussels, Belgium), or 2 g/ml phytohemagglutinin (Remel, Lenexa, KS). IFN- concentrations were measured after 4 d by ELISA as described (3). Blocking anti transforming growth factor (TGF)- 1, 2, 3, or anti IL- 10 (R&D Systems Europe, Ltd., Abingdon, UK) antibodies were added at 5 g/ml. CD25 high T cells were depleted from PBMC by positive immunomagnetic selection using anti-cd25 antibodies (EasySep Stemcell, Grenoble, France). Total PBMCs and CD25 high T-cell depleted PBMCs were in vitro cultured in parallel under the same conditions ( PBMC/ml). the suppression of the T-cell immune responses during active TB, we investigated the role of these factors in the downmodulation of the HBHA-induced IFN- secretion by the PBMCs from patients with active TB. Incubation of the PBMC from patients with active TB with blocking anti TGF- or anti IL-10 antibodies during HBHA stimulation did not significantly enhance the IFN- responses to HBHA (Figure 1), indicating that these regulatory cytokines were not the key factors of the low IFN- response to HBHA in patients with active TB. When these antibodies were added during PPD stimulation as a positive control of effectiveness of the antibodies, the IFN- response to PPD was significantly increased (p ; data not shown), as previously described (15). In contrast, depletion of the CD4 CD25 high T cells from the PBMCs of the patients with active TB resulted in a strong increase in the HBHA-induced IFN- secretion (p ; median fold increase in IFN- concentrations, 5.24) (Figure 2A), which reached levels similar to those observed for LTBI, whereas their depletion had no effect on the HBHA-induced IFN- secretion by the PBMCs from subjects with LTBI (p ) (Figure 2B). In patients with active TB, no significant effect of the CD25 high T-cell depletion was noted on the IFN- secretion induced by ESAT-6 (p ) (Figure 3A), whereas the effect on the IFN- secretion induced by PPD was significant but modest in comparison to that for HBHA (p ; median fold increase in IFN- concentrations, 1.38) (Figure 3B). PPD is a mixture of antigens containing, among many others, the antigen 85 complex. The latter is a group of strongly protective protein antigens Phenotypic and Proliferation Analyses CD25 and FOXP3 expression by CD3 CD4 lymphocytes was analyzed on a Canto Flow Cytometer (BD Biosciences, San José, CA) after staining with PerCP-labeled anti-cd3, FITC-labeled or APC-Cy7- labeled anti-cd4, PE-labeled anti-cd25, and APC-labeled anti- FOXP3. All reagents were from BD Biosciences, except for the anti- FOXP3 antibodies (ebioscience, San Diego, CA) and were used according to the manufacturers instructions. CD3 CD4 T-cell proliferation, within the total PBMC or the CD25 high T-cell depleted PBMC, was analyzed by flow cytometry after labeling the PBMC for 10 minutes with 0.5 M CFSE (carboxy fluoresceine diacetate succinimidyl ester) (Vybrant CFDA SE Cell Tracer Kit; Molecular Probes, Eugene, OR) in phosphate-buffered saline bovine serum albumin 0.1%, followed by washing with fresh culture medium and antigenic stimulation for 6 days with 10 g/ml HBHA, 4 g/ml PPD, 10 g/ml ESAT-6, or 5 IC/ml candidin (Stallergènes, Antony Cedex, France). The data were analyzed using the Flow JO software (version 6.3.4; Tree Star, Inc., Ashland, OR). Statistical Analyses Differences between two groups were assigned by the nonparametric Mann-Whitney U test (the Wilcoxon signed rank test for paired values). Correlation was analyzed by the nonparametric Spearman test and by linear regression analysis. A value of p 0.05 was considered significant. All results were obtained with the GraphPad Prism software (GraphPad Inc., San Diego, CA), version 4.0b. RESULTS Role of CD4 CD25 high T Cells in the Downmodulation of HBHA-specific IFN- Responses in Patients with Active TB In view of the reported role of the regulatory cytokines IL-10 (11) or TGF- (12) or of the CD4 CD25 high T cells (13, 14) in Figure 1. Effect of transforming growth factor (TGF)- and IL-10 neutralization on heparin-binding hemagglutinin (HBHA) specific IFN- production in patients with active tuberculosis. Neutralizing anti TGF- or anti IL-10 antibodies were added to the peripheral blood mononuclear cells from 12 patients with active tuberculosis during HBHA stimulation, and HBHA-induced IFN- concentrations were measured in the cell culture supernatants and compared with the IFN- concentrations of the peripheral blood mononuclear cells incubated in the absence of antibodies. Individual values for each patient are linked. The dotted horizontal line represents the limit of the sensitivity of the IFN- detection. All values below this line are not significant.

3 Hougardy, Place, Hildebrand, et al.: Regulatory T Cells in Human Tuberculosis 411 Figure 2. Effect of CD25 T-cell depletion on heparin-binding hemagglutinin (HBHA) induced IFN- secretion in individuals infected with Mycobacterium tuberculosis.peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis (A ) or latent tuberculosis infection (B) were stimulated with HBHA before and after CD25 T-cell depletion, and the IFN- concentrations were measured in the culture supernatants. Individual values for each patient are linked. The dotted horizontal line represents the limit of the sensitivity of the IFN- detection. All values below this line are not significant. nhbha native methylated HBHA. (16) that, similarly to HBHA, have been shown to be better recognized by T cells from subjects with LTBI than by T cells from patients with active TB (17). The CD25 high T-cell depletion also resulted in a significant increase of the IFN- response to antigen 85 during active TB (p ; median fold increase in IFN- concentrations, 2.69) (Figure 3C). These findings indicate that the CD4 CD25 high T cells in the PBMC from patients with active TB are functional regulatory T (Treg) cells that play a major role in the immune suppression of T-cell responses to protective antigens during TB and display antigen specificity, the strongest relative effect being observed for HBHA. Although we cannot exclude that the CD25 T-cell depletion resulted in the depletion of some CD8 CD25 Tcells,the CD4 CD25 T cells were the most abundant types among the depleted cells (96.45%, as estimated by flow cytometry). The depleted cell population occasionally contained some contaminating monocytes. However, their percentages were always low (median, 2%) in these populations, and there was no correlation between the amounts of contaminating monocytes in the CD25 T-cell population and the suppressive effects. Role of CD4 CD25 high T Cells in the Downmodulation of the HBHA-induced Lymphocyte Proliferation in TB Although antigen-induced IFN- secretion is more relevant to protection and/or the pathogenesis of TB, we investigated whether the results obtained on the IFN- secretion were in line with results from T-cell proliferation assays because this readout is often used for the demonstration of the presence of functional Treg cells. Seven patients with active TB identified as having Treg cells on the basis of the results of the IFN- secretion were tested in T-cell proliferation assays and were compared with two patients with LTBI and two uninfected control subjects. The CD4 CD25 high T-cell depletion from PBMCs of patients with active TB resulted in a significant increased CD4 T-cell proliferation upon stimulation with HBHA, as compared with undepleted PBMCs from the same patients (p ; median fold increase in proliferation, 3.8) (Figure 4). No effect of the CD4 CD25 high T-cell depletion was noted on the ESAT-6 induced CD4 T-cell proliferation (p 1.0; data not shown) or on the CD4 T-cell proliferation mediated by nonmycobacterial antigens, such as candidin (p 0.875; data not shown), further confirming the antigen specificity of the Treg cells in patients with active TB. No effect of the depletion was noted on the HBHA-induced proliferation in the LTBI subjects or in the uninfected control subjects (data not shown). Phenotypic Analysis of the CD4 CD25 high Treg Cells in Patients with Active TB The proportion of CD25 high T cells among CD4 lymphocytes was analyzed by flow cytometry. The CD25 high population could be easily differentiated from the CD25 medium and the CD25 low CD4 T cells on the basis of the fluorescence intensity of the CD25 labeling, accompanied by a slightly lower fluorescence intensity for the expression of CD4 (Figure 5A). As previously reported (13, 14), the proportion of CD4 CD25 high T cells was significantly higher among the PBMCs from patients with active TB compared with LTBI (medians, 6.08% and 4.53% of the CD4 T lymphocytes; 25th to 75th percentiles, for TB and for LTBI, respectively; p ). The FOXP3 transcription factor is considered to be the most reliable molecular marker of the CD4 CD25 high Treg cells (18 20). We therefore investigated its expression in the CD4 CD25 high T cells. As evidenced by flow cytometry analysis, a substantial fraction of the CD4 T cells from patients with active TB expressed CD25 high at their surface and FOXP3 intracellularly (Figures 5B, upper panel, and 5C), whereas significantly less CD25 high FOXP3 cells were found among the CD4 cells from patients who had LTBI (Figures 5B, middle panel, and 5C). The median of the fluorescence intensity of the FOXP3 marker was similar in the two groups (data not shown), indicating that there was no increased FOXP3 expression per cell in TB compared with LTBI, but there was a significant rise in the proportion of CD4 CD25 high FOXP3 Treg cells in TB compared with LTBI (p ) (Figure 5C). To investigate whether these differences resulted from a rise of

4 412 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 3. Effect of CD25 T-cell depletion on early-secreted antigen target (ESAT)-6, purified protein derivative (PPD), Ag85- induced IFN- secretions in patients with active tuberculosis. Peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis were stimulated with ESAT-6 (A ), PPD (B), or Ag85 (C) before and after CD25 T-cell depletion, and the IFN- concentrations were measured in the culture supernatants. Individual values for each patient are linked. The dotted horizontal line represents the limit of the sensitivity of the IFN- detection. All values below this line are not significant. this subpopulation in patients with active TB or from a decrease in LTBI subjects, the proportion of these cells was analyzed in healthy noninfected and nonvaccinated control subjects. The proportion of the Treg cells was similar in LTBI subjects and in the control subjects (Figures 5B, lower panel, and 5C). A comparison of the percentages of the CD4 CD25 high, the CD4 FOXP3, and the CD4 CD25 high FOXP3 T lymphocytes before and after the depletion procedure indicated that the CD4 CD25 high lymphocytes and most of the CD4 FOXP3 T cells had been successfully removed (Figure 6A) The most important effect of the depletion procedure was the nearly complete disappearance of the CD4 CD25 high FOXP3 T lymphocytes (Figures 6A and B), suggesting that these cells were involved in the suppression of the HBHA-induced IFN- secretion and proliferation in patients with active TB. Characterization of the HBHA-induced T-Cell Responses in TB after Removal of Treg Cells HBHA is a methylated protein, and subjects with LTBI preferentially respond to the methylated form, whereas they poorly recognize the nonmethylated form, so that the IFN- secretion is significantly higher upon stimulation with methylated than with

5 Hougardy, Place, Hildebrand, et al.: Regulatory T Cells in Human Tuberculosis 413 Figure 4. Effect of CD25 T-cell depletion on antigen-specific T-cell proliferation. Peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis were incubated before and after CD25 T-cell depletion with heparin-binding hemagglutinin (HBHA), and the CD4 T-cell proliferation was measured using the carboxy fluoresceine diacetate succinimidyl ester (CFSE) labeling method. Individual values for each individual are linked. nhbha native methylated HBHA. nonmethylated HBHA. Linear regression analysis indicated no relationship between the IFN- concentrations induced by both forms of HBHA (4). We therefore examined the IFN- responses to both forms of HBHA in patients with active TB before and after CD4 CD25 high T-cell depletion. Linear regression analyses indicated that the IFN- concentrations induced by native methylated HBHA (nhbha) correlated linearly with those induced by recombinant nonmethylated HBHA (rhbha) in patients with active TB before (r ; p ; data not shown) and after (r ; p ) (Figure 7) depletion. In addition, the IFN- concentrations induced by both forms of HBHA were not significantly different from each other before (medians of and pg/ml for nhbha and rhbha, respectively) and after depletion (medians of and pg/ml, respectively). In contrast, results obtained in subjects with LTBI who were tested with the same antigenic batches confirmed previous data (4) (i.e., higher IFN- responses to nhbha compared with rhbha and absence of a linear correlation between the values obtained with both forms of HBHA; data not shown). Noninfected control subjects were tested in parallel, and their PBMCs did not secrete significant IFN- levels in response to either form of HBHA before and after CD4 CD25 high T-cell depletion (data not shown). These results indicate that the immune response in patients with active TB is not preferentially directed to the methylated part of HBHA, in contrast to what is observed for LTBI. DISCUSSION During persistent infections, a complex interplay is established between the immune system s capacity to rid the host of the Figure 5. Flow cytometry analyses of CD25 high and FOXP3 expression by the CD4 T cells. (A ) Representative flow cytometric analysis of CD4 and CD25 expression on peripheral blood mononuclear cells from a patient with active tuberculosis ( TB). CD4 CD25 high T cells are shown gated. (B) Representative flow cytometric analysis of intracellular FOXP3 and surface CD25 expressions on peripheral blood mononuclear cells from a patient with active TB (upper panel ), a patient with latent TB infection (LTBI; middle panel), and a noninfected control subject (CTRL; lower panel). The numbers indicate the percentages of the different cell subsets among the CD3 CD4 lymphocytes. (C) The frequencies of the CD25 high FOXP3 cells among the CD3 CD4 cells from 18 patients with active TB, 12 patients with LTBI, and 11 noninfected control subjects were measured by flow cytometry. The horizontal bars represent the medians of the results. infectious agent and the ability of the microorganism to escape from this protective immune response to sustain infection. Because immune responses are tightly controlled by regulatory mechanisms involving specialized T-cell subsets named Treg cells (20, 21), it is not surprising that some microbial pathogens make use of these cells to evade protective immunity (22). TGF- mediated (12) and IL-10 mediated (11) regulatory mechanisms that downmodulate Th1 responses during active TB in humans have previously been described. Recently, CD4 CD25 high T cells have been reported to be present at elevated levels in TB and to be able to depress T-cell mediated IFN- production in these patients (13, 14). We show here that this is the mechanism involved in the decreased IFN- response to HBHA in patients with active TB compared with LTBI. HBHA is a recently described antigen that provides strong protection in mouse models against M. tuberculosis challenge (4, 23) and is considered to be

6 414 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 7. Correlation between the native methylated heparin-binding hemagglutinin (nhbha) and recombinant nonmethylated HBHA (rhbha) induced IFN- concentrations. After depletion of the CD25 high FOXP3 T cells, the peripheral blood mononuclear cells from patients with active tuberculosis were in vitro stimulated with nhbha or with rhbha, and the IFN- concentrations were measured in the cell culture supernatants after 96 hours of culture. Figure 6. Phenotypic characterization of the peripheral blood mononuclear cells (PBMC) before and after CD25 high T-cell depletion. (A ) Representative flow cytometric analysis of the intracellular FOXP3 and surface CD25 staining on PBMCs from a patient with active tuberculosis (TB) before (upper panel) and after (lower panel) the CD25 high T-cell depletion procedure. (B) The frequencies of the CD25 high FOXP3 cells among the CD3 CD4 cells from six patients with active TB (circles) and from six patients with latent TB infection (inverted triangles) were measured by flow cytometry before and after the CD25 high T-cell depletion procedure. Individual values for each subject are linked. one of the promising new vaccine candidates against TB (24). It is also able to distinguish LTBI from active TB in humans by the higher levels of HBHA-induced IFN- in LTBI compared withtb(3,4)andmaybeconsideredasadiagnosticantigenfor the detection of LTBI. Although neutralization of TGF- or IL-10 had no effect, depletion of the CD4 CD25 high T cells from the PBMCsofpatientswithactiveTBresultedinamorethanfivefold median increase in the IFN- response to HBHA. The relative effect of the CD4 CD25 high T-cell depletion on the IFN- responses to PPD and antigen 85 were lower, and no significant effect was observed on the ESAT-6 induced and the candidin-induced T-cell responses, indicating a certain degree of antigen specificity of the Treg cells. Although the CD4 CD25 high T-cell depletion resulted occasionally in the depletion of small amounts of monocytes, it is unlikely that the suppressive effects observed here are due to the contaminating monocytes. We observed no correlation between the amounts of contaminating monocytes in the CD4 CD25 high T-cell population and the suppressive effect. Furthermore, if monocytes were the suppressor cells, they would be expected to exert suppressive effects on the IFN- responses to ESAT-6 and candidin, which was not observed. The lack of effect on the ESAT-6 response found here is in contrast to the observations reported by Guyot-Revol and colleagues (13), who found a less than 50% median increase in the ESAT-6/CFP-10 response after CD25 high T-cell depletion. Occasionally, we also found a slight increase in the ESAT-6 response after CD4 CD25 high T-cell depletion in some patients (Figure 3A), but this increase was not statistically significant. Antigen specificity of CD25 high T-cell suppressor activity has been reported in several systems (25, 26), and the T-cell receptor repertoire of these cells has been proposed to be as diverse as that of the CD4 CD25 cells (26), allowing for immunosuppression to a wide variety of antigens. In addition to the development of Treg cells in the periphery that reduce HBHA-specific IFN-, it is possible that during active TB a subset of CD4 effector T cells, producing antigenspecific IFN-, is sequestered at the tissue site of the infection. The presence of antigen-specific IFN- producing cells at the site of infection has been shown for ESAT-6 (27) and for HBHA (our unpublished results). The results described in this study show that the presence of effector T cells at the site of infection does not preclude their presence in the peripheral blood because their IFN- secretion and proliferation can be unmasked by the depletion of CD4 CD25 Treg cells. By using intracellular staining we found that the CD25 high Treg cells depleted in this study express FOXP3 and can thus be defined as the CD25 high FOXP3 subpopulation of the CD4 lymphocytes. The frequency of this subpopulation was markedly increased in patients with active TB compared with patients with LTBI or uninfected control subjects. Guyot-Revol and colleagues (13) noted a slight (2.2-fold) increase in the expression of FOXP3 mrna in patients with active TB compared with noninfected control subjects. Based on these observations, they concluded that the CD4 CD25 high T cells may be Treg cells, although they could not demonstrate direct FOXP3 expression by individual CD4 CD25 high T cells. They suggested that the enhanced levels of FOXP3 mrna in patients with active TB was due to increases in Treg cell frequency. Conversely, Gazzola and colleagues (28) recently reported an overexpression of the FOXP3 gene among the CD4 CD25 high T cells from patients with active TB. The results from double staining for CD25 and FOXP3 described here conclusively indicate that during active TB the frequency of Treg cells expressing CD25 and FOXP3 are elevated in the peripheral blood. Although CD4 CD25 high FOXP3 T cells are also present in the peripheral blood of subjects with LTBI and in uninfected subjects, albeit at lower levels than in patients with active TB, suppression of the immune responses to HBHA occurs only in patients with active TB and not in patients with LTBI or in control subjects. This is likely to be due to an imbalance in the specific effector/regulatory T cell as a consequence of the significant rise of the Treg cells in TB compared with LTBI. After CD25 high Treg cell depletion, the HBHA-stimulated IFN- responses by the PBMCs of patients with active TB reached levels similar to those of undepleted PBMCs in LTBI. A qualitative difference in HBHA T-cell responses remained between the two groups. HBHA is methylated in its C-terminal region, which contains several mono- and dimethyl-lysines (29), and the methylation has been shown to play an important role

7 Hougardy, Place, Hildebrand, et al.: Regulatory T Cells in Human Tuberculosis 415 in protective immunogenicity (4, 23). Patients with LTBI mount a strong Th1 response to the methylated, native form of the protein and respond poorly to nonmethylated, recombinant HBHA. Furthermore, the relation between the response to nhbha and to rhbha in LTBI is not linear (4). In contrast, we found that even after CD25 high Treg cell depletion, the PBMCs from patients with active TB produced rhbha-induced IFN- at levels that equaled those of the nhbha-induced IFN- and that the responses to rhbha and nhbha were linearly correlated. These observations indicate that in active TB the IFN- response is directed against the N-terminal, nonmethylated moiety of the protein, in contrast to the IFN- response previously described in patients with LTBI (4). HBHA is a surface-associated protein involved in binding of the mycobacteria to epithelial cells and in extrapulmonary dissemination (30), but it can also be released into the culture supernatant in late stage cultures (9). In its surface-associated form, only the C-terminal, methylated end of the protein is exposed (9), whereas the N-terminal domain is hidden within the bacterial cell wall. The differences in immune responses between LTBI and TB may thus be due to thereleaseofhbhaduringactivetbwithahighbacterialload, whereas during latent infection, the protein is not released in sufficient quantities for its N-terminal domain to be immunogenic. Altogether, these results show that, during active TB, elevated levels of CD4 CD25 high FOXP3 Treg cells present in the peripheral blood suppress T-cell responses to protective antigens, such as HBHA, which may represent an important strategy used by M. tuberculosis to evade protective immunity of the host. The fact that these cells display antigen specificity provides hope for novel targeted immune interventions to circumvent these pathogenic strategies. Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment : The authors thank A. Libois, D. Lauwers, and M. Rosen for the recruitment of patients with active TB; M. Doherty for providing ESAT-6; K. Huygen for providing antigen 85; and V. Verscheure for excellent technical assistance. References 1. Dye C, Scheele S, Dolin P, Pathania P, Raviglione MC. Consensus statement: global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 1999;282: Flynn JA, Chan R. Immunology of tuberculosis. Annu Rev Immunol 2001;19: Masungi C, Temmerman S, Van Vooren JP, Drowart A, Pethe K, Menozzi FD, Locht C, Mascart F. 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