Clinical Immunology (2011) 138, 50–59 available at www.sciencedirect.com Clinical Immunology www.elsevier.com/locate/yclim Specific cytokine patterns of pulmonary tuberculosis in Central Africa Johannes Nemeth b,c,d , Heide-Maria Winkler a,b , Lucas Boeck f , Ayola A. Adegnika b,c , Ella Clement e , Toung M. Mve e , Peter G. Kremsner b,c , Stefan Winkler a,b,⁎ a Department of Internal Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Austria b Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon c Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Germany d Department of Medicine, University Hospital Basel, Basel, Switzerland e Hôpital de Nkembo, Libreville, Gabon f Clinic of Pneumology, University Hospital Basel, Basel, Switzerland Received 19 September 2009; accepted with revision 14 September 2010 Available online 15 October 2010 KEYWORDS T cell; Tuberculosis; Cytokines; FoxP3 Abstract Different cytokines have been suggested to be involved in the pathogenesis of pulmonary tuberculosis (TB). The frequencies of Mycobacterium tuberculosis (MTB) specific CD4+ and CD8+ T cells, CD4+CD25+ Forkhead Box Protein (FoxP)3+ T cells, interleukin (IL)-6, interferon (IFN)-γ, Tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β and IL-10 were assessed in HIV-negative, pulmonary tuberculosis (TB) patients (n = 30) and in healthy controls (n = 23) in Gabon. Peripheral blood mononuclear cells (PBMC) were stimulated with purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6). In patients, a pronounced pro-inflammatory cytokine response with highly significant increased levels of IL-6 and TNF-α accompanied by increased TGF-β was detectable. Differences in IFN-γ responses between patients and healthy individuals were less pronounced than expected. FoxP3 expression did not differ between groups. A distinct cytokine pattern is associated with active pulmonary TB in patients from Central Africa. © 2010 Elsevier Inc. All rights reserved. Introduction ⁎ Corresponding author. Department of Internal Medicine I, Division of Infectious Diseases and Tropical Medicine, Medical University of Vienna, Waehringerguertel 18-20, A-1090 Vienna, Austria. Fax: +43 1404004418. E-mail address: stefan.winkler@meduniwien.ac.at (S. Winkler). Tuberculosis (TB) is one of the most important infectious diseases with nearly 10 million new cases of active TB occurring every year and about two billion people infected with Mycobacterium tuberculosis (MTB) globally [1]. The 1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2010.09.005 Specific cytokine patterns of PT in Central Africa majority of infected individuals never develop active disease since invading pathogens are controlled by immune system. Patients undergoing depletion of CD4+ T cells in the course of human immunodeficiency virus infection or functional neutralization of tumor necrosis factor (TNF)-α due to antiTNF-α-antibodies are at markedly increased risk to develop active TB [2,3]. Similarly, disorders of IFN-γ receptors predispose for severe courses of mycobacterial infections [4]. Type 1 biased CD4+ T cells play a central role in protection against TB by promoting activation of macrophages with the production of IFN-γ and TNF-α [5]. Thus, it has been suggested that a defective type 1 activation is a possible mechanism to explain the progression of latent MTB infection to active disease. However, studies assessing IFN-γ production capacity in active TB yielded contradictory results. Decreased IFN-γ production after stimulation with mycobacterial antigens was reported in TB patients, underlining the assumption that active disease may be associated with defective or suppressed type 1 responses [6–8]. In contrast, increased IFN-γ production in peripheral blood as well as at the site of infection was shown to be a hallmark of active TB, a fact which can even be used for diagnostic purposes [9–13]. A possible simultaneous up-regulation of inhibitory cytokines and regulatory T cells could explain this discrepancy. The regulatory capability of T cells has been linked to the expression of the transcription factor Forkhead Box Protein (FoxP)-3 [14–16]. Previous studies suggested that T cell derived over-regulation could promote suppression of cytokine responses in active TB, allowing thereby progression of disease [17–24]. In the recent study, we assumed that stimulation of T cells with purified protein derivative (PPD) reflects global cellular responsiveness in this population from Central Africa with high prevalence of latent TB, high BCG coverage and endemic non-tuberculous mycobacteria [1]. In contrast, early secretory antigenic target-6 (ESAT-6) induced cytokine responses were assumed to be highly specific for MTB. Thus, this experimental setting enabled us to compare global cellular responsiveness with highly MTB-specific cytokine production in patients with active TB and healthy individuals. Moreover, measuring multiple pro- and anti-inflammatory mediators should reflect as closely as possible the immune network underlying the development of active, pulmonary TB. Patients and methods Patients This cross sectional study was conducted at the Albert Schweitzer Hospital in Lambaréné and at the Hospital N´ Kembo, Libreville, Gabon, from January to November 2006. TB rates in Gabon are expected to be between 100 and 299 per 100,000, according to current WHO estimation [1]. Besides MTB, other mycobacterial infections such as Mycobacterium ulcerans are endemic. BCG vaccination is free of charge and recommended for all neonates. Vaccine coverage is high in our study area. Adult patients with active pulmonary TB seeking medical care at the Albert Schweizer Hospital in Lambarènè and the 51 Hospital N´Kembo as well as healthy volunteers were invited to participate within the study. The diagnostic laboratories of the Albert Schweizer Hospital and the Hospital N´Kembo were screened on a daily basis. If a newly detected, positive smear was reported, an investigator approached the respective patient to ask whether he or she was willing to participate in the study. Active pulmonary TB was defined by sputum-positive smears by Ziehl–Neelsen staining, chest-X rays consistent with TB, and clinical history, including at least three of the following symptoms: night sweat, cough lasting for more then 3 weeks, unintended weight loss, malaise, fever, lassitude, known exposure to open tuberculosis. If the patient was not clinically ill according to the stated criteria or had no radiographic changes consistent with TB, the patient was not included. Smear negative patients with the clinical diagnosis of active TB were not included. Exclusion criteria of both, patients and healthy subjects, were age younger than 16 years (1), HIV antibodies detectable in sera (2), pregnancy (3) and any other acute illness (4) as assessed by clinical examination. Noteworthy, healthy volunteers were also excluded if they had a history of active TB or if somebody with open TB was living in the same household. At inclusion participants were screened for malaria and HIV (Determine HIV-1/2® test, Abbott Laboratories®, Tokyo, Japan). Demographic data and clinical history were recorded at inclusion and 45 ml blood was drawn from a cubital vein. No further follow up was conducted in the course of this study. Written informed consent was obtained from all participants. The study protocol was approved by the ethics committee of the International Foundation for the Albert Schweitzer Hospital in Lambaréné. Methods Flow cytometry for intracellular staining of cytokines Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by ficoll-diatrizoate centrifugation and plated out in 24-well plates (2 × 106 cells per well). Cells were cultured in 1 ml ultra culture medium (UCM) (Bio Whittaker, Walkersville, MD) supplemented with L-glutamine (2 mM/l; Sigma, St. Louis, MI), gentamicin (170 mg/l; Sigma) and 2-mercaptoethanol (3.5 μl/l; Merck, Darmstadt, Germany) for 18 h at 37 °C in 5% CO2 and stimulated with purified protein derivate PPD (Statens Serum Institute, Copenhagen, Denmark) at a final concentration of 10 μg/ ml or with ESAT6 (Statens Serum Institute, Copenhagen, Denmark) with a final concentration of 5 μg/ml. In order to amplify TCR signalling and to facilitate the initial phase of Tcell activation, the co-stimulatory MAb CD28 (Pharmingen San Diego, CA) was added at a final concentration of 5 μg/ml. Brefeldin A (10 μg/ml final concentration, Sigma) was added after 6 h to block protein secretion. After 18 h, cells were harvested on ice, washed twice in phosphate-buffered saline (PBS), and fixed with 2% formaldehyde (1 ml per 2 × 106 cells) for 20 min. After two additional washes in PBS, the cells were re-suspended in Hank's balanced salt solution (supplemented with 0.3% bovine serum albumin and 0.1% sodium-azide). The cells were washed twice with PBS and made permeable with saponin (0.1%; Sigma), re-suspended with 50 μl of saponin 52 buffer diluted antibodies and incubated for 25 min in the dark. The following monoclonal antibodies were used: antiCD4, PerCP and APC labelled, anti-CD8, PerCP labelled (Becton and Dickinson, Mountain View, CA); anti-CD25, APC labelled (Becton and Dickinson); anti-FoxP3, phytoerythrin (PE) labelled (eBioscience, San Diego, CA); anti-TGF-β, PE labelled (IQProducts, Groningen, The Netherlands); MAb IFNγ (clone: B 27), fluorescein-isothiocyanate (FITC) labelled; MAb IL–2 (MQ1-17H12), PE labelled; IL-10 (JES3-9D7), PE labelled; TNF-α (MAB-11), PE labelled (all Becton and Dickinson). Four-color staining was performed, and at least 105 cells were analyzed on a FACS-Calibur (Becton Dickinson) equipped with a two-laser system (488- and 630-nm wavelength, respectively). The data were analyzed with CELL-Quest Pro software (Becton and Dickinson) and results were expressed as the percentage of cytokine-producing cells in each population. Detection of cytokines in plasma and supernatants Supernatants were collected from wells stimulated for 18 h as described above but without addition of Brefeldin A and frozen at −20° until assayed. In both plasma and supernatants IL-6, IL-10, TNF-α, IFN-γ and TGF-β were quantified using pre-validated commercially available ELISAs (R&D Systems, Minneapolis, MN). All tests were performed in duplicate, following the instructions of the manufacturer. Isolation of CD4+CD25+ and CD4+CD25− cells PBMC were prepared as described above. CD25high cells (1–2% of PBMC) were isolated by positive selection from PBMC with anti-CD25 magnetic microbeads (2 μl per 107 PBMC) (Miltenyi Biotec, Bergisch-Gladbach, Germany), and separated over an LS column (Miltenyi Biotec, BergischGladbach, Germany). For highest purity the CD25high cells were applied to a second, freshly prepared LS column and washed with PBS (including 0.5% BSA, 2 mM EDTA). FACS analysis proved purity greater than 96%. FoxP3 gene expression by qPCR Total RNA was isolated from purified CD4+CD25high and PBMC cells using TRI REAGENT (Molecular Research Center, Inc., Ohio, USA) following the recommendations of the supplier. 500 ng of total RNA was reverse transcribed into first-strand cDNA using Transcriptor-RT (Roche Diagnostics, Basel, Switzerland), dNTPs (10 μM each) and random hexamer primers. Quantitative RT-PCR was performed by LightCycler technology using the Fast Start SYBR Green I Kit (Roche Diagnostics) for amplification and detection. RT-PCR conditions for FoxP3 expression were a 10 min 95 °C denaturation step, amplification with 60 cycles of 5 min at 95 °C, 5 min at 68 °C and 15 min at 72 °C and finally melting point analysis in 0.2 °C steps using 10 μM of the following primers: FoxP3_ forward (F), 5'-TCAAGCACTGCCAGGCG-3'; FoxP3_reverse (R), 5'-CAGGAGCCCTTGTCGGAT-3'. The primer pair span intron/exon boundaries to minimize amplification of genomic DNA. In all assays each LightCycler capillary was loaded with 2 μl DNA Master Mix, 2.4 μl MgCl2 (25 mM), 12.6 μl H2O, 0.5 μl of each primer (10 μM) and 2 μl first-strand cDNA of the sample to a total volume of 20 μl. All samples were run J. Nemeth et al. in duplicate. Target gene expression was normalized to either β2-microglobulin (β2M) or 5-aminolevulinic acid synthase (ALAS). Primer sequences of ALAS: F, 5'-CCACTGGAAGAGCTGTGTGA-3'; R, 5'-ACCCTCCAACACAACCAAAG-3'. RT-PCR efficiency was determined for each primer pair using cDNA dilutions. Primer specificity was tested with melting point analysis, gel electrophoresis and subsequently DNA sequencing. Statistics Statistical analysis was performed using a standard statistical package (SPSS 15.0 for Windows; SPSS Inc., Chicago, IL). The Mann–Whitney U-test was applied for group differences. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. Results Participants 30 patients and 23 healthy individuals were included in this study. Both groups were comparable regarding age and gender. Body temperature was significantly higher and hemoglobin concentrations were significantly lower in TB patients when compared to healthy controls (p b 0.001 and p = 0.033, respectively; Table 1). Levels of circulating cytokines in peripheral plasma Plasma levels of circulating cytokines except for IL-6 were similar in patients and healthy subjects. IL-6 plasma levels (p = 0.012) were significantly increased (corrected for multiple comparisons) in TB patients (Fig. 1: plasma). Levels of cytokines released in the supernatant Following cytokines were increased in the supernatant of TB patients when compared to healthy individuals (corrected for multiple comparisons): TNF-α, spontaneously secreted in the control well (p = 0.002) and after ESAT-6 stimulation (p = 0.002; Fig. 1A). The same pattern was observed for IL-6 (control well: p = 0.012; ESAT-6: p = 0.002; Fig. 1E). TGF-β secretion was increased among TB patients, both in the control well as after stimulation (control well: p = b 0.001; Table 1 Demographic characteristics and results of laboratory testing for study participants. Study participants TB (n = 30) Sex, number of male/female Age, median years (range) Admission temperature median °C (range) WBC count 10x9 cells per l 18/12 29 (16–79) 38 (36.5–39.0)* Hemoglobin concentration, g/dl Control (n = 23) 15/8 28 (20–31) 36.5 (36.1–37.0) 6.6 (3–8.8) 5.0 ( 2.8–7.9) 10.2 (7.3–15.3)* 13.3 (8.9–16.9) Specific cytokine patterns of PT in Central Africa 53 Figure 1 (plasma): Plasma levels of different cytokines in TB patients (n = 30) and controls (n = 21) from Central Africa. (A, B, C, D, E) Tumour necrosis factor (TNF)-α (A), interleukin (IL)-10 (B), transforming growth factor (TGF)-β (C), interferon (IFN)-γ (D) and IL-6 (E) in supernatants following stimulation with purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6), respectively. White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. Comparability of groups was analyzed by Mann–Whitney U-test. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. Significant differences are marked by *. PPD: p = b 0.001; ESAT-6: p = 0.002). Remarkably, TGF-β secretion appeared not to be influenced by short time stimulation. + + Frequency of PPD specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-10, IL-2 and TGF-β Differences between healthy individuals and patients after PPD stimulation were minor. TNF-α producing CD4+ T cells (p b 0.001) and IL-10 producing CD8+ T cells (p = 0.003) were significantly increased in patients (Figs. 2, 3). Frequency of ESAT-6 specific CD4+ and CD8+ T cells expressing IFN-γ, TNF-α, IL-10, IL-2 and TGF-β After stimulation with the MTB specific antigen ESAT-6 significant differences between patients and healthy individuals were restricted to CD4+ T cells. TNF-α producing CD4+ T 54 J. Nemeth et al. 100 100 101 102 TGFb PE 101 102 TNFa PE 103 103 104 104 100 100 101 101 102 102 TN NFa PE 103 104 103 104 100 101 102 TGFb PE 103 104 100 101 102 TNFa PE 103 104 Figure 2 Representative two-parameter dot plots indicating the frequency of PPD and ESAT-6-specific CD4+ T cells expressing IFN-γ and/or TNF-α or TGF-β, respectively. PBMC were incubated with medium alone (A), with PPD (B) and ESAT-6 (C). cells (p = b 0.001) and CD4+ T cells (p = 0.003) co-producing IL2 and IFN-γ were significantly increased in patients when compared with healthy individuals. CD4+ T cells co-producing TNF-α and IFN-γ appeared to be increased in TB patients. Differences, however, were not significant after correction for multiple analysis (Figs. 2, 4). Levels of FoxP3 in PBMCs, CD25high T cells and after stimulation with mycobacterial antigens FoxP3 mRNA from PBMCs was assessed in 19 participating individuals. Target gene expression was normalized to 5aminolevulinic acid synthase (ALAS). No differences between patients and controls were detected (Fig. 4; Table 2). Furthermore, CD25high T cells were sorted with magnetic banding and analyzed for FoxP3mRNA expression. Similarly, no difference between patients and healthy individuals was found. FoxP3mRNA appeared to be inducible by stimulation. However, after correction differences did not reach statistical significance. Expression of FoxP3+ in CD4+CD25+ T cells was analyzed on a single cell level by flow cytometry after stimulation with mycobacterial antigens. Again, no significant differences were found between patients and healthy individuals (Fig. 5B, Table 2). Discussion In the present work, we report a distinct cytokine profile consistent of simultaneously increased TNF-α, IL-6 and TGFβ in a TB patient cohort from Central Africa. Spontaneous TNF-α secretion as well as MTB specific TNFα production in CD4+T cells was increased in patients with active TB. TNF-α blocking therapy is a risk factor to reactivate latent TB infection [2], highlighting the role of TNFα in granuloma maintenance during latent TB. The role of TNF-α during active TB is less clear and might not only be beneficial. Overproduction of TNF-α could be responsible for immunopathology and contributive to the progression of disease [25]. In the recent study-cohort, MTB specific TNF-α appeared to be highly specific for active TB, a fact which might be useful for diagnostic purposes in the future. In contrast, IFN-γ, which is now extensively used for immune-based diagnostics [12,13], was not significantly increased in patients when compared to healthy individuals. After ESAT-6 stimulation only CD4+T cells co-producing IL-2 and IFN-γ were significantly increased in patients. This is in line with a recent study, which reported increased IL-2 and IFN-γ co-producing T cells in TB patients [26]. Moreover, measurement of IL-2 together with IFN-γ has been shown to increase the discriminatory power of immune based Specific cytokine patterns of PT in Central Africa 55 Figure 3 Frequency of cytokine expressing CD4+ T and CD8+ T cells obtained after stimulation of peripheral blood mononuclear cells (PBMC) with purified protein derivative (PPD) in TB patients ( n = 30) and in healthy individuals (n = 23) from Central Africa. White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. Each figure indicates the frequency of the T cell subclass producing either one cytokine or co-producing the respective cytokine with IFN-γ. Significant differences are marked by *. The Mann– Whitney U-test was used to test for significance. A Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. diagnostics [27]. T cells capable of producing more than one cytokine were described to be advantageous during human HIV infection [28,29]. Our data suggest that T cells capable to produce two cytokines are a marker of active TB, a finding which is in line with the studies mentioned above [26,27]. 56 J. Nemeth et al. Figure 4 Frequency of cytokine expressing CD4+ T and CD8+ T cells obtained after stimulation of peripheral blood mononuclear cells (PBMC) with early secretory antigenic target-6 (ESAT-6) in TB patients ( n = 30) and in healthy individuals (n = 23) from Central Africa. Boxes (line indicates the median percentage) and whiskers (displaying the range). White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. Each figure indicates the frequency of the T cell subclass producing either one cytokine or coproducing the respective cytokine with IFN-γ. Significant differences are marked by *. The Mann–Whitney U-test was used to test for significance, a Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. TGF-β was secreted at higher levels in patients compared with healthy controls and was poorly influenced by specific stimulation. The T cell independent TGF-β secretion in TB patients was not influenced by antigenic stimulation. Increased levels of TGF-β in the supernatants without an increased expression of TGF-β within the T cell subsets suggest that the cytokine is mainly monocyte-derived. PBMCs contain between 10% and 15% of monocytes, which have Specific cytokine patterns of PT in Central Africa Table 2 Induction of FoxP3 after stimulation with MTB specific antigens on mRNA level. Induction of FoxP3 mRNA after stimulation medium PPD ESAT-6 patients (n = 8) controls (n = 10) 1 (0) 1.9 (0.38) 1.4 (0.18) 1 (0) 1.5 (0.14) 0.8 (0.24) been shown to produce spontaneously TGF-β in peripheral blood of patients suffering from TB [30]. TGF-β is known as inhibitor of most effector functions of T cells, limiting the acquisition of CTL function of CD8+ T cells [31] and impairing the development of naïve CD4+ T cells to type 1 or type 2 cells [32]. In active TB, inhibition of TGF-β was associated with increased MTB specific T cell responses [33]. 57 IL-6, in contrast, showed an increased spontaneous production in control wells and was inducible by ESAT-6 stimulation. This expression-pattern was comparable to the pattern of TNF-α. IL-6 plays a central role during inflammation in general and, more specifically, during MTB infection [34]. MTB itself has been shown to induce IL-6 [35], which, in turn, has been described to impair macrophage responses to IFN-γ [36]. We report an excess of IL-6, TNF-α and TGF-β spontaneously secreted in the supernatant, suggesting that these cytokines are in part or–in case of IL-6–completely produced by monocytes. This observation suggests that during active, pulmonary TB a rather unspecific inflammation takes place which is absent in latent infection. Hence, the combination of MTB specific cytokines with markers of inflammation could lead to immune based diagnostics from peripheral blood which is able to discriminate between latent infection and active disease. In line, IL-6 together with TNF-α and IFN-γ have recently been used as markers to monitor TB treatment success [37]. Figure 5 (A) Frequency of PPD and ESAT-6-specific CD4+ T cells expressing CD25 and FoxP3 obtained after stimulation of peripheral blood mononuclear cells (PBMC) with early secretory antigenic target-6 (ESAT-6) boxes (line indicates the median percentage) and whiskers (displaying the range). White boxes indicate tuberculosis (TB) patients, grey boxes indicate healthy individuals. The Mann– Whitney U-test was used to test for significance, a Bonferroni–Holm procedure was used to correct for multiple comparisons between groups. (B) Representative two-parameter dot plots indicating the frequency of PPD and ESAT-6-specific CD4+ T cells expressing CD25 and FoxP3. PBMC were incubated with medium alone (A) with PPD (B) and ESAT-6 (C). For measurement purposes, CD4+CD25+ T cells were divided into FoxP3 high and FoxP3 low positive T cells. Upper numbers indicate the percentage of CD4+CD25+ T cells classified as expressing high FoxP3. The lower number indicates the percentage of CD4+CD25+ T cells with low FoxP3 expression. However, all analyses were done on total FoxP3, i.e. in this example the following numbers were used for calculation: 0.08%, 0.26%, and 0.21%, respectively. Table 2: left column displays the mean (range) induction of FoxP3 mRNA after stimulation as measured by PCR. FoxP3 expression was normalized to FoxP3 mRNA levels in the control well. Differences are statistically not different. 58 After PPD stimulation, CD8+T cells producing IL-10 were significantly increased. Given the very small absolute amount of CD8+ T cells producing IL-10 the biological relevance of this finding remains unclear. A link between T cell mediated anergy through IL-10 during active TB has been published earlier [38]. We were unable to detect differences of FoxP3 mRNA expression in peripheral blood in patients with active TB when compared to healthy individuals. This was shown not only for PBMCs but also for CD25high T cells. These findings differ from previous studies, describing increased levels of FoxP3 in peripheral blood of patients with active TB [17–23]. In line with our findings, a recent study failed to detect regulatory T cells solely based on their FoxP3 expression [39]. Additionally and in contrast to some of the other studies on FoxP3 in human TB, we included only patients with open pulmonary TB [22,23]. Lower FoxP3 levels in open pulmonary TB compared with non-pulmonary TB have been suggested earlier [23]. Different clinical manifestations of active TB appear to have different levels of FoxP3, as a comprehensive study on miliary TB suggests [40]. The lack of systemic immune suppression and elevated FoxP3 levels could be related to the inclusion of subjects with non-tuberculous mycobacterial pneumonitis. Mycobacterium avium and Mycobacterium kansasii can cause pulmonary disease indistinguishable from MTB infection without mycobacterial culture [41]. This also could explain differences to other studies showing elevated FoxP3 levels in pulmonary TB. Summarized, we identified a highly specific cytokine pattern indicative of active TB in patients Gabon. Surprisingly, FoxP3 was not increased, a finding which does not support a central role of regulatory T cells in the pathogenesis of active, pulmonary TB. Differences of MTB specific IFNγ levels comparing TB patients and healthy individuals were less pronounced than expected. However, IFN-γ production was not suppressed in patients, a fact arguing against a defective type 1 response during active TB. A significant amount of IL-6, TNF-α and TGF-β levels was spontaneously produced by cells from TB patients, reflecting most probably a systemic, unspecific inflammation. This observation could provide the rationale for novel immunological approaches to detect active TB. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments The investigators want to thank the study participants. Peter Pongratz is acknowledged for the timely delivery of samples. References [1] World Health Organization. Tuberculosis facts 2007. http:// www.who.int/tb/en/, last assessed 20.7.10 [2] S.H. Kaufmann, A.J. McMichael, Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis, Nat. Med. 11 (2005) 33–44 Review. [3] Food and Drug Administration. Safety update on TNF-α Antagonists. www.fda.gov last assessed 20.7.10. J. Nemeth et al. [4] S.E. Dorman, C. Picard, D. Lammas, K. Heyne, J.T. van Dissel, R. Baretto, S.D. Rosenzweig, M. Newport, M. Levin, J. Roesler, D. Kumararatne, J.L. Casanova, S.M. Holland, Clinical features of dominant and recessive interferon gamma receptor 1 deficiencies, Lancet 364 (2004) 2113–2121. [5] S.H. Kaufmann, Tuberculosis: back on the immunologists' agenda, Immunity 24 (2006) 351–357 Review. [6] C.S. Hirsch, Z. Toossi, C. Othieno, J.L. Johnson, S.K. Schwander, S. Robertson, R.S. Wallis, K. Edmonds, A. Okwera, R. Mugerwa, P. Peters, J.J. Ellner, Depressed T-cell interferongamma responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy, J. Infect. Dis. 180 (1999) 2069–2073. [7] C.S. Hirsch, Z. Toossi, C. Othieno, J.L. Johnson, S.K. Schwander, S. Robertson, R.S. Wallis, K. Edmonds, A. Okwera, R. Mugerwa, P. Peters, J.J. Ellner, Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis, J. Clin. Immunol. 6 (1986) 146–151. [8] E.K. Jo, H.J. Kim, J.H. Lim, D. Min, Y. Song, C.H. Song, T.H. Paik, J.W. Suhr, J.K. Park, Dysregulated production of interferon-gamma, interleukin-4 and interleukin-6 in early tuberculosis patients in response to antigen 85B of Mycobacterium tuberculosis, Scand. J. Immunol. 51 (2000) 209–217. [9] S. Winkler, M. Necek, H. Winkler, A.A. Adegnika, T. Perkmann, M. Ramharter, P.G. Kremsner, Increased specific T cell cytokine responses in patients with active pulmonary tuberculosis from Central Africa, Microbes Infect. 7 (2005) 1161–1169. [10] P. Ravn, M.E. Munk, A.B. Andersen, B. Lundgren, J.D. Lundgren, L.N. Nielsen, A. Kok-Jensen, P. Andersen, K. Weldingh, Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP10 for diagnosis of active tuberculosis, Clin. Diagn. Lab. Immunol. 12 (2005) 491–496. [11] C. Lange, M. Pai, F. Drobniewski, G.B. Migliori, Interferongamma release assays for the diagnosis of active tuberculosis: sensible or silly? Eur. Respir. J. 33 (6) (2009) 1250–1253. [12] C. Jafari, S. Thijsen, G. Sotgiu, D. Goletti, J.A. Benítez, M. Losi, R. Eberhardt, D. Kirsten, B. Kalsdorf, A. Bossink, I. Latorre, G. B. Migliori, A. Strassburg, S. Winteroll, U. Greinert, L. Richeldi, M. Ernst, C. Lange, Tuberculosis Network European Trialsgroup, Bronchoalveolar lavage enzyme-linked immunospot for a rapid diagnosis of tuberculosis: a Tuberculosis Network European Trialsgroup study, Am. J. Respir. Crit. Care Med. 180 (7) (2009) 666–673. [13] J. Nemeth, H.M. Winkler, R.H. Zwick, R. Rumetshofer, P. Schenk, O.C. Burghuber, W. Graninger, M. Ramharter, S. Winkler, Recruitment of Mycobacterium tuberculosis specific CD4(+) T cells to the site of infection for diagnosis of active tuberculosis, J. Intern. Med. 265 (2009) 163–168. [14] S. Hori, T. Nomura, S. Sakaguchi, Control of regulatory T cell development by the transcription factor Foxp3, Science 299 (2003) 1057–1061. [15] M.A. Gavin, J.P. Rasmussen, J.D. Fontenot, V. Vasta, V.C. Manganiello, J.A. Beavo, A.Y. Rudensky, Foxp3-dependent programme of regulatory T-cell differentiation, Nature 445 (2007) 771–775. [16] S. Mendez, S.K. Reckling, C.A. Piccirillo, D. Sacks, Y. Belkaid, Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity, J. Exp. Med. 200 (2004) 201–210. [17] J.M. Hougardy, V. Verscheure, C. Locht, F. Mascart, In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans, Microbes Infect. 9 (2007) 1325–1332. [18] L. Li, S.H. Lao, C.Y. Wu, Increased frequency of CD4(+)CD25 (high) Treg cells inhibit BCG-specific induction of IFN-gamma Specific cytokine patterns of PT in Central Africa [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] by CD4(+) T cells from TB patients, Tuberculosis 87 (2007) 526–534. J.M. Hougardy, S. Place, M. Hildebrand, A. Drowart, A.S. Debrie, C. Locht, F. Mascart, Regulatory T cells depress immune responses to protective antigens in active tuberculosis, Am. J. Respir. Crit. Care Med. 176 (2007) 409–416. T. Roberts, N. Beyers, A. Aguirre, G. Walzl, Immunosuppression during active tuberculosis is characterized by decreased interferon-gamma production and CD25 expression with elevated forkhead box P3, transforming growth factor-beta, and interleukin-4 mRNA levels, J. Infect. Dis. 195 (2007) 870–878. X. Chen, B. Zhou, M. Li, Q. Deng, X. Wu, X. Le, C. Wu, et al., CD4(+)CD25(+)FoxP3(+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease, Clin. Immunol. 123 (2007) 50–59. L. Gazzola, C. Tincati, A. Gori, M. Saresella, I. Marventano, F. Zanini, FoxP3 mRNA expression in regulatory T cells from patients with tuberculosis, Am. J. Respir. Crit. Care Med. 174 (2006) 356. V. Guyot-Revol, J.A. Innes, S. Hackforth, T. Hinks, A. Lalvani, Regulatory T cells are expanded in blood and disease sites in patients with tuberculosis, Am. J. Respir. Crit. Care Med. 173 (2006) 803–810. S.M. Wahl, Transforming growth factor-beta: innately bipolar, Curr. Opin. Immunol. 19 (2007) 55–62. V.F. Quesniaux, M. Jacobs, N. Allie, S. Grivennikov, S.A. Nedospasov, I. Garcia, M.L. Olleros, Y. Shebzukhov, D. Kuprash, V. Vasseur, S. Rose, N. Court, R. Vacher, B. Ryffel, TNF in host resistance to tuberculosis infection, Curr. Dir. Autoimmun. 11 (2010) 157–179 Review. N. Caccamo, G. Guggino, S.A. Joosten, G. Gelsomino, P. Di Carlo, L. Titone, D. Galati, M. Bocchino, A. Matarese, A. Salerno, A. Sanduzzi, W.P. Franken, F. Dieli, Multifunctional CD4+T cells correlate with active Mycobacterium tuberculosis infection, Eur. J. Immunol. 40 (2010) 2211–2220. R. Biselli, S. Mariotti, V. Sargentini, I. Sauzullo, M. Lastilla, F. Mengoni, V. Vanini, E. Girardi, D. Goletti, R. D' Amelio, R. Nisini, Detection of interleukin-2 in addition to interferongamma discriminates active tuberculosis patients, latently infected individuals, and controls, Clin. Microbiol. Infect. 16 (8) (2010) 1282–1284. A. Harari, G.P. Rizzardi, K. Ellefsen, D. Ciuffreda, P. Champagne, P.A. Bart, D. Kaufmann, A. Telenti, R. Sahli, G. Tambussi, L. Kaiser, A. Lazzarin, L. Perrin, G. Pantaleo, Analysis of HIV-1- and CMV-specific memory CD4 T-cell responses during primary and chronic infection, Blood 100 (2002) 1381–1387. M.J. Boaz, A. Waters, S. Murad, P.J. Easterbrook, A. Vyakarnam, Presence of HIV-1 Gag-specific IFN-gamma+IL-2+ and CD28+IL-2+ CD4 T cell responses is associated with nonprogression in HIV-1 infection, J. Immunol. 169 (2002) 6376–6385. 59 [30] Z. Toossi, P. Gogate, H. Shiratsuchi, T. Young, J.J. Ellner, Enhanced production of TGF-beta by blood monocytes from patients with active tuberculosis and presence of TGF-beta in tuberculous granulomatous lung lesions, J. Immunol. 154 (1) (1995) 465–473. [31] G.E. Ranges, I.S. Figari, T. Espevik, M.A. Palladino Jr., Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha, J. Exp. Med. 166 (1987) 991–998. [32] S.L. Swain, G. Huston, S. Tonkonogy, A. Weinberg, Transforming growth factor-beta and IL-4 cause helper T cell precursors to develop into distinct effector helper cells that differ in lymphokine secretion pattern and cell surface phenotype, J. Immunol. 147 (1991) 2991–3000. [33] C.S. Hirsch, J.J. Ellner, R. Blinkhorn, Z. Toossi, In vitro restoration of T cell responses in tuberculosis and augmentation of monocyte effector function against Mycobacterium tuberculosis by natural inhibitors of transforming growth factor beta, Proc. Natl Acad. Sci. USA 94 (1997) 3926–3931. [34] J.L. Flynn, Immunology of tuberculosis and implications in vaccine development, Tuberculosis 84 (2004) 93–101. [35] Y. Zhang, M. Broser, W. Rom, Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF IL 6 and NF-kappa B, Proc. Natl Acad. Sci. USA 92 (8) (1995) 3632. [36] V. Nagabhushanam, A. Solache, L.M. Ting, C.J. Escaron, J.Y. Zhang, J.D. Ernst, Innate inhibition of adaptive immunity: Mycobacterium tuberculosis-induced IL-6 inhibits macrophage responses to IFN-gamma, J. Immunol. 171 (9) (2003) 4750–4757. [37] A.M. Mattos, C.D. Almeida, K.L. Franken, C.C. Alves, C. Abramo, M.A. Souza, M. L'hotellier, M.J. Alves, A.P. Ferreira, S.C. Oliveira, T.H. Ottenhoff, H.C. Teixeira, Increased IgG1, IFN-{gamma}, TNF-{alpha} and IL-6 responses to Mycobacterium tuberculosis antigens in patients with Tuberculosis are lower after chemotherapy, Int. Immunol. 22 (2010) 775–782. [38] V.A. Boussiotis, E.Y. Tsai, E.J. Yunis, S. Thim, J.C. Delgado, C.C. Dascher, A. Berezovskaya, D. Rousset, J.M. Reynes, A.E. Goldfeld, IL-10-producing T cells suppress immune responses in anergic tuberculosis patients, J. Clin. Invest. 105 (2000) 131.-25-30. [39] T. Chiacchio, R. Casetti, O. Butera, V. Vanini, S. Carrara, E. Girardi, D. Di Mitri, L. Battistini, F. Martini, G. Borsellino, D. Goletti, Characterization of regulatory T cells identified as CD4 (+)CD25(high)CD39(+) in patients with active tuberculosis, Clin. Exp. Immunol. 156 (2009) 463–470. [40] P.K. Sharma, P.K. Saha, A. Singh, S.K. Sharma, B. Ghosh, Mitra DK.FoxP3+ regulatory T cells suppress effector T-cell function at pathologic site in miliary tuberculosis, Am. J. Respir. Crit. Care Med. 179 (2009) 1061–1070. [41] D.E. Griffith, Nontuberculous mycobacterial lung disease, Curr. Opin. Infect. Dis. 23 (2) (2010) 185–190 Review.