International Immunology, Vol. 22, No. 9, pp. 775–782 doi:10.1093/intimm/dxq429 Advance Access publication 11 July 2010 ª The Japanese Society for Immunology. 2010. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org Increased IgG1, IFN-g, TNF-a and IL-6 responses to Mycobacterium tuberculosis antigens in patients with Tuberculosis are lower after chemotherapy 1 Department of Parasitology, Microbiology and Immunology, Biological Sciences Institute, Federal University of Juiz de Fora, 36036-900 Juiz de Fora, Brazil 2 Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands 3 Respiratory Diseases Division, Central Public Health Clinic, Juiz de Fora, Brazil 4 Department of Public Health, Faculty of Medicine, Federal University of Juiz de Fora, 36036-900 Juiz de Fora, Brazil 5 Departament of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil *These authors contributed equally to this work Correspondence to: H. C. Teixeira; E-mail: henrique.teixeira@ufjf.edu.br Received 15 July 2009, accepted 16 June 2010 Abstract Detection of specific antibodies may represent an additional tool in diagnosis of tuberculosis (TB). Herein, levels of serum IgG antibodies against early secreted antigenic target (ESAT-6), culture filtrate antigen-10 (CFP-10) and 16kDa Mycobacterium tuberculosis antigens were measured in 33 active pulmonary TB patients (0M-TB), in 47 patients after 1–3 months of treatment (3M-TB) and in 22 patients who had completed 6 months of chemotherapy (6M-TB). The control group consisted of 38 BCG-vaccinated healthy controls (HC). In addition, IFN-g, tumor necrosis factor (TNF)-a, IL-6, IL-2, IL-4 and IL-10 production in PBMC cultures from 20 patients were measured following stimulation with the M. tuberculosis-specific fusion protein ESAT-6/CFP-10. Elevated levels of IgG against ESAT-6, CFP-10 and 16kDa antigens were detected in 0M-TB and 3M-TB patients in comparison to the HC and 6M-TB groups. Receiver operating characteristic analysis indicated sensitivity of 85, 94 and 61% and specificity of 89, 87 and 89% for serum IgG against ESAT-6, CFP-10 and 16kDa, respectively. A predominant IgG1 response to ESAT-6 and CFP-10 was observed in 0M-TB patients, together with ESAT-6/CFP-10-specific IFN-g, TNF-a and IL-6 that were produced at lower levels in the 6M-TB group. These data indicate that a Th1 phenotype against early phase Mtb antigens appears to be dominant in the peripheral blood of patients with active pulmonary TB that is reduced after chemotherapy. Taken together, ESAT-6/CFP-10 cytokine tests together with detecting IgG antibodies specific to ESAT-6 and CFP-10 may be the useful TB disease biomarkers in monitoring treatment success. Keywords: antibody response, chemotherapy, cytokines, ESAT-6/CFP-10, tuberculosis Introduction The global burden of tuberculosis (TB) is falling slowly, TB remaining a major health problem worldwide. The number of new cases of TB yearly is estimated at ;9.4 million, 80% of which is concentrated in only 22 countries. Brazil, with an estimated 100 000 new cases yearly, occupies the 19th position among the countries with highest incidence of TB in the world (1). The diagnosis of pulmonary TB is based on the identification of the bacillus by microscopic examination of sputum smear or by culture, however, such methods present certain limitations. Around 30–50% of TB patients are negative in the bacilloscopic examination, and the culture, when necessary, requires a long time for the growth of Mycobacterium tuberculosis, which delays diagnosis (2–4). Since early diagnosis and efficient treatment are the basis for interrupting the transmission chain of TB and for controlling disease (3), TB patients with negative bacilloscopy represent a great problem for TB control programs. Although none of the diagnostic tests reviewed in 2007 by Steingart et al. (5) performed satisfactorily to replace sputum smear microscopy, the Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 Ana Márcia M. Mattos1,*, Caroline de S. Almeida1,*, Kees L. M. C. Franken2, Caio César de S. Alves1, Clarice Abramo1, Maria Aparecida de Souza1, Marcos L’Hotellier3, Márcio J. M. Alves4, Ana Paula Ferreira1, Sérgio C. Oliveira5, Tom H. M. Ottenhoff2 and Henrique C. Teixeira1 776 IgG and cytokine responses in human TB anti-TB antibody response and cytokine production was analyzed. Methods Population studied One hundred and twenty-two patients with pulmonary TB from the Respiratory Diseases Division of the Central Public Health Clinic in Juiz de Fora, State of Minas Gerais, Brazil, were selected. Only those patients with detectable acid-fast bacilli in the sputum bacilloscopy or culture-confirmed TB, and who had undergone clinical and chest X-ray examinations prescribed by the Brazilian Ministry of Health, were included in the study. For the antibody/ELISA study, patients with pulmonary TB were divided into three groups in accordance with the stage of treatment: (i) 33 patients with active TB (0M group), patients without prior treatment (n = 24) or with up to 15 days of treatment (n = 9), (ii) 47 patients with 1–3 months of anti-TB chemotherapy (1-3M group) and (iii) 22 patients who had completed 6 months of treatment (6M group). The age of the patients varied between 13 and 79 years, average age was 35 years. For the cytokine/CBA study, 20 pulmonary TB patients were divided into two groups: 0M group (n = 10) and 6M group (n = 10), the age of these patients varied between 21 and 73 years. Average age was 40 years. AIDS, diabetes, hepatitis, hypertension, pregnancy, malnutrition, malignances, treatment failure, corticosteroids use and alcoholism were exclusion criteria. The treatment for all patients was 2 months of rifampicin/isoniazid/pyrazinamide followed by 4 months of rifampicin/isoniazid anti-TB treatment. There was no primary resistance to isoniazid in the group of patients studied. Thirty-eight healthy BCG-vaccinated individuals, most of whom were university medical students from Juiz de Fora, without prior history of mycobacterial disease, were included in the control group. None of the healthy controls (HC) was individuals in direct contact with TB patients. Most of the patients were of the masculine gender (66%) and presented a vaccination scar (75%). The age of the controls varied between 13 and 44 years. Average age was 24 years. All patients and controls gave informed consent for blood sampling after written information was provided. The Medical Ethics Committee of the Federal University of Juiz de Fora approved the study protocol (no. 174/2006 and no. 256/2008). Antigens Antigens 16kDa, ESAT-6, CFP-10 and a fusion protein of ESAT-6 and CFP-10 were produced and quality controlled by Leiden University Medical Center. Briefly, genes were amplified by PCR and cloned by Gateway Technology (Invitrogen, San Diego, CA, USA) in a bacterial expression vector containing an N-terminal histidine tag. The proteins were over-expressed in Escherichia coli BL21(DE3) and purified, as described previously (26). Purity and size were checked by gel electrophoresis and western blotting with anti-His antibodies and anti-E. coli antibodies. Residual endotoxin levels were determined with a Limulus amoebocyte lysate assay (Cambrex) and were found to be <50 IU mg 1 recombinant protein. Recombinant antigens were freeze-dried, Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 development of more efficient methods that may complement microscopic examination is necessary. A serological test based on the detection of circulating antibodies against M. tuberculosis-specific antigens could be a good alternative. This method is quite attractive because of its easy application, low cost testing many serum samples in parallel and relatively low invasiveness (6). Besides, it can help in the study of immunological response during infection. However, low sensitivity and low specificity to the antigens limit the application of serological tests for TB (7, 8). A possibility for the development of more specific tests involves M. tuberculosis antigens that are absent in Mycobacterium bovis BCG vaccine strain and in environmental mycobacteria. Early secreted antigenic target-6kDa (ESAT-6) and culture filtrate antigen-10 (CFP-10) have been shown to be good markers of infection by M. tuberculosis (9, 10). ESAT-6 and CFP-10 are immunodominant proteins, encoded by the region of difference 1, a chromosomal region preserved in virulent strains, such as M. bovis and M. tuberculosis (11, 12). Tests based on the detection of IFN-c by T cells in response to ESAT-6 and CFP-10 antigens have presented elevated sensitivity (73%) and specificity (93%) in TB diagnosis (13). However, these tests fail to differentiate disease from infection and cannot be used for case detection in endemic settings; this has implications for detecting latently infected subjects in low or non-endemic settings (14, 15). Another promising antigen in the development of new tests for TB diagnosis is the 16kDa, a protein detected in latent TB individuals more than in active TB patients, expressed by the bacillus principally when subjected to anaerobic conditions (6, 16). ESAT-6, CFP-10 and the 16kDa antigens are immunogenic for B cells, as shown by the presence of antibodies specific to these proteins in patients with pulmonary and extra pulmonary TB (6, 17). Control of active M. tuberculosis infection is mediated by T cells and toll-like receptor-driven Th1 cytokines, such as IL12/IFN-c and tumor necrosis factor (TNF)-a, but these cytokines on their own are not enough (18–22). In the mouse, an immune stimulus that predominantly activates Th1 cells (IL12 and IFN-c) stimulates mostly IgG2a response, while a stimulus that activates Th2 cells (IL-4 and IL-10) generates a predominant IgG1 and IgE response (23). The human counterparts of murine IgG subclasses are based on similarities in biological and functional activities. Murine IgG2a and IgG2b and human IgG1 and IgG3 share the ability to fix complement and bind to protein antigens. Murine IgG3 and human IgG2 recognize predominantly carbohydrate epitopes (24). Human IgG1 may enhance bacterial uptake and the clearance of pathogens via the Fc receptor on macrophages, while IgG2 mediates immunity by non-phagocytic effectors cells (25). Correlation between active and treated TB and the levels of Th1/Th2 cytokines and IgG isotypes has not been clearly defined. In this study, serum levels of IgG and IgM antibodies specific to ESAT-6, CFP-10 and 16kDa antigens were evaluated in patients with pulmonary TB and in BCG-vaccinated healthy individuals. We also measured the production of IFN-c, TNF-a, IL-6, IL-2, IL-4 and IL-10 by PBMC of TB patients and controls in response to the fusion molecule ESAT-6/CFP-10 using the cytometric bead array (CBA) method. The effect of the TB chemotherapy on levels of IgG and cytokine responses in human TB 777 shipped to Juiz de Fora, reconstituted in PBS and stored at 70°C until testing in ELISA and CBA. Enzyme-linked immunosorbent assay Separation and culture of PBMCs Cryopreserved PBMC samples obtained after density centrifugation on ficoll-plaque plus (Amersham Bioscienes, Uppsala, Sweden) were thawed, washed and re-suspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 2 mM L-glutamine, 100 U ml 1 penicillin, 100 lg ml 1 streptomycin, 10 ll ml 1 non-essential amino acids and 10% heat-inactivated normal human AB plasma (Hemominas, Juiz de Fora, Brazil). The cells were added to 96-well plates in a volume of 200 ll (2 3 105 cells per well) and incubated in the presence or absence of recombinant ESAT-6/CFP-10 (5 lg ml 1), for 96 h at 37°C in 5% CO2, in duplicates. The culture supernatants were harvested and stored at 20°C. Detection of cytokines by CBA The CBA technique was performed for the detection of cytokines in PBMC culture supernatants (5 ll per sample) using the human Th1/Th2 cytokine kit II, as described by the manufacturer (Becton Dickinson, San Jose, CA, USA). Acquisition and analysis of standards and samples were performed using the FACScalibur flow cytometer and BD software CBA Isotype Analysis (Becton Dickinson). The positive response to ESAT-6/CFP-10 antigen was obtained by subtracting the unstimulated culture values from those obtained from cultures stimulated with ESAT-6/CFP-10. Statistical analysis The comparison between the groups was carried out through the non-parametric Mann–Whitney U-test. The receiver oper- Results Detection of specific IgG antibodies in active TB In this study, levels of IgG antibodies against ESAT-6, CFP10 and 16kDa antigens were shown to be significantly elevated in sera of patients with active TB when compared with the HC group (P < 0.001, Fig. 1). The IgG response to ESAT-6 and CFP-10 was lower after 6 months of successful anti-TB chemotherapy. Our results indicate that patients with pulmonary TB have strong responses to ESAT-6 and CFP-10 in the active phase of the disease and even after the initiation of treatment. In contrast, IgG levels against 16kDa antigen peaked after 1–3 months of treatment (P < 0.01 versus active TB group), suggesting different humoral responses to Mtb cytosolic (16kDa) and secreted (ESAT-6 and CFP-10) antigens following initiation of anti-TB chemotherapy. Having observed elevated levels of IgG antibodies against ESAT-6 and CFP-10 in serum of patients with active TB, we further determined the IgG subclasses involved. The results in Fig. 2 show that serum levels of ESAT-6- and CFP-10-specific IgG1 antibodies were higher than IgG2 (P < 0.001 for ESAT-6 and P < 0.05 for CFP-10) in active TB. Lower levels of IgG4 against ESAT-6 and CFP-10 were detected when compared with levels observed for IgG1 and IgG2 (P < 0.001). The production of IgG4 was, however, higher than that observed in the HC in case of ESAT-6 (Fig. 2). Levels of IgG3 anti-ESAT-6 antibodies were very low and did not differ between the studied groups (data not shown). Figure 3 shows that serum IgM antibodies to ESAT-6 were significantly lower in comparison to ESAT-6/ CFP-10 (P < 0.01) for TB patients and control. IgG response to ESAT-6/CFP-10 compared with ESAT-6 alone was not significantly different but was always greater than IgM responses. ROC analysis (Fig. 4) using data from the active TB group showed that the values of the area under the curve (AUC) for IgG responses to ESAT-6 [AUC = 0.946, 95% CI: 0.865– 0.985] and CFP-10 (AUC = 0.942, 95% CI: 0.859–0.983) were significantly higher than the AUC value for the 16kDa antigen (AUC = 0.809, 95% CI: 0.699–0.893, P < 0.05). There was no significant difference between ESAT-6 and CFP-10 (P > 0.05). The evaluation of sensitivity and specificity for the ROC curve showed, at the optimal cutoff point (located nearest to the left upper corner of the ROC curve Cartesian space), that ESAT-6, CFP-10 and 16kDa antigenspecific IgG levels presented sensitivities of 85, 94 and 61%, respectively, and specificities of 89, 87 and 89%, respectively. In terms of likelihood ratios, values were, respectively, 7.73, 7.23 and 5.55 for the positive likelihood ratio and 0.17, 0.07 and 0.44 for the negative likelihood ratio (Table 1). These results confirm the better performance of the antigens ESAT-6 and CFP-10 in comparison to the antigen 16kDa. Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 Polystyrene 96-well microplates were coated overnight with 100 ll per well of ESAT-6, CFP-10, ESAT-6/CFP-10 and 16kDa antigens (2 lg ml 1) diluted in 0.06 M carbonate buffer (pH 9.6) solution and then washed with PBS containing 0.05% Tween 20 (PBS-T). Serum samples, previously collected (from consecutive patients within the recruitment period) and stored at 20°C, were thawed and added in duplicates (100 ll per well) and were diluted 1:20 in PBS-T containing 10% normal goat serum. Hundred microliters of PBS-T was used as negative control. After 1 h incubation at 37°C, plates were washed and goat anti-human IgG (1:1000) (Invitrogen Corporation, Carlsbad, CA, USA) or mouse antihuman IgG1 (1:1000), anti-IgG2 and anti-IgG4 (1:2000) (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) conjugated with HRP were added. Plates were incubated at 37°C for 1 h and washed, and a substrate solution containing 0.5 mg ml 1 ortho-phenylenediamine in sodium citrate buffer, pH 5.0, and 0.03% H2O2 was used. The reaction was stopped with 2N H2SO4 and the optical density measured at 492 nm (Spectramax-190, Molecular Devices, Sunnyvale, CA, USA). The results were expressed by ELISA index (EI), calculated by the formula EI = S/(B + 3 SD), where S is the average optical density value of the duplicate test samples and B corresponds to the average optical density value of the duplicate negative controls plus three times the SD. ating characteristic (ROC) curve was used for analysis of the accuracy values: area under the ROC curve, sensitivity, specificity and likelihood ratios were obtained using MedCalc Statistical (Version 5.00.020, Brussels, Belgium). Statistical significance was set at 5% (P < 0.05). 778 IgG and cytokine responses in human TB Fig. 2. Levels of serum IgG1, IgG2 and IgG4 against ESAT-6 (A) and CFP-10 (B) in patients with pulmonary TB. TB = patients having received no >15 days anti-TB chemotherapy (n = 33) and HC (n = 38). Bars represent the median. Fig. 3. Levels of serum IgM (A) and IgG (B) against ESAT-6/CFP-10 and ESAT-6 in patients with pulmonary TB and HC. 0M = patients having received no >15 days anti-TB chemotherapy (n = 33 for ESAT-6/CFP-10, n = 10 for ESAT-6), 6M = patients after completion of 6 months of treatment (n = 22 for ESAT-6/CFP-10, n = 10 for ESAT-6) and HC (n = 38 for ESAT-6/CFP-10, n = 10 for ESAT-6). Each bar represents mean 6 SEM. *p < 0.05. Production of cytokines in response to the fusion protein ESAT-6/CFP-10 The fusion protein ESAT-6/CFP-10 was used to induce the production of cytokines by PBMC from TB patients. IFN-c, TNF-a, IL-2, IL-6, IL-10 and IL-4 production were evaluated in supernatants of PBMC cultures using the CBA method. Figure 5 shows that the ESAT-6/CFP-10 protein was able to stimulate a higher production of IFN-c, TNF-a, IL-2 and IL-6 from PBMC of patients with active TB in comparison with the response detected in the control group (P < 0.05). Patients who completed the anti-TB treatment had a marked reduction in the production of IFN-c, TNF-a and IL-6, showing levels of these cytokines similar to those observed in HC. The purified protein derivative (PPD) skin test (RT-23 SSI 2TU) status of this control group was evaluated using a 10-mm cutoff (38% PPD+ and 62% PPD ). Similar results were obtained for the PPD and PPD+ groups (data not shown) because of this they were grouped together. The groups studied did not differ in the production of IL-10 and IL-4 after ESAT-6/CFP-10 stimulation (Fig. 5). In addition, although the TB and control groups Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 Fig. 1. Levels of serum IgG against ESAT-6 (A), CFP-10 (B) and 16kDa (C) in patients with pulmonary TB. 0M = patients having received no >15 days anti-TB chemotherapy (n = 33), 1–3M = patients with 1–3 months of treatment (n = 47), 6M = patients after completion of treatment (n = 22) and HC (n = 38). Bars represent the median. *p < 0.05. IgG and cytokine responses in human TB 779 Table 1. Sensitivity and specificity of the ESAT-6, CFP-10 and 16kDa antigens in the serodiagnosis of pulmonary TB ESAT-6 CFP-10 16kDa Cutoffa Sensitivityb (%) Specificityb (%) +LRc 3.399 3.691 3.000 85 94 61 89 87 89 8.06 7.14 5.76 LRc 0.17 0.07 0.44 a The cutoff, sensitivity and specificity data were determined based on the analysis of ROC curves. The values of sensitivity and specificity presented were those corresponding to the point of the ROC curve nearer to the point of sensitivity and specificity equal to 100%. c +LR and LR = positive and negative likelihood ratio (LR). b were of different mean ages, which could possibly affect cytokine production, no correlation was detected between age and either cytokine production or IgG serum levels when the Spearman rank correlation coefficient was used (data not shown). Discussion The most important results obtained from this study indicate the following: (i) patients with active TB present high serum levels of IgG1 antibodies against ESAT-6, CFP-10 and 16kDa; (ii) ROC analysis showed that IgG levels against ESAT-6 and CFP-10 antigens presented higher accuracy in discriminating individuals infected with M. tuberculosis than the 16kDa antigen; (iii) high levels of IFN-c and IL-6 were produced by PBMC of patients with active TB after stimulation with ESAT-6/CFP-10 and (iv) levels of IgG1 antibodies and IFN-c, TNF-a and IL-6 production in response to ESAT6/CFP-10 were lower in the treated group. Several studies have detected antibodies in sera of patients with active TB against a variety of M. tuberculosis antigens (8, 27, 28). In this study, patients with active disease presented elevated levels of IgG antibodies against ESAT-6 and CFP-10 compared with the BCG-vaccinated HC group. Analysis of the ROC curve showed sensitivities of 85 and 94% and specificities of 89 and 87% for ESAT-6 and CFP-10, respectively. In contrast to our results, Greenaway et al. (29), while evaluating patients with active TB in Gambia, detected anti-ESAT-6 and anti-CFP-10 IgG antibodies with corresponding sensitivities of 67 and 63% and specificities of 51 and 55%, respectively. This variability in sensitivity and specificity may be related to the level of exposure of the population to a pathogen. Hoff et al. (30) noticed that individuals in endemic areas for TB, such as Ethiopia, presented higher levels of M. tuberculosis-specific antibodies compared with individuals in non-endemic areas, such as Denmark, and areas of intermediate endemicity, such as Brazil. These results suggest that serological tests using ESAT-6 and CFP-10 may be useful in diagnosing TB in non-endemic areas or in areas of low endemicity, such as observed in Juiz de Fora, Brazil. The 16kDa is a cytosolic protein of the M tuberculosis complex (17). Its specificity in serological tests has been high enough to qualify 16kDa as a strong candidate for TB diagnosis (17, 31). However, it is produced mainly during the stationary phase of mycobacteria growth (32), which compromises its capacity for diagnosing active disease and favors the diagnosis of latent TB (16). Higher levels of anti-16kDa IgG were observed during the first 3 months of chemotherapy. Elevated levels of antibodies against filtered M. tuberculosis antigens in the first 2 months of chemotherapy have been associated with intense stimulation of the humoral response by antigens released from killed bacteria combined with the disappearance of circulating mycobacterial antigens so that specific antibodies are no longer trapped in immune complexes (33). In this context, large amounts of IgG antibodies against the secreted ESAT-6 and CFP-10 antigens appear to be associated with viable and metabolically active bacilli. We speculate that chemotherapy kills bacteria leading to release of a great amount of cytosolic antigens increasing specific antibody levels to 16kDa antigen after the first term of treatment. In this work, 16kDa had a sensitivity of 61% and a specificity of 89% in the diagnosis of the active TB. In accordance with our findings, Raja et al. (17) observed low sensitivity (62%) and high specificity (100%) when detecting anti-16kDa IgG in sera from children with positive bacilloscopy and culture. The analysis of the ROC curve confirmed the superior performance of ESAT-6 and CFP-10 compared with 16kDa in diagnosing patients with active TB. Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 Fig. 4. ROC curves for ESAT-6 versus CFP-10 (A) and ESAT-6 versus 16kDa (B). 780 IgG and cytokine responses in human TB Little attention has been given to research into the subclasses of antibodies involved in TB (34). In this study, the analysis of IgG subclasses against ESAT-6 and CFP-10 revealed a predominance of IgG1 antibodies in relation to IgG2 and IgG4 in serum of patients with active TB. IgM was also low and detected best with the fusion protein ESAT-6/CFP-10. Our results are consistent with other studies that also observed predominance of IgG1 antibodies and low IgM production in the serum of patients with TB (24, 34). We notice here that the increased IgG1 was coincident with augmented levels of IFN-c and IL-6 detected in PBMC of patients with active TB stimulated with the fusion protein ESAT-6/CFP-10. TNF-a and IL-2 were also higher in patients with active TB but the measurements were near the limit of detection. Harvesting supernatants following 96 h of cell culture seems to be the best time for IFN-c production but may not be the best culture period for the detection of other cytokines, such as TNF-a and IL-2. A longitudinal study with data within the same group demonstrated the decline of IgG1, IFN-c and TNF-a following TB treatment (Supplementary data). These results suggest a possible correlation of IgG1 production with Th1 and inflammatory responses in TB. In favor of this possibility, human antibodies induced by BCG vaccination enhanced IFN-c production and improved bacteria internalization and growth inhibition (35). Hussain et al. (36) showed that opsonization by IgG1 regulated the production of inflammatory cytokines such as TNF-a and IL-6, without altering the production of anti-inflammatory cytokines such as IL-10 in monocytes when they were stimulated by mycobacterial antigens. A positive feedback loop between IgG1 and IL-6 production may occur since PBMC stimulated with pokeweed mitogen produced increased IgG1 when cultured with IL-6, a crucial co-stimulator of T-cell responses (37, 38). In addition, IL-10 knockout mice infected with M. tuberculosis strain Erdman had an increase in Th1-type immunity that did not appear to be capable of enhancing the antimicrobial activity. Long-term expression of the inflammatory properties associated with Th1 cytokines can result in increased pathology associated with lung inflammation (39). On the other hand, a predominant Th1 phenotype in the peripheral blood of TB patients may favor restriction of disease dissemination to other sites. In accordance, active pulmonary TB patients from Juiz de Fora, Brazil, also showed high plasma levels of the IFN-c-induced CXCchemokines CXCL9 and CXCL10 detected by CBA (40). Using a commercial Luminex kit, Djoba Siawaya et al. (41) showed that plasma levels of CXCL10 and IL-6 were significantly higher in active TB patients compared with PPD+ healthy community controls and dropped during therapy. In contrast, no significant differences in levels of cytokines, such as IFN-c and IL-2, were detected before and during TB treatment, which could be associated with a compartmentalized expression of some cytokines (41). Besides the reduction of antibody titers by 6 months of anti-TB chemotherapy, the present study demonstrated that the production of IFN-c, TNF-a, IL-6 and IL-2 that was higher in PBMC cultures from patients with active TB in response to ESAT-6/CFP-10 was lower after chemotherapy. According to Ribeiro-Rodrigues et al. (42), the cells obtained in the sputum of TB patients after anti-TB treatment presented low production of IFN-c and TNF-a that correlates with a reduction in bacillary burden. In addition, number of IFN-c-secreting cells induced by PPD and ESAT-6 increased in the first month of TB chemotherapy and decreased after 3 and 6 months of treatment in children with TB (43). Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 Fig. 5. IFN-c (A), TNF-a (B), IL-6 (C), IL-2 (D), IL-4 (E) and IL-10 (F) production by PBMC stimulated with ESAT-6/CFP-10 (5 lg ml 1) in patients with pulmonary TB. 0M = patients with active TB (n = 10), 6M = patients after completion of 6 months of treatment (n = 10) and HC (n = 13). Each bar represents mean 6 SEM. *p < 0.05. IgG and cytokine responses in human TB 781 Supplementary data Supplementary Figures 1 and 2 are available at International Immunology Online. Funding Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq; 310912/2006-7, 305941/2009-7); Fundacxão de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG; CBB PPM 0247/08); Coordenac xão de Aperfeic xoamento de Pessoal de Nı́vel Superior (CAPES; Procad 642/2008, 720/ 2009), Brazil. Acknowledgements We are grateful for the co-operation of the staff of the Respiratory Diseases Division of the Central Public Health Clinic of Juiz de Fora. Conflict of Interest: The authors have no conflicting financial interests. References 1 WHO. 2009. Global tuberculosis control: a short update to the 2009 report. WHO Press, Geneve. 2 Pilheu, J. A. 1998. Tuberculosis 2000: problems and solutions. Int. J. Tuberc. Lung Dis. 2:696. 3 American Thoracic Society, Center for Disease Control and Prevention. 2000. Diagnostic standards and classification of tuberculosis in adults and children. Am. J. Respir. Crit. Care Med. 161:1376. 4 Teixeira, H. C., Abramo, C. and Munk, M. E. 2007. Immunological diagnosis of tuberculosis: problems and strategies for success. J. Bras. Pneumol. 33:323. 5 Steingart, K. R., Ramsay, A. and Pai, M. 2007. Commercial serological tests for the diagnosis of tuberculosis: do they work? Future Microbiol. 2:355. 6 Abebe, F., Holm-Hansen, C., Wiker, H. G. and Bjune, G. 2007. Progress in serodiagnosis of Mycobacterium tuberculosis infection. Scand. J. Immunol. 66:176. Downloaded from http://intimm.oxfordjournals.org/ at Universidade Federal de JuÃ-z de Fora on May 6, 2013 High levels of IL-10 and IL-4 have been detected in patients with severe TB (44). In this study, levels of IL-10 and IL-4 were low in patients with active TB in response to the ESAT-6/CFP-10 fusion protein. We suggest that the low IL-4 and IL-10 response in this study may be associated with a mild form of the disease in the group of TB patients studied. Low IL-4 and IL-10 production may favor the dominant Th1 phenotype described here since IL-10 can indirectly inhibit the polarization toward Th1 by repressing the secretion of IL-12 by activated macrophages, and IL-4 represses the expression of the IL-12 receptor on developing cells, skewing them toward Th2 cells (39, 45). Moreover, it was described that IFN-c, a hallmark of Th1 cells, directly down-regulates IL-4 expression in human T lymphocytes via interferon regulatory factor (IRF)-1 and IRF-2 (46). Herein, a Th1 phenotype has been shown to be dominant in the peripheral blood of patients with active pulmonary TB and to be reduced after chemotherapy. 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