T-Cell Responses to the Mycobacterium tuberculosis-specific Antigen ESAT-6 in Brazilian Tuberculosis Patients

Transcription

1 INFECTION AND IMMUNITY, Dec. 2002, p Vol. 70, No /02/$ DOI: /IAI Copyright 2002, American Society for Microbiology. All Rights Reserved. T-Cell Responses to the Mycobacterium tuberculosis-specific Antigen ESAT-6 in Brazilian Tuberculosis Patients Fernando L. L. Cardoso, 1,2 Paulo R. Z. Antas, 1 Alexandre S. Milagres, 3 Annemieke Geluk, 4 Kees L. M. C. Franken, 4 Eliane B. Oliveira, 1 Henrique C. Teixeira, 5 Susie A. Nogueira, 2 Euzenir N. Sarno, 1 Paul Klatser, 6 Tom H. M. Ottenhoff, 4 and Elizabeth P. Sampaio 1 * Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ, 1 and Department of Infectious and Parasitic Diseases, Hospital Universitário Clementino Fraga Filho, Federal University of Rio de Janeiro, 2 and Department of Lung Diseases, Hospital Municipal Raphael de Paula e Souza, 3 Rio de Janeiro, and Laboratory of Immunology, Federal University of Juiz de Fora, Juiz de Fora, 5 Brazil, and Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, 4 and Department of Biomedical Research, Royal Tropical Institute, Amsterdam, 6 The Netherlands Received 28 January 2002/Returned for modification 3 April 2002/Accepted 11 September 2002 The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN- ), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN- secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-tb controls. Nevertheless, the levels of IFN- in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN- in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN- production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity. Tuberculosis (TB) remains a major public health problem in the 21st century. A third of the world s population is infected with Mycobacterium tuberculosis, and 5 to 10% of the infected population will develop the disease during their lifetime. TB is responsible for more than 2 million deaths per year worldwide (9). The situation is exacerbated by coinfections with human immunodeficiency virus (HIV) and the emergence of multidrug-resistant strains of M. tuberculosis. The Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine available against TB, and yet its efficacy is controversial. BCG has shown extremely variable levels of protection in different populations (12), ranging from 0% (38) in the study of Chingleput, South India, to 80% in The British MRC BCG Trial, Great Britain (37). A cell-mediated immune (CMI) response of the Th1 type, characterized by elevated production of gamma interferon (IFN- ) and interleukin-12 (IL-12), is essential to mount a protective immunity against M. tuberculosis (8, 13, 26). Besides, a basic principle for selecting novel antigen candidates for subunit vaccine design is their capacity to induce a protective Th1 response. The definition of new antigens to be applied in an immune-based diagnostic assay for early detection of TB also has a high priority. Purified protein * Corresponding author. Mailing address: Leprosy Laboratory, Oswaldo Cruz Institute, FIOCRUZ-Av. Brasil, 4365 Manguinhos, Rio de Janeiro, Brazil Phone: Fax: (021) esampaio@gene.dbbm.fiocruz.br. derivative (PPD) from M. tuberculosis is a mixture of complex antigens that has been long used as a skin test for TB diagnosis. The tuberculin skin test (TST) is technically simple, but it lacks diagnostic specificity in BCG-vaccinated individuals. ESAT-6, the early secreted antigenic target, is a low-molecular-weight protein essentially present in pathogenic mycobacteria (17, 22), including members of the mycobacterium complex (M. tuberculosis, M. bovis, and Mycobacterium africanum) and Mycobacterium leprae. The ESAT-6 gene is located within the RD1 region (region of differences), a chromosome region present mainly in pathogenic mycobacteria and deleted from all M. bovis BCG strains examined so far (17, 22). In previous studies, analysis of T-cell responses to M. tuberculosis ESAT-6 showed a range of recognition of between 35 and 92% (3, 24, 25, 29, 30, 34, 40). The majority of TB patients were able to recognize ESAT-6, as estimated by the induction of IFN-, while healthy unrelated controls (from regions of low TB endemicity) did not (3, 24, 25, 29, 30, 34, 40). Consequently, the possible use of ESAT-6 as a marker of M. tuberculosis infection has been proposed. In addition, Pais and colleagues (27) demonstrated that in memory-immune mice, protective T-cell responses against M. tuberculosis were associated with a very high frequency of T cells directed to ESAT-6. Antigen 85B (Ag85B) is a secreted mycobacterial protein that belongs to the Ag85 complex, a major group of related proteins that can be detected in large quantities in cell wall extracts and in culture filtrate protein (CFP) preparations from 6707

2 6708 CARDOSO ET AL. INFECT. IMMUN. TABLE 1. Clinical characteristics of TB patients and controls in this study Group (n) Cavitary No. with TB Noncavitary Mean SD age (yr) Pleural TB (10) Pulmonary TB Untreated (26) Treated (24) Controls (20) a a The control group represents healthy donors with a negative TST (n 13) and patients with pulmonary disorders other than TB (n 7). mycobacteria (1, 43). Such secreted antigens are likely to play a vital role in the induction of protective immunity (2, 6, 39). Indeed, Ag85B induces strong T-cell proliferation and IFN- secretion in most healthy individuals exposed to M. tuberculosis and in BCG-vaccinated donors (20). Moreover, Ag85B DNA vaccine (39) and subunit vaccines expressing Ag85B and ESAT-6 (6) were able to induce protection in a murine model of TB, and coimmunization with these proteins resulted in a greater degree of protection (18), suggesting that multisubunit vaccination may represent a promising strategy against TB. For the development of new vaccines and diagnostic reagents, there is an urgent need for assessment of immune responses to M. tuberculosis antigens in areas of TB endemicity. In the present study, T-cell responses to recombinant ESAT-6 (resat-6), 85Ag, and PPD were therefore investigated in Brazilian TB patients and controls from the city of Rio de Janeiro, an area of high endemicity for TB and leprosy. IFN- release in vitro was correlated to clinical groups, pleural and pulmonary TB, and the stage of TB treatment and compared to the responses to Ag85 and PPD in the same population. Furthermore, the possible influence of environmental mycobacterial exposure on the T-cell response to M. tuberculosis ESAT-6 is discussed. MATERIALS AND METHODS Population studied. Patients with pulmonary (n 50) and pleural (n 10) TB diagnosed at the outpatient unit of a district hospital, Raphael de Paula e Sousa, Rio de Janeiro, Brazil, were enrolled into the study. The criteria for TB diagnosis were in accordance with those of the World Health Organization and the Ministry of Health, Brazil. Pulmonary TB diagnosis was defined by at least one sputum-positive smear by Ziehl-Neelsen staining or a sputum culture positive for M. tuberculosis, respiratory symptoms for 4weeks, and suggestive lesions on the chest X ray. Patients with pulmonary TB (35 males and 15 females) were categorized into the following groups: (i) according to the presence of lung cavitation (n 18) at radiographic examination and (ii) according to the anti-tb chemotherapy phase as untreated ( 7 days of chemotherapy; n 26) or treated ( 30 days of chemotherapy; n 24). Anti-TB chemotherapy regimens were performed in accordance with the recommendations of the Ministry of Health, Brazil. Diagnosis of pleural TB (eight males and two females) was defined by histopathological examination of pleural biopsy compatible with granuloma formation, positive Ziehl-Nielsen staining, or culture isolation of M. tuberculosis from pleural effusion or a pleural biopsy fragment, suggestive clinical symptoms, and clinical response to TB treatment. The clinical characteristics of all groups are shown in Table 1. The control group (n 20; 12 males and 8 females) comprised individuals with other pulmonary non-tb diseases (n 7) and no history of TB in the past, including pneumonia (n 2), chronic obstructive pulmonary disease (n 2), asthma (n 2), or silicosis (n 1), and healthy donors (n 13) with negative TST (skin induration, 4 mm). Six healthy individuals (all males) with a strong positive TST (Mantoux test) ( 10 mm) and no history of TB and 14 leprosy patients (Leprosy Laboratory, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) were also evaluated (32). BCG vaccination status (presence of BCG vaccination scar) was checked in all individuals. None of the TB patients needed hospital admission to receive treatment or had clinical evidence of anti-tb chemotherapy failure or miliary TB. Pregnant or breastfeeding women and individuals with comorbidity, such as diabetes and cancer, were not included. All enrolled TB patients tested negative by enzyme-linked immunosorbent assay (ELISA) for HIV. Ethics committees. All patients gave permission for blood sampling after written consent, and the Ethics Committees of Hospital Raphael de Paula Sousa and FIOCRUZ approved the research protocol. Mycobacterial antigens. M. tuberculosis resat-6 and 85B antigens were produced as described previously (14). Preparations were kept frozen in phosphatebuffered saline (PBS) at 20 C until use. Proteins generated from the vector were used as a negative control. PPD RT23 was obtained from the Statens Serum Institute (Copenhagen, Denmark). resat-6 and PPD were used in the in vitro assays at a final concentration of 5 g/ml, and Ag85 was used at a final concentration of 2.5 g/ml. Cell culture and stimulation. After written consent, heparinized venous blood was obtained from all patients and controls, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N.J.) density centrifugation. A total of PBMCs per well were plated in 96-well round-bottom plates (Costar Corporation, Cambridge, Mass.) in 200 l of RPMI 1640 medium supplemented with 20% autologous plasma, 100 U of penicillin per ml, 100 g of streptomycin per ml, and 2 mm L-glutamine (Gibco BRL) and incubated at 37 C in a humidified 5% CO 2 incubator. Antigens or mitogen were added to the wells in duplicate, and after 5 days, supernatants were recovered, pooled, and kept frozen ( 20 C) until use. Control wells comprised cells cultured in medium alone. IFN- detection by ELISA. The concentration of IFN- in cell-free culture supernatants was determined by using a commercial ELISA with specific pairs of monoclonal antibodies (MAbs) (Pharmingen, Inc., San Diego, Calif.) according to the manufacturer s specifications. Cytokine levels are expressed as picograms of protein per milliliter, and the detection limit of the assay was 8 pg/ml. IFN- levels in control cultures were mostly undetectable unless otherwise indicated. Cytokine values in the experimental wells are expressed subtracted from the values obtained in the control wells. The levels of the in vitro response obtained from the PPD-negative healthy donors and those of the patients with pulmonary disorders other than TB were very similar and were pooled together as the control group. Intracellular cytokine staining. For intracellular cytokine detection, PBMCs were seeded (10 6 cells per ml) into 24-well plates (Costar), incubated in the presence or absence of antigens (PPD or resat-6) for 16 h when anti-hcd28 MAb at 3 g/ml (Becton & Dickinson Co., Mountain View, Calif.) was added to the wells 6 h prior to the end of the incubation period, and 1 h later, brefeldin A (10 g/ml) (Sigma Chemical Company, St. Louis, Mo.) was added to the same cultures (23). After the incubation period, the cells were harvested, resuspended in PBS-fluorescence-activated cell-sorting (FACS) buffer, and labeled with fluorescein isothiocyanate (FITC)-conjugated anti-hcd4 or anti-hcd8 antibody. For detection of intracytoplasmatic IFN-, the cells were fixed in 4% paraformaldehyde (PFA), washed in PBS, and permeabilized with Hanks balanced salt solution (HBSS) plus 0.1% saponin and 0.05% sodium azide (HBSS/SAP). Cells were then incubated with phycoerythrin (PE)-conjugated anti-hifn- or the isotype control immunoglobulin G1 (IgG1) MAb (Pharmingen) for 30 min in HBSS/SAP at room temperature. After being washed in PBS, cells were fixed in 1% PFA for immediate analysis. Labeled cells were run as described above, and a total of 50,000 events in the lymphocyte region were recorded for each sample. Thresholds for positivity were set by using the irrelevant isotype IgG1 antibody (negative control) as reference. All data were expressed as a percentage of cells double stained for IFN- and for CD4 or CD8 T-cell markers. Statistical analysis. Results are reported as means standard errors, and differences between responses from TB patient groups and controls were analyzed with nonparametric Kruskall-Wallis and Mann-Whitney tests (EpiInfo 6, version 6.04d, Database and Statistics Program for Public Health) whenever appropriate. The level of statistical significance adopted was P RESULTS IFN- production in response to M. tuberculosis resat-6 is enhanced in Brazilian TB patients compared to that in controls. Production of IFN- in response to resat-6 was evaluated in 60 TB patients. The specificity of the antigen response

3 VOL. 70, 2002 T-CELL RESPONSE TO resat-6 IN BRAZILIAN TB PATIENTS 6709 TABLE 2. Rate of positive antigen-induced IFN- response to ESAT-6, PPD, and Ag85B Group (n) a % with positive response b % with BCG vaccination scar ESAT-6 PPD Ag85 Pulmonary TB (50) Pleural TB (10) Controls c (20) NA d Healthy controls TST positive (6) TST negative (10) BCG positive (44) a Total number of individuals assayed. b Positive antigen-induced IFN- response ( 100 pg/ml). c Non-TB patients (n 7) and TST-negative healthy donors (n 13). d NA, not available. to ESAT was evaluated and compared to that of the control group (healthy individuals and non-tb pulmonary patients; n 20). A total of 60% of all TB patients (36 individuals) and 60% of controls responded to the recombinant antigen (IFN-, 100 pg/ml) in vitro (Table 2). Although no major differences were noted in the percentage of responders among the groups (Table 2), comparison of IFN- levels showed a significant difference (P 0.01) between the treated TB patients (mean, 1,241 pg/ml; 25th to 75th percentile, 499 to 1,906 pg/ml) and the controls (mean, 491 pg/ml; 25th to 75th percentile, 354 to 586 pg/ml), but not versus the untreated patients (mean, 1,124 pg/ml; 25th to 75th percentile, 301 to 2,291 pg/ml; P 0.2) (Fig. 1A). Patients, independent of treatment stage (Table 1), were further categorized as having pleural (n 10) or pulmonary TB with the presence (n 18) or absence (n 32) of lung cavities on chest X-ray examination. As shown in Fig. 1B, the response to ESAT-6 in pleural TB patients (mean IFN- level, 1,279 pg/ml; 25th to 75th percentile, 417 to 2,190 pg/ml) was significantly elevated compared to that of controls (P 0.03). In addition, pulmonary TB with lung cavity presented higher IFN- levels (mean, 1,498 pg/ml; 25th to 75th percentile, 582 to 2,291 pg/ml; P 0.02), whereas no significant differences (P 0.2) were observed in the response of noncavitary pulmonary TB (mean, 975 pg/ml) compared to that of controls (Fig. 1B). In order to determine further whether ESAT-6 discriminates the response among different groups of controls from an area of TB and leprosy endemicity, six healthy individuals with a strong positive TST and 14 leprosy patients were also assayed in vitro (Fig. 1C). IFN- values measured in 5-day culture supernatants in response to resat-6 showed in the former group the highest IFN- production (mean, 1,553 pg/ml; 25th to 75th percentile, 1,128 to 1,529 pg/ml). For the leprosy patients, seven individuals (50%) demonstrated positive responses (mean, 426 pg/ml; 25th to 75th percentile, 158 to 626 pg/ml), which were not significantly different (P 0.05) from the values released by healthy controls with a negative TST (n 10; mean IFN- level, 381 pg/ml; 25th to 75th percentile, 154 to 518 pg/ml). Accordingly, the response of TST-positive controls (n 6) was significantly enhanced (Fig. 1C) compared to FIG. 1. Evaluation of IFN- production in response to resat-6 antigen in vitro. (A) PBMCs obtained from Brazilian TB patients with either untreated (n 26) or treated (n 24) pulmonary TB and controls (n 20) were cultured in the presence of ESAT-6 (5 g/ml), and after 5 days, supernatants were harvested and assayed by ELISA. (B) Cells obtained from pleural TB patients (n 10) and from pulmonary TB patients with (n 18) and without (n 32) lung cavity versus controls (n 20) were also assayed in vitro in response to ESAT as described above. (C) The responses of healthy controls (HC) who presented a positive ( 10 mm induration, n 6) or negative ( 4 mm induration, n 10) TST and the responses of leprosy patients (n 14) were evaluated in vitro after PBMC stimulation with ESAT-6 and compared to those of TB patients. Results are mean picograms of protein per milliliter standard error for each group of individuals with positive responses (IFN-, 100pg/ml) to the antigen. PBMC responses to recombinant protein controls (vector controls) were mostly below 30 pg/ml., significant difference compared to controls or to leprosy patients;, significant difference compared to TST-negative healthy controls. Differences in response between TB patients and TST-negative healthy controls were marginally significant (P 0.05). the results from the 10 TST-negative healthy donors and from the leprosy patients as well (P and 0.002, respectively). Moreover, IFN- release in response to ESAT-6 in all TB patients (n 60) was elevated compared to those in leprosy patients (P 0.03) and TST-negative individuals (P 0.05). IFN- production in response to PPD and Ag85B in TB

4 6710 CARDOSO ET AL. INFECT. IMMUN. patients. In order to determine whether PPD and Ag85B could discriminate the groups mentioned above, PBMCs were stimulated in vitro, and supernatants were assayed for IFN- by ELISA. Following PPD stimulation, levels of IFN- were not different in any of the groups studied, even compared to those in the controls (Fig. 2A). PBMCs isolated from patients with pulmonary (mean IFN- level, 1,564 pg/ml) and pleural (1,803 pg/ml) TB demonstrated similar IFN- values (Fig. 2A), as did those from patients with a lung cavity (1,558 pg/ml) or without a lung cavity (1,450 pg/ml) in the treated and untreated TB groups (data not shown). The six healthy TST-positive donors showed the highest IFN- response (mean, 2,192 pg/ml; 25th to 75th percentile, 1,629 to 2,756 pg/ml), which was significantly elevated compared to the results from 20 controls (1,185 pg/ml; 25th to 75th percentile, 523 to 1,791 pg/ml; P 0.05) and from the 10 healthy TST-negative individuals (782 pg/ml; 25th to 75th percentile, 345 to 743 pg/ml; P 0.01) (Fig. 3A). In all groups, PPD induced higher IFN- levels than were induced by ESAT-6. However, only in the controls (n 20) were the amounts of IFN- released in response to ESAT-6 (mean, 491 pg/ml) and PPD (mean, 1,254 pg/ml) significantly different (P 0.03). The T-cell response to Ag85B (Fig. 2B) was also assessed in the PBMCs from 32 TB patients, representing 14 untreated TB patients (mean IFN- level, 503 pg/ml; 25th to 75th percentile, 263 to 804 pg/ml), 18 treated TB patients (mean, 891 pg/ml; 25th to 75th percentile, 395 to 1,454 pg/ml), and 17 controls (mean, 614 pg/ml; 25th to 75th percentile, 331 to 840 pg/ml). In contrast to the response to ESAT-6, no major differences in IFN- production among the groups were noted. When the T-cell response to the mycobacterial antigens was evaluated in the TST-positive versus TST-negative healthy donors (Fig. 2C), the IFN- values were statistically different (P 0.05) from those for all antigens tested (ESAT-6, PPD, and Ag85B) (Fig. 2C). A high rate of responders to PPD and Ag85B was evident in all groups (Table 2). All patients with pleural TB (100%) released IFN- in response to PPD in the cultures, as did 85% of the controls. For Ag85B, the rates were 88 and 90%, respectively. More interestingly, among the 10 TST-negative donors, only 1 presented a negative PPD response in vitro (Table 2). Correlation of BCG vaccination and T-cell response to M. tuberculosis antigens in vitro. The relationship between the in vitro immune response and BCG vaccination was assessed by scoring the presence of the BCG immunization scar in all individuals enrolled in this study. The percentages of individuals with a BCG scar were similar in patients with pleural TB (60%), pulmonary TB with lung cavity (72%), and pulmonary TB without lung cavity (78%). As expected, in the negative TST control group (n 10), only 5 individuals presented a BCG scar. Evaluation of IFN- production by PBMCs in response to PPD in those individuals showed only 1 patient (not BCG vaccinated) out of the 10 who did not respond to PPD in vitro. Positive responses to ESAT-6 were detected in 59% of the TB patients with a BCG scar, while 100% of those patients responded to PPD in vitro (Table 2). Moreover, from the TST-negative healthy control group without BCG vaccination, four of the five individuals responded to ESAT-6. When ana- FIG. 2. T-cell responses of TB patients and controls to mycobacterial antigens. (A) Secretion of IFN- by PBMCs was assessed after in vitro stimulation with PPD, when after 5 days, supernatants were harvested and assayed by ELISA. The values shown are mean IFN- level standard error for all groups studied: pleural TB (n 10), pulmonary TB (n 50), TST-positive healthy controls (HC; n 6), and TST-negative healthy controls (n 10). No major differences were found in the IFN- levels among the groups. (B) The PBMC response to the 85B antigen (2.5 g/ml) was also evaluated in TB patients (treated TB, n 18; untreated TB, n 14) and in the control group (healthy TST-negative donors and non-tb patients; n 17). Horizontal bars represent mean IFN- values that were not significantly different among the groups; (C) The in vitro T-cell responses to mycobacterial antigens of the TST-positive healthy controls and the TSTnegative donors were compared. Following in vitro stimulation of PBMCs with either ESAT-6 (5 g/ml), PPD (5 g/ml), or Ag85B (2.5 g/ml), culture supernatants were assayed by ELISA. A total of 6 TST-positive healthy controls and 10 TST-negative healthy controls were evaluated. The values shown represent the mean level of IFN- standard error and were significantly higher in the former group for all the antigens tested (, P 0.05). All results are expressed as already subtracted from the values obtained in the unstimulated cultures. PBMC responses to recombinant protein controls were mostly below 30 pg/ml.

5 VOL. 70, 2002 T-CELL RESPONSE TO resat-6 IN BRAZILIAN TB PATIENTS 6711 FIG. 3. Intracellular flow cytometric detection of IFN- production in CD4 and CD8 T cells. PBMCs obtained from TB patients were stimulated with ESAT-6 (5 g/ml) and anti-cd28 antibody as described in Materials and Methods. After the culture period, cells were harvested and evaluated by flow cytometry. Cells were acquired in the gated lymphocyte population, and the quadrants were set up with an isotype-matched control. The numbers in the figure represent the percentage of positive cells in each quadrant. The results are presented as already subtracted from the values obtained in nonstimulated control cultures with anti-cd28 and brefeldin A treatments. Data from one representative experiment are shown. lyzing the immune response induced by the Ag85B, 78% of the BCG-vaccinated and 87% of the non-bcg-vaccinated TB patients were responsive in vitro. Therefore, BCG vaccination did not seem to interfere with the T-cell response to ESAT-6, according to a previous study (31), while the opposite occurred in response to PPD, because all vaccinated TB patients and vaccinated healthy controls responded to PPD in vitro, in line with the fact that ESAT-6 is not present in the M. bovis BCG vaccine (17, 22). Detection of intracellular cytokine staining in CD4 and CD8 T cells in response to ESAT-6 in vitro. In order to determine whether CD4 and/or CD8 T cells were able to release IFN- in response to ESAT-6 and PPD in the TB population, primary PBMCs were stimulated in vitro with the mycobacterial antigens and anti-cd28 MAb, and the cells were processed as described in Materials and Methods. Double-stained cells were then analyzed by flow cytometry. A total of five TB patients were assayed, and the mean T-cell responses to PPD for CD4 and CD8 T cells (percentage of IFN- -positive cells) were 1.82% 0.23% and 1.72% 0.27%, respectively. The responses to ESAT-6 showed, in the primary stimulated PBMCs, a total of 0.28% 0.13% IFN- CD4 double-stained T cells and 0.55% 0.14% IFN- CD8 cells. The results in response to ESAT-6 and PPD are presented as already subtracted from the levels in the nonstimulated control cultures with anti-cd28 and brefeldin A treatments. Figure 3 demonstrates that in one representative experiment, both CD4 (1.4%) and CD8 (0.6%) T-cell subsets produced IFN- in response to ESAT-6, and in anti-cd28 and brefeldin-nonstimulated control cells, the results were 0.3% for CD4 cells and 0.1% for CD8 cells (data not shown). DISCUSSION Since the identification of M. tuberculosis ESAT-6 (35), there has been growing interest in the use of this antigen to diagnose TB. As demonstrated by Ravn et al. (30), the immune systems of TB patients from Ethiopia (35%) and Denmark (56%) were able to recognize ESAT-6, and 58% of the healthy related contacts of TB patients from Ethiopia also produced IFN- in response to the antigen, whereas only 2 out of the 36 BCG-vaccinated and nonvaccinated healthy donors from Denmark were responsive. In the present study, the percentage (on average 60%) was similar to that described in previous studies. However, a high rate of responders (60%) was also found among controls with non-tb contact history. Interestingly, in recent studies from India and The Gambia, two countries with ethnically diverse populations and, like Brazil, having TB and leprosy coendemicity, the rates of non-tb contact responders were 80% (21) and 30% (41), respectively. However, the levels of IFN- in response to ESAT-6 in Brazilian controls were much lower than those in TB patients, enough to differentiate both categories (Fig. 1), while the responses to PPD and Ag85B could not (Fig. 2). Clearly, patients with pleural and cavitary pulmonary TB showed the highest IFN- values in response to both ESAT-6 and PPD. In addition, the response to ESAT-6 was significantly different when comparing leprosy patients to the TB group (Fig. 1C). To our knowledge, this is the first report investigating T-cell responses to ESAT-6 in Brazilians. Brazilians represent an ethnically diverse population compared with those in the previous works, who showed a high rate of responders among controls, including the group with negative TST: on the other hand, the group with a negative TST had significantly lower IFN- values in vitro. All TB patients assessed herein were receiving ambulatory care, and none had clinical evidence of disseminated TB or comorbidity, including AIDS. The trend of higher IFN- release by PBMCs in the treated TB patients compared to the level in controls, as opposed to the untreated TB patients (Fig. 1A), has also been confirmed by others (25, 42). There is

6 6712 CARDOSO ET AL. INFECT. IMMUN. increasing interest in investigating T-cell responses by using the enzyme-linked immunospot (ELISPOT) assay, a very sensible and specific method that detects the frequency of antigenspecific T cells in PBMCs. A group of Brazilian TB patients had their PBMCs tested for ESAT-6 by IFN- ELISPOT, and as expected, the frequencies of ESAT-6-specific T cells were able to discriminate TB-treated patients from controls, but not from the untreated TB group (data not shown), supporting the present data. This shift in the CMI response during chemotherapy may be explained by a number of factors, including sequestration of M. tuberculosis-specific T cells at the site of disease, leading to reduced frequency in peripheral blood in the untreated TB; the release of anti-inflammatory cytokines by PBMCs, particularly IL-10; and the temporary depression of T-cell responsiveness (42). The Th1 response associated with increased lung destruction, like the presence of the lung cavity at chest X-ray examination, was also evaluated. Although, chest computed tomography was not available to exclude the presence of a small cavity, individuals with a lung cavity shown in the chest X-ray had significantly higher IFN- levels than did controls (Fig. 1B). These data could suggest an increased inflammatory response associated with immunopathology. The enhanced IFN- values detected in the T cells from pleural TB patients provide evidence that an increased delayed-type hypersensitivity reaction may be associated with pleurisy formation and self-limited disease (27, 28). On the other hand, Yamamura et al. (44) have already proposed that hypersensitive reactions to M. tuberculosis could play a significant role in the immunopathogenesis of lung cavity formation. Additionally, M. tuberculosis-infected animals treated with azathioprine combined with anti-tb chemotherapy could prevent the formation of lung cavities (44). This is the first study in humans to observe an association between higher IFN- production in patients with pulmonary TB and lung tissue necrosis and cavitation, independent of anti-tb chemotherapy (Table 1). The consequences of such destructive immunopathology might result in bronchogenic dissemination of bacilli to other parts of the lungs, outside environment contamination, hemoptysis, and irreparable limitation of quality of life due to lung destruction and fibrosis. Even more, tumor necrosis factor alpha and IL-10 are two important cytokines that modulate CMI and must play an additional role in this phenomenon (4, 5). A better understanding of the immunoregulatory mechanisms in human TB that are involved in immunopathology is then vital to explain the pathogenesis of mycobacterial infection and to further elaborate immunotherapeutic proposals to control infection and immunopathologic damage. The importance of CD4 T cells in the protective immune response against M. tuberculosis has long been established as being associated with the production of IFN-, which is essential for macrophage activation and control of mycobacterial replication (8, 13, 26). Recent studies have emphasized a role for CD8 T cells in the immune response to TB (15, 19, 33, 34) that seems to require the production of IFN- by such major histocompatibility complex class I-restricted cells. Through intracellular cytokine staining and flow cytometry, the production of IFN- induced by ESAT-6 among both CD4 and CD8 T lymphocytes was evaluated in the patient population. Although the rate of positive cells detected in the periphery through this methodology is low, it may represent a relevant occurrence that corresponds to specific clonally activated circulating antigen-responsive T cells in the patient population. Accordingly, the rate of cells positive to PPD detected by flow cytometry was higher than that of cells positive to ESAT-6, a fact that corroborates the ELISA data. Interestingly, both CD4 and CD8 T cells were able to release IFN- at similar rates, albeit its functional relevance to the pathogenesis of disease still needs to be established. As related to PPD, Converse and colleagues (7) have showed that the PBMC response to PPD in vitro is more sensitive than the TST at detecting T cells primed with mycobacterial antigens, even in HIV-positive individuals. Our results confirm these data, since 90% of the TST-negative healthy donors released IFN- following in vitro stimulation with PPD (Table 2). These results reinforce that the lack of PPD specificity is a major limitation to its use as diagnostic reagent for M. tuberculosis infection in areas of TB endemicity and where environmental mycobacterium exposure is potentially elevated. Exposure to environmental mycobacteria seems to be higher in tropical countries than in countries with a temperate climate. Additionally, exposure occurs not only through the respiratory tract, but also through the digestive tract and minor skin lesions. These multiple forms of mycobacterial exposure permit T cells to be primed for mycobacterial antigens by diverse types of antigen-presenting cells. Subsequently, environmental mycobacteria must cause a potential modulation in the immune response to M. tuberculosis. In the same way, the exposure to environmental mycobacteria and M. leprae in tropical areas could also play a role in the T-cell responses to ESAT-6. Brazil is a country with high leprosy endemicity. In this study, 7 of the 14 leprosy patients tested were able to mount a positive response to ESAT-6 (Fig. 1C). The M. leprae and M. tuberculosis ESAT-6 amino acid sequences were identical in 36% (36). Consequently, T-cell cross-reactive immune responses to both antigens may be a relevant event in areas of leprosy and tuberculosis endemicity and could explain in part the positive immune response to ESAT-6 in healthy controls (16). The specificity of the ESAT response is further indicated by the fact that 100% of the BCG-vaccinated TB patients responded to PPD, while only 59% responded to ESAT-6 (Table 2). Likewise, among the healthy TST-negative donors, 9 of 10 individuals did respond to ESAT. Nevertheless, the responses to ESAT-6 in the TST-positive individuals parallel those of the TB patients and were significantly different from those of the controls and the TST-negative group (Fig. 1C). Thus, it is suggested further that in an area of endemicity, ESAT-6 does not discriminate between latent infection and disease; the same result was found for the TB patient contacts (41). In this scenario, the experience developed with the application of TST (10) can help us to analyze the possible factors that may influence the responses to ESAT-6. In a population from an area of TB endemicity that has also been exposed to other mycobacteria, the specificity of a skin test depends on the criteria used to define a positive result. The utility of TST depends on the prevalence of infection with M. tuberculosis and the relative cross-reactions with nontuberculous mycobacteria. Similar to the interpretation of TST, an in vivo skin test show-

7 VOL. 70, 2002 T-CELL RESPONSE TO resat-6 IN BRAZILIAN TB PATIENTS 6713 ing a strong reaction in response to ESAT-6 could mostly detect individuals who present high T-cell responses, albeit with a decrease in sensitivity compared to that obtained with the in vitro assay. ESAT-6 has been already used as a skin test for diagnosis of mycobacterial infection in animals (11). Finally, the use of ESAT-6 as a skin test reagent to detect active or latent M. tuberculosis infection that is not influenced by BCG vaccination should be investigated. ACKNOWLEDGMENTS We are grateful to P. K. C. Gomes, K. C. Pereira for the graphic work, and E. R. Leite for efficient secretarial help. We also thank K. S. Cunha and the nursing staff of the TB ward (Hospital Municipal Raphael de Paula e Souza) for helping with patient recruitment and sample collection. This work was supported by EEC (INCO-DC), grant no. ERBIC 18CT , The Netherlands Royal Academy of Arts and Sciences, The Netherlands Leprosy Foundation (NLR), CNPq, and FIOCRUZ, Brazil. REFERENCES 1. Andersen, P The T cell response to secreted antigens of M. tuberculosis. Immunobiol. 191: Andersen, P., and I. Heron Specificity of protective memory immune response against Mycobacterium tuberculosis. Infect. Immun. 61: Arend, S. M., P. Andersen, K. E. van Meijaarden, R. L. Skjot, Y. W. Subronto, J. T. van Dissel, and T. H. Ottenhoff Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6kDa protein and culture filtrate protein 10. J. Infect. Dis. 181: Barnes, P. F., J. S. Abrams, S. Lu, P. A. Sieling, T. H. Rea, and R. L. Modlin Patterns of cytokine production by mycobacterium-reactive human T-cell clones. Infect. Immun. 61: Boggdan, C., Y. Vodovotz, and C. Nathan Macrophage deactivation by interleukin 10. J. Exp. Med. 174: Brandt, L., M. Elhay, I. Rosenkrands, E. B. Lindblad, and P. Andersen ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect. Immun. 68: Converse, P. J., S. L. Jones, J. Astemborski, D. Vlahov, and N. M. H. Graham Comparison of tuberculin interferon- assay with the tuberculin skin test in high-risk adults: effect of human immunodeficiency virus infection. J. Infect. Dis. 176: Cooper, A., D. Dalton, T. Stewart, J. Griffin, D. Russel, and I. Orme Disseminated tuberculosis in interferon- gene-disrupted mice. J. Exp. Med. 178: Dolin, P. J., M. C. Raviglione, and A. Kochi Global tuberculosis incidence and mortality during Bull. W. H. O. 72: Edwards, L. B., G. W. Comstock, and C. E. Palmer Contributions of Northern populations to the understanding of tuberculin sensitivity. Arch. Environ. Health 17: Elhay, M. J., T. Oettinger, and P. Andersen Delayed type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect. Immun. 66: Fine, P. E. M Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346: Flynn, J. L., J. Chan, K. J. Triebold, D. K. Dalton, T. A. Stewart, and B. R. Bloom An essential role for interferon in resistance to M. tuberculosis infection. J. Exp. Med. 178: Franken, K. L., H. S. Hiemstra, K. E. van Meijgaarden, Y. Subronto, J. den Hartigh, T. H. Ottenhoff, and J. W. Drijfhout Purification of histagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr. Purif. 18: Geluk, A., K. E. Meijgaarden, K. L. M. C. Franken, J. W. Drijfhout, S. D Souza, A. Necker, K. Huygen, and T. H. Ottenhoff Identification of major epitopes of Mycobacterium tuberculosis Ag85B that are recognized by HLA-A 0201-restricted CD8 T cells in HLA-transgenic mice and humans. J. Immunol. 165: Geluk, A., K. E. van Meijgaarden, K. L. M. C. Franken, Y. W. Subronto, B. Wieles, S. M. Arend, E. P. Sampaio, T. de Boer, W. R. Faber, B. Naafs, and T. H. M. Otthenhoff Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell reactivity with Mycobacterium tuberculosis. Infect. Immun. 70: Harboe, M., T. Oettinger, H. G. Wiker, I. Rosenkrands, and P. Andersen Evidence for occurrence of the ESAT-6 protein in Mycobacterium tuberculosis and virulent Mycobacterium bovis and for its absence in Mycobacterium bovis BCG. Infect. Immun. 64: Kamath, A. T., C. G. Feng, M. MacDonald, H. Briscoe, and W. J. Britton Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect. Immun. 67: Lalvani, A., R. Brookes, R. J. Wilkinson, A. S. Malin, A. A. Pathan, P. Andersen, H. Dockrell, G. Pasvol, and A. V. S. Hill Human cytolytic and interferon -secreting CD8 T lymphocytes specific for Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95: Lalvani, A., P. Nagvenkar, Z. Udwadia, A. A. Pathan, K. A. Wlkinson, J. S. Shastri, K. Ewer, A. V. Hill, A. Mehta, and C. Rodrigues Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J. Infect. Dis. 183: Launois, P., R. DeLeys, M. N. Niang, A. Drowart, M. Andrien, P. Dierckx, J.-L. Cartel, J.-L. Sarthou, J.-P. Van Vooren, and K. Huygen T-cellepitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy. Infect. Immun. 62: Mahairas, G. G., P. J. Sabo, M. J. Hickey, D. C. Singh, and C. K. Stover Molecular analyses of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178: Maino, V. C., and L. J. Picker Identification of functional subsets by flow cytometry: intracellular detection of cytokine expression. Cytometry 34: Mustafa, A. S., H. A. Amoudy, H. G. Wiker, A. T. Abal, P. Ravns, F. Oftung, and P. Andersen Comparison of antigen-specific T cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis. Scand. J. Immunol. 48: Mustafa, A. S., F. Oftung, H. A. Amoudy, N. M. Madi, A. T. Abal, F. Shaban, I. Rosenkrands, and P. Andersen Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 are recognized by antigen-specific human T cell lines. Clin. Infect. Dis. 30(Suppl. 3):S201 S Newport, M. J., C. M. Huxley, S. Huston, C. M. Hawrylowicz, B. A. Oostra, R. Williamson, and M. Levin A mutation in the interferon- receptor gene and susceptibility to mycobacterial infection. N. Engl. J. Med. 335: Pais, T. F., R. A. Silva, R. Smedegaard, R. Appelberg, and P. Andersen Analysis of T cells during delayed-type hypersensitivity to purified protein derivative (PPD) versus challenge with tuberculosis infection. Immunol. 95: Phalen, S. W., and D. N. McMurray T-lymphocyte response in guinea pig model of tuberculous pleuritis. Infect. Immun. 61: Pollock, J., and P. Andersen The potential of the ESAT-6 antigen secreted by virulent mycobacteria for specific diagnosis of tuberculosis. J. Infect. Dis. 175: Ravn, P., A. Demissie, T. Eguale, H. Wondwosson, D. Lein, H. A. Amoudy, A. S. Mustafa, A. K. Jensen, A. Holm, I. Rosenkrands, F. Oftung, J. Olobo, F. von Reyn, and P. Andersen Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis. J. Infect. Dis. 179: Ravn, P., H. Boesen, B. K. Pedersen, and P. Andersen Human T cell responses induced by vaccination with Mycobacterium bovis Bacillus Calmette-Guérin. J. Immunol. 158: Sampaio, E. P., M. O. Moraes, J. A. C. Nery, A. R. Santos, H. J. Matos, and E. N. Sarno Pentoxifylline decreases in vivo and in vitro tumour necrosis factor-alpha (TNF-alpha) production in lepromatous leprosy patients with erythema nodosum leprosum (ENL). Clin. Exp. Immunol. 111: Serbina, N. V., and J. L. Flynn Early emergence of CD8 T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect. Immun. 67: Smith, S. M., M. R. Klein, A. S. Malin, J. Sillah, K. Huygen, P. Andersen, K. P. W. J. McAdam, and H. M. Dockrell Human CD8 T cells specific for Mycobacterium tuberculosis secreted antigens in tuberculosis patients and healthy BCG-vaccinated controls in The Gambia. Infect. Immun. 68: Sorensen, A. L., S. Nagai, G. Houen, P. Andersen, and Å. B. Andersen Purification and characterization of a low-molecular-mass antigen secreted by Mycobacterium tuberculosis. Infect. Immun. 63: Spencer, J. S., M. A. M. Marques, M. C. B. S. Lima, A. P. Junqueira-Kipnis, B. C. Gregory, R. W. Truman, and P. J. Brennan Antigenic specificity of Mycobacterium leprae homologue of ESAT-6. Infect. Immun. 70: Sutherland, I., and V. H. Sprinett Effectiveness of BCG vaccination in England and Wales in Tubercle 68: Tuberculosis Research Centre (ICMR), Chennai Fifteen year follow up of trial of BCG vaccines in south India for tuberculosis prevention. Indian J. Med. Res. 110: Ulmer, J. B., M. A. Liu, D. L. Montgomery, A. M. Yawman, R. R. Deck, C. M. DeWitt, J. Content, and K. Huygen Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination. Vaccine 15: Ulrichs, T., M. E. Munk, H. Mollenkopf, S. Behr-Perst, R. Colangeli, M. L. Genaro, and S. H. E. Kaufmann Differential T cell responses to Mycobacterium tuberculosis ESAT-6 in tuberculosis patients and healthy donors. Eur. J. Immunol. 28:

8 6714 CARDOSO ET AL. INFECT. IMMUN. 41. Vekemans, J., C. Lienhardt, J. S. Sillah, J. G. Wheeler, G. P. Lahai, M. T. Doherty, T. Corrah, P. Andersen, K. P. MacAdam, and A. Marchant Tuberculosis contacts but not patients have higher gamma interferon responses to ESAT-6 than do community controls in The Gambia. Infect. Immun. 69: Wilkinson, R. J., H. M. Vordermeier, K. A. Wilkinson, A. Sjölund, C. Moreno, G. Pasvol, and J. Ivanyi Peptide-specific T cell response to Mycobacterium tuberculosis: clinical spectrum, compartmentalization, and effect of chemotherapy. J. Infect. Dis. 178: Winker, H. G., and M. Harboe The antigen 85 complex: a major secreted product of Mycobacterium tuberculosis. Microbiol. Rev. 56: Yamamura, Y., Y. Ogawa, H. Yamagata, and Y. Yamamura Prevention of tuberculous cavity formation by immunosuppressive drugs. Am. Respir. Dis. 98: Editor: S. H. E. Kaufmann