INFECTION AND IMMUNITY, Dec. 2002, p. 6707–6714 Vol. 70, No.

12
0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.12.6707–6714.2002
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. Sampaio1*
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

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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 produc-
tion 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 multi-
drug-resistant strains of M. tuberculosis. The Mycobacterium
bovis Bacillus Calmette-Guérin (BCG) vaccine is the only vac-
cine available against TB, and yet its efficacy is controversial.
BCG has shown extremely variable levels of protection in dif-
ferent 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) re-
sponse 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. tubercu-
losis (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, Os-
waldo Cruz Institute, FIOCRUZ-Av. Brasil, 4365 Manguinhos, Rio de
Janeiro, Brazil 21045-900. Phone: 55 21 598-4287. Fax: (021) 270-9997.
E-mail: 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-molec-
ular-weight protein essentially present in pathogenic mycobac-
teria (17, 22), including members of the mycobacterium com-
plex (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 en-
demicity) 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) dem-
onstrated that in memory-immune mice, protective T-cell re-
sponses 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
6708 CARDOSO ET AL.
TABLE 1. Clinical characteristics of TB patients and controls in
this study
No. with TB
Group (n) Mean ⫾ SD age (yr)
Cavitary Noncavitary
Pleural TB (10) 31.5 ⫾ 7.7
Pulmonary TB
Untreated (26) 11 15 33.9 ⫾ 13.4
Treated (24) 7 17 32.9 ⫾ 10.5
Controls (20)a 32 ⫾ 14.4
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 re-
agents, there is an urgent need for assessment of immune
responses to M. tuberculosis antigens in areas of TB endemic-
ity. In the present study, T-cell responses to recombinant
ESAT-6 (rESAT-6), 85Ag, and PPD were therefore investi-
gated 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 com-
pared to the responses to Ag85 and PPD in the same popula-
tion. Furthermore, the possible influence of environmental
mycobacterial exposure on the T-cell response to M. tubercu-
losis 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 Min-
istry 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 ⱖ4weeks, and suggestive lesions on the
chest X ray. Patients with pulmonary TB (35 males and 15 females) were cate-
gorized into the following groups: (i) according to the presence of lung cavitation
(n ⫽ 18) at radiographic examination and (ii) according to the anti-TB chemo-
therapy phase as untreated (⬍7 days of chemotherapy; n ⫽ 26) or treated (ⱖ30
days of chemotherapy; n ⫽ 24). Anti-TB chemotherapy regimens were per-
formed 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 for-
mation, 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, ⱕ4 mm). Six healthy
individuals (all males) with a strong positive TST (Mantoux test) (ⱖ10 mm) and
INFECT. IMMUN.
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 breast-
feeding 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 pro-
duced as described previously (14). Preparations were kept frozen in phosphate-
buffered 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 concen-
tration of 2.5 ␮g/ml.
Cell culture and stimulation. After written consent, heparinized venous blood
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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 2 ⫻ 105 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% CO2 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 com-
prised 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 (106 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 flu-
orescein isothiocyanate (FITC)-conjugated anti-hCD4 or anti-hCD8 antibody.
For detection of intracytoplasmatic IFN-␥, the cells were fixed in 4% parafor-
maldehyde (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 ana-
lyzed 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 ⬍ 0.05.
RESULTS
IFN-␥ production in response to M. tuberculosis rESAT-6 is
enhanced in Brazilian TB patients compared to that in con-
trols. Production of IFN-␥ in response to rESAT-6 was eval-
uated in 60 TB patients. The specificity of the antigen response
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
% with positive responseb % with BCG
Group (n)a
ESAT-6 PPD Ag85 vaccination scar

Pulmonary TB (50) 60 95 88 82

Pleural TB (10) 70 100 100 60

c
Controls (20) 60 85 100 NAd

Healthy controls
TST positive (6) 100 100 100 100
TST negative (10) 80 90 90 50

BCG positive (44) 59 100 78 100

a
Total number of individuals assayed.

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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 discrimi-
nates 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 pa-
tients, seven individuals (50%) demonstrated positive re-
sponses (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 con-
trols (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 pul-
monary 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 (ⱕ4 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. ⴱ, significant difference compared to controls or
to leprosy patients; ⴱⴱ, significant difference compared to TST-nega-
tive 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 ⫽ 0.001 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
6710 CARDOSO ET AL. INFECT. IMMUN.

patients. In order to determine whether PPD and Ag85B could

discriminate the groups mentioned above, PBMCs were stim-
ulated 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 signifi-
cantly 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)

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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%, re- FIG. 2. T-cell responses of TB patients and controls to mycobac-
spectively. More interestingly, among the 10 TST-negative do- terial antigens. (A) Secretion of IFN-␥ by PBMCs was assessed after in
nors, only 1 presented a negative PPD response in vitro (Table vitro stimulation with PPD, when after 5 days, supernatants were
2). harvested and assayed by ELISA. The values shown are mean IFN-␥
level ⫾ standard error for all groups studied: pleural TB (n ⫽ 10),
Correlation of BCG vaccination and T-cell response to M. pulmonary TB (n ⫽ 50), TST-positive healthy controls (HC; n ⫽ 6),
tuberculosis antigens in vitro. The relationship between the in and TST-negative healthy controls (n ⫽ 10). No major differences were
vitro immune response and BCG vaccination was assessed by found in the IFN-␥ levels among the groups. (B) The PBMC response
scoring the presence of the BCG immunization scar in all 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
individuals enrolled in this study. The percentages of individ- (healthy TST-negative donors and non-TB patients; n ⫽ 17). Horizon-
uals with a BCG scar were similar in patients with pleural TB tal bars represent mean IFN-␥ values that were not significantly
(60%), pulmonary TB with lung cavity (72%), and pulmonary different among the groups; (C) The in vitro T-cell responses to my-
TB without lung cavity (78%). As expected, in the negative cobacterial antigens of the TST-positive healthy controls and the TST-
TST control group (n ⫽ 10), only 5 individuals presented a negative donors were compared. Following in vitro stimulation of
PBMCs with either ESAT-6 (5 ␮g/ml), PPD (5 ␮g/ml), or Ag85B (2.5
BCG scar. Evaluation of IFN-␥ production by PBMCs in re- ␮g/ml), culture supernatants were assayed by ELISA. A total of 6
sponse to PPD in those individuals showed only 1 patient (not TST-positive healthy controls and 10 TST-negative healthy controls
BCG vaccinated) out of the 10 who did not respond to PPD in were evaluated. The values shown represent the mean level of IFN-␥
vitro. ⫾ standard error and were significantly higher in the former group for
all the antigens tested (ⴱ, P ⬍ 0.05). All results are expressed as already
Positive responses to ESAT-6 were detected in 59% of the subtracted from the values obtained in the unstimulated cultures.
TB patients with a BCG scar, while 100% of those patients PBMC responses to recombinant protein controls were mostly below
responded to PPD in vitro (Table 2). Moreover, from the 30 pg/ml.
TST-negative healthy control group without BCG vaccination,
four of the five individuals responded to ESAT-6. When ana-
VOL. 70, 2002
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 pa-
tients 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 anti-
gens 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 re-
sponses 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 re-
sponse to ESAT-6 and PPD are presented as already sub-
tracted from the levels in the nonstimulated control cultures
with anti-CD28 and brefeldin A treatments. Figure 3 demon-
strates 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-non-
stimulated 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
T-CELL RESPONSE TO rESAT-6 IN BRAZILIAN TB PATIENTS 6711
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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 Den-
mark 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 coende-
micity, 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 cate-
gories (Fig. 1), while the responses to PPD and Ag85B could
not (Fig. 2). Clearly, patients with pleural and cavitary pulmo-
nary 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. Brazil-
ians represent an ethnically diverse population compared with
those in the previous works, who showed a high rate of re-
sponders 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-␥ re-
lease 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
6712 CARDOSO ET AL.
increasing interest in investigating T-cell responses by using
the enzyme-linked immunospot (ELISPOT) assay, a very sen-
sible and specific method that detects the frequency of antigen-
specific 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, partic-
ularly IL-10; and the temporary depression of T-cell respon-
siveness (42).
The Th1 response associated with increased lung destruc-
tion, like the presence of the lung cavity at chest X-ray exam-
ination, was also evaluated. Although, chest computed tomog-
raphy 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 re-
sponse associated with immunopathology. The enhanced
IFN-␥ values detected in the T cells from pleural TB patients
provide evidence that an increased delayed-type hypersensitiv-
ity 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 immuno-
pathogenesis of lung cavity formation. Additionally, M. tuber-
culosis-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 conse-
quences 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 under-
standing 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 essen-
tial 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 in-
tracellular cytokine staining and flow cytometry, the produc-
tion of IFN-␥ induced by ESAT-6 among both CD4⫹ and
CD8⫹ T lymphocytes was evaluated in the patient population.
INFECT. IMMUN.
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 cir-
culating 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 myco-
bacterial antigens, even in HIV-positive individuals. Our re-
sults confirm these data, since 90% of the TST-negative
Downloaded from http://iai.asm.org/ on January 20, 2018 by guest
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 poten-
tially 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 myco-
bacterial exposure permit T cells to be primed for mycobacte-
rial antigens by diverse types of antigen-presenting cells. Sub-
sequently, 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 se-
quences 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 re-
sponded 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 applica-
tion 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-
VOL. 70, 2002 T-CELL RESPONSE TO rESAT-6 IN BRAZILIAN TB PATIENTS
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). Fi-
nally, 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 980377, The Netherlands Royal Academy of Arts and Sciences,
The Netherlands Leprosy Foundation (NLR), CNPq, and FIOCRUZ,
Brazil.
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