See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/51454159
Effect of Chemotherapy on Whole-Blood
Cytokine Responses to Mycobacterium
tuberculosis Antigens in a Small Cohort of
Patients with Pulmonary Tuberculosis
ARTICLE in CLINICAL AND VACCINE IMMUNOLOGY: CVI · JUNE 2011
Impact Factor: 2.47 · DOI: 10.1128/CVI.05037-11 · Source: PubMed
CITATIONS
READS
6
55
8 AUTHORS, INCLUDING:
Sylvie Bertholet
David J Horne
65 PUBLICATIONS 2,756 CITATIONS
26 PUBLICATIONS 213 CITATIONS
GSK Vaccines S.r.l., Italy
SEE PROFILE
University of Washington Seattle
SEE PROFILE
Rhea N Coler
Masahiro Narita
77 PUBLICATIONS 3,180 CITATIONS
51 PUBLICATIONS 1,149 CITATIONS
Infectious Disease Research Institute
SEE PROFILE
University of Washington Seattle
SEE PROFILE
Available from: Sylvie Bertholet
Retrieved on: 05 February 2016
CLINICAL AND VACCINE IMMUNOLOGY, Aug. 2011, p. 1378–1386
1556-6811/11/$12.00 doi:10.1128/CVI.05037-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 8
Effect of Chemotherapy on Whole-Blood Cytokine Responses to
Mycobacterium tuberculosis Antigens in a Small Cohort of
Patients with Pulmonary Tuberculosis䌤†
Sylvie Bertholet,1‡ David J. Horne,2 Elsa M. Laughlin,1 Margery Savlov,2 Ines Tucakovic,1
Rhea N. Coler,1 Masahiro Narita,2,3 and Steven G. Reed1*
Infectious Disease Research Institute, 1124 Columbia Street, Ste. 400, Seattle, Washington 981041; Division of Pulmonary and
Critical Care Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, Washington 981042; and
Public Health—Seattle & King County, Tuberculosis Control Program, Seattle, Washington 981043
Received 17 March 2011/Returned for modification 18 April 2011/Accepted 15 June 2011
The development of genomic and proteomic tools has enabled studies that begin to characterize the
molecular targets of an effective host immune response to Mycobacterium tuberculosis, including understanding
the specific immune responses associated with tuberculosis (TB) disease progression, disease resolution, and
the development of latency. One application of such tools is the development of diagnostic reagents and assays
useful as a test of cure. Such a test could be of considerable importance for the evaluation of new therapeutics.
We and others have previously described immunodominant proteins of M. tuberculosis, including both vaccine
and diagnostic candidates. In the present study, we describe the changes in immune responses to a panel of 71
M. tuberculosis antigens in six patients during the course of therapy. The levels of six cytokines were measured
in 24-h whole-blood assays with these antigens, revealing that gamma interferon (IFN-␥), tumor necrosis factor
(TNF), and interleukin-10 (IL-10) were differentially regulated in response to a subset of antigens. Therefore,
measuring the production of these three cytokines in response to a panel of carefully selected M. tuberculosis
proteins during the course of TB therapy might be a promising path toward the development of a test of cure
and warrants further validation in larger cohorts of pulmonary TB patients.
Tuberculosis (TB) caused by Mycobacterium tuberculosis
complex bacilli is one of the leading causes of death worldwide
(42). Upon exposure to M. tuberculosis, 30 to 40% of close
contacts will develop TB infection, of whom 5% would be
expected to develop active disease within a 24-month period
while the other 95% enter a state of controlled latent TB
infection (LTBI), which can reactivate later in life following
decreased immunocompetence of the host. T cells, both CD4⫹
and CD8⫹, and the cytokines gamma interferon (IFN-␥) and
tumor necrosis factor (TNF) play important roles in the prevention of active disease and the control of LTBI, as demonstrated by gene-knockout animal models and human subjects
with mutations affecting the expression of these two cytokines
(17).
The traditional diagnosis of active TB disease relies on positive identification of acid-fast bacilli (AFB) in a sputum smear
or M. tuberculosis identified in culture and is supported by
delayed-type hypersensitivity reactions to intradermal injection
of M. tuberculosis-specific and nonspecific purified protein derivative (PPD). The tuberculin skin test (TST) utilizes PPD
and has a number of drawbacks, notably that TST cross-reacts
with the Mycobacterium bovis vaccine strain bacillus Calmette-
Guérin (BCG) and other environmental mycobacteria, increasing the number of false positives (37). As IFN-␥ is required for a T helper 1 (Th1) response to M. tuberculosis, this
cytokine has been measured ex vivo in serum (35, 39), sputum
(35), bronchoalveolar lavage fluid (4), pleural effusions of TB
patients (21, 25), or culture supernatants of peripheral blood
mononuclear cells (PBMC) after in vitro stimulation with M.
tuberculosis antigens (2, 18, 40). IFN-␥ responses to antigen
stimulation are easily induced in PBMC or whole blood and
can be detected using simple technologies, such as enzymelinked immunosorbent assay (ELISA) or enzyme-linked immunosorbent spot assay (ELISPOT), since IFN-␥ is not labile
and is usually produced in measurable quantities. Differences
in the levels of IFN-␥ measured in culture supernatants of
stimulated lymphocytes from TB patients and controls varied
considerably depending on the study and were inconclusive as
a diagnostic tool (40), probably due to nonstandardized sample
handling and variable duration of stimulation with M. tuberculosis antigens (13).
More recently, standardized short (⬍24 h) peripheral blood
T-cell IFN-␥ responses to three M. tuberculosis-specific antigens, early secreted antigenic target 6 (ESAT-6), culture
filtrate protein 10 (CFP-10), and antigen TB7.7 (Rv2654),
have been investigated for the management of TB. Two
assays, the ELISPOT assay T-SPOT.TB (Oxford Immunotec,
Oxford, United Kingdom) that uses PBMC and the ELISA
QuantiFERON-TB gold (Cellestis, Victoria, Australia) that
uses whole blood, measure IFN-␥ responses to overlapping
ESAT-6/CFP-10 peptides and ESAT-6/CFP-10/TB7.7, respectively, in less than 24 h. It was reported that the number of
ESAT-6/CFP-10-specific IFN-␥-producing cells in circulating
* Corresponding author. Mailing address: Infectious Disease Research Institute, 1124 Columbia Street, Ste. 400, Seattle, WA 98104.
Phone: (206) 419-7870. Fax: (206) 381-3678. E-mail: sreed@idri.org.
‡ Present address: Novartis Vaccines and Diagnostics, via Fiorentina
1, Siena 53100, Italy.
† Supplemental material for this article may be found at http://cvi
.asm.org/.
䌤
Published ahead of print on 29 June 2011.
1378
VOL. 18, 2011
T-CELL CYTOKINE RESPONSES IN TUBERCULOSIS PATIENTS
PBMC reflects bacterial load and the relative risk for pathology and illness. While both assays have advanced the diagnosis
of latent TB infection, their utility in monitoring immune responses during the course of chemotherapy for active TB disease and in correlating IFN-␥ levels after antigen stimulation
with the decrease in bacterial load has yet to be confirmed (12).
The results obtained to date from a small number of studies
using these two assays are controversial for both active TB and
LTBI. Some of these studies report decreased IFN-␥ responses
to specific mycobacterial antigens (1, 8, 10, 20, 26, 28, 33, 34,
36) during and after chemotherapy, while others have shown
persistently positive or even stronger responses with chemotherapy (3, 16, 31, 38, 41, 43). Decreases in IFN-␥ responses
to CFP-10 during treatment appeared to correlate better
with reduction in bacterial loads than analogous responses
to ESAT-6 or ESAT-6 and CFP-10 combined (9, 14, 27). Host
immune responses to M. tuberculosis antigens during therapy
are complex, dynamic events that will likely require the
measure of more than one parameter—cytokine and/or antigen. It has been suggested that the IFN-␥/IL-10 ratio might
be used to successfully predict the development of active
disease after exposure to TB (24). In addition, Eum et al.
recently showed that the regulation of TNF during therapy
might be better than IFN-␥ in predicting sputum conversion
at 6 months (15).
The identification of surrogate markers of bacterial clearance in TB patients receiving chemotherapy is important for a
number of reasons, including early diagnosis of treatment efficacy, prevention of relapse due to incomplete cure, and as
endpoints in clinical trials evaluating new tuberculosis medications (22). The objective of this study was to identify in
pulmonary TB patients surrogate markers for successful response to therapy by characterizing the dynamic of cytokine
responses to a large array of M. tuberculosis antigens before,
during, and after completion of treatment. For that purpose,
cytokines associated with Th1, Th2, and Th17 were analyzed. The levels of IFN-␥, TNF, interleukin-10 (IL-10),
IL-5, IL-2, and IL-17 were characterized in whole blood
after ⬍24 h of stimulation with PPD, M. tuberculosis lysate
(MtbL), ESAT-6, and 70 additional recombinant M. tuberculosis proteins, including both secreted and latency antigens.
MATERIALS AND METHODS
Study subjects. TB patients (n ⫽ 6), recruited at the TB Control Program,
Public Health—Seattle & King County (Seattle, WA), were assessed for cytokine
profiles during therapy using M. tuberculosis antigen-stimulated whole-blood
cultures. The study was approved by the Human Subjects Review Committee of
the University of Washington, and written informed consent was obtained from
each study subject. All six patients were diagnosed with active pulmonary TB by
sputum culture, were PPD positive (indurations of ⬎10 mm), and were negative
for human immunodeficiency virus (HIV) and hepatitis B and hepatitis C viruses.
Four patients had a history of BCG vaccination, one did not, and the status of
one patient was unknown. The mean age was 32.8 years (range, 19 to 49), and the
male/female ratio was 5:1. Each patient was given the standard 6 months of
directly observed therapy (DOT) for TB, which consisted of a combination of at
least two of the following drugs: isoniazid, rifampin, ethambutol, pyrazinamide or
rifamate (a combination of isoniazid and rifampin). Each patient was initially
treated with standard short-course therapy according to the guidelines of the
American Thoracic Society and Centers for Disease Control and Prevention (7),
consisting of a 2-month intensive phase with four drugs (isoniazid, rifampin,
ethambutol, and pyrazinamide) and 4 months of isoniazid and rifampin. One
patient (TBC-04) harbored a multidrug-resistant M. tuberculosis strain that was
1379
TABLE 1. Categories of selected M. tuberculosis antigens
Category
Antigens
Secreted/membrane ................Rv0125, Rv0153c, Rv0164, Rv0390,
Rv0455c, Rv0496, Rv0577, Rv0733,
Rv0909, Rv1411, Rv1511, Rv1626,
Rv1827, Rv1860, Rv1926c, Rv1980c,
Rv1984c, Rv2031, Rv2220, Rv2873,
Rv2875, Rv3029c, Rv3881, Ag85B
PE/PPE protein family...........Rv0915, Rv1789, Rv1818c, Rv2608, Rv3478
EsX protein family .................Rv0287, Rv1793, Rv3619, Rv3620c, ESAT-6
Database ..................................Rv0952, Rv1009, Rv1174, Rv1211, Rv1253,
Rv1270c, Rv1288, Rv1884c, Rv2389c,
Rv2450, Rv3204, Rv3407
Latency
Growth in
macrophage .....................Rv0467, Rv0523c, Rv0655, Rv0716, Rv1099,
Rv1397c, Rv1410c, Rv1569, Rv1589,
Rv1590, Rv3541c, Rv3587
Hypoxia ................................Rv1240, Rv1738, Rv1813c, Rv2032, Rv2428,
Rv2558, Rv2624c, Rv2626c, Rv2801c,
Rv3044, Rv3129c, Rv3130, Rv3810
resistant to both isoniazid and rifampin and was treated with an alternative
regimen for 18 months. All patients had converted to culture negative at the end
of the treatment and were considered adequately treated. In addition, healthy
PPD-negative subjects (n ⫽ 7) with no history of BCG vaccination were included
as controls.
Reagents. M. tuberculosis PPD (lot P A0814-1) was obtained from Mycos
Research LLC (Loveland, CO). Phytohemagglutinin (PHA) was purchased from
Sigma (Sigma-Aldrich, St. Louis, MO). Target M. tuberculosis recombinant
His-tagged proteins were prepared in Tris buffer as previously described (5),
and all showed residual endotoxin levels of ⬍100 endotoxin units (EU)/mg of
protein.
Whole-blood assay. At the time of enrollment in the study, patients had been
started on chemotherapy for ⬍2 weeks. Blood samples were drawn into heparinized Vacutainers (BD Biosciences, San Jose, CA) between the hours of
10 a.m. and 2 p.m. at the initial visit (time zero [T0]) and at 2 weeks (T0.5) and
1, 3, 6, 9, and 12 months (T1, T3, T6, T9, and T12) thereafter. Blood (20 ml) was
diluted 1:1 with sterile RPMI 1640 (Invitrogen) tissue culture medium. Diluted
blood (450 l/well) was plated in 48-well tissue culture plates within 2 h of
collection. Blood cultures were stimulated with 50 l/well of Tris buffer, 10 g/ml
PPD, 10 g/ml PHA, 10 g/ml MtbL, or 10 g/ml of each recombinant protein
of M. tuberculosis (Table 1) for 20 to 22 h at 37°C in 5% CO2. Culture supernatants were harvested and stored for later use in separate polypropylene plates
as three 100-l aliquots at ⫺80°C.
Cytokine determination by Luminex. Supernatants were filtered through
1.2-m and 0.2-m filters to remove possible M. tuberculosis bacilli. The filtrates
were analyzed for IL-2, IL-5, IL-10, IL-17, IFN-␥, and TNF cytokine production
using a customized human 6-plex Procarta cytokine assay kit (Affymetrix, Santa
Clara, CA), following the directions of the manufacturer. Samples were read on
a Luminex 200 (Luminex Corporation, Austin, TX) machine powered by the
MasterPlex Software Suite program and analyzed using the MasterPlex QT 3.0
(MiraiBio, Inc., Alameda, CA) quantification software in order to obtain cytokine concentration values. The sensitivities and ranges of the assays were determined by running a dose-response curve of a reference standard for each cytokine from the manufacturer on each plate.
Data analyses. All data were analyzed by using MasterPlex QT 3.0 and Microsoft Excel. Antigen-specific cytokine responses are shown after subtraction of
the spontaneous cytokine secretion that occurs in the absence of a stimulant. A
macro was written with Microsoft Excel to convert cytokine levels from pg/ml or
fold change to colored heat maps. Comparison of the levels of IFN-␥ and IL-10
or those of TNF and IL-10 were provided as differences rather than ratios to
account for zero values where present. Intra-assay variation was initially calculated for each cytokine at the T0 and T6 time points on duplicate wells of cells
stimulated with PHA, PPD, ESAT-6, or antigen 85B (Ag85B). The intra-assay
cytokine variation averaged 1.1 ⫾ 0.3 (mean ⫾ standard deviation [SD]; range,
1.0 to 1.5). There were no statistically significant differences observed between
stimulants or cytokines, as determined using a 1-way analysis of variance
(ANOVA) test. A cutoff for fold-change cytokine expression compared to the
1380
BERTHOLET ET AL.
CLIN. VACCINE IMMUNOL.
FIG. 1. Heat map of whole-blood cytokine responses to a subset of M. tuberculosis antigens recognized by two-thirds of the subjects. Whole
blood was obtained from six TB patients at time 0 (T0) and 6 months (T6) of antibiotic treatment and stimulated with Tris buffer, PHA, PPD,
MtbL, or an individual recombinant M. tuberculosis protein for 24 h at 37°C. Plasma levels of IFN-␥, TNF, and IL-10 were measured using
multiplexing ELISA. Antigen-specific cytokine concentrations are represented in a heat map from blue (⬎1 pg/ml) to dark brown (⬎1,500 pg/ml).
expression at T0 was considered different when greater than the absolute value
of 2 (averagevariation ⫹ 3 ⫻ SDvariation).
RESULTS
To explore whether changes in cytokine responses to defined
M. tuberculosis antigens would provide a surrogate marker for
sputum smear and culture conversion, whole blood was obtained
from six pulmonary TB subjects during the course of chemotherapy. The blood samples were stimulated for 24 h with an extensive
panel of 71 single M. tuberculosis proteins, including ESAT-6 and
Ag85B, and PPD. The levels of cytokines associated with Th1/
effector (IFN-␥ and TNF), memory (IL-2), regulatory (IL-10),
Th2 (IL-5), and Th17 (IL-17) cells were measured in culture
supernatant with the Luminex technology.
Profile of cytokine responses to M. tuberculosis antigens at
initiation and end of therapy. Whole-blood cells’ cytokine responses to M. tuberculosis antigen stimulation were initially
compared at the beginning (T0) and end (T6) of therapy (see
Fig. S1 in the supplemental material). While the six cytokines
were upregulated in all patients in response to PHA stimulation, indicating that active disease did not inhibit cytokine
responses, only IFN-␥, TNF, IL-10, and IL-2 were increased in
response to PPD and MtbL. Furthermore, cytokine responses
to single M. tuberculosis antigens were mostly restricted to
IFN-␥, TNF, and IL-10. Based on these observations, we focused our analyses on a subset of M. tuberculosis antigens that
induced antigen-specific cytokine responses of ⬎25 pg/ml in
four of six patients (67%) at T6 for one or more of the latter
three cytokines (Fig. 1). Nineteen, 33, and 31 M. tuberculosis
antigens met these criteria for IFN-␥ (Fig. 1A), TNF (Fig. 1B),
and IL-10 (Fig. 1C), respectively. Interestingly, among these
M. tuberculosis antigens, 17 upregulated all three cytokines, 12
more were associated with increases in TNF and IL-10 responses, and one was associated with IFN-␥ and TNF responses. When concentrations of effector (IFN-␥ and TNF)
versus regulatory (IL-10) cytokines were compared, the IL-10
levels were generally higher than the IFN-␥ and TNF levels for
most M. tuberculosis antigens and TB subjects at both T0 and
T6, resulting in net negative values (Table 2). In addition, there
VOL. 18, 2011
T-CELL CYTOKINE RESPONSES IN TUBERCULOSIS PATIENTS
1381
TABLE 2. Differences between plasma levels of IFN-␥ effector and IL-10 regulatory cytokines with stimulation
by various M. tuberculosis antigens
Amt of IFN-␥ or TFN ⫺ amt of IL-10 (pg/ml) in patient at indicated time pointa
Cytokine pair
measured and
M. tuberculosis
antigen used
T0
T6
T0
T6
T0
T6
T0
T6
T0
T6
T0
T6
IFN-␥ ⫺ IL-10
PHA
PPD
MtbL
ESAT-6
Ag85B
Rv0287
Rv0455
Rv0467
Rv0523
Rv0655
Rv1174
Rv1253
Rv1397
Rv1626
Rv1738
Rv1818
Rv1884
Rv2428
Rv2624
Rv3478
Rv3619
Rv3620
580
600
620
7
0
⫺139
⫺114
⫺62
⫺84
0
⫺18
⫺35
⫺42
⫺37
⫺88
⫺50
⫺24
⫺130
⫺57
0
⫺24
⫺79
1182
110
315
69
0
⫺387
⫺406
⫺83
⫺356
6
204
⫺395
⫺90
⫺175
⫺519
⫺178
⫺85
⫺182
⫺298
⫺80
⫺173
⫺302
⫺761
3714
4402
324
24
⫺76
⫺243
20
⫺200
11
⫺44
⫺549
32
⫺84
⫺410
85
⫺62
⫺250
⫺226
⫺146
⫺137
28
⫺556
1895
1726
179
25
⫺46
⫺491
⫺15
⫺367
27
83
⫺783
5
⫺193
⫺570
⫺184
⫺54
⫺378
⫺334
⫺44
⫺264
⫺295
⫺223
⫺331
⫺25
0
0
⫺488
⫺594
0
⫺174
0
0
⫺647
0
0
⫺286
0
0
⫺72
0
0
0
0
456
⫺462
⫺187
0
0
⫺249
⫺445
⫺86
⫺317
⫺19
⫺125
⫺624
⫺20
⫺151
⫺630
⫺115
⫺23
⫺459
⫺256
⫺55
⫺234
⫺246
⫺261
⫺1166
⫺612
⫺2
5
⫺498
⫺993
⫺116
⫺553
3
⫺267
⫺801
⫺64
⫺215
⫺662
⫺918
⫺568
⫺289
⫺322
⫺215
⫺254
⫺163
4380
⫺1046
⫺915
⫺19
⫺71
⫺1400
⫺1703
⫺156
⫺779
⫺109
⫺418
⫺1417
⫺239
⫺381
⫺1575
⫺823
⫺241
⫺775
⫺789
⫺316
⫺905
⫺869
⫺542
49
551
250
⫺58
⫺853
⫺1030
⫺76
⫺232
⫺24
65
⫺1136
⫺83
⫺127
⫺831
⫺164
⫺328
⫺217
⫺352
⫺30
⫺393
⫺190
⫺565
⫺1781
⫺1327
8
⫺91
⫺1209
⫺1608
⫺163
⫺574
⫺165
⫺147
⫺1668
⫺247
⫺381
⫺1981
⫺297
⫺621
⫺461
⫺671
⫺222
⫺443
⫺583
⫺2933
⫺641
⫺428
16
23
58
⫺107
65
86
5
105
⫺203
15
75
⫺351
50
80
⫺75
13
51
63
⫺180
⫺3350
⫺1412
⫺1380
36
15
82
⫺267
⫺132
⫺438
⫺69
⫺117
⫺189
⫺54
⫺245
⫺473
⫺85
⫺82
⫺319
⫺170
⫺42
⫺312
⫺749
TNF ⫺ IL-10
PHA
PPD
MtbL
Ag85B
Rv0164
Rv0287
Rv0455
Rv0467
Rv0496
Rv0523
Rv0655
Rv0716
Rv0915
Rv1174
Rv1211
Rv1253
Rv1288
Rv1397
Rv1511
Rv1626
Rv1738
Rv1789
Rv1818
Rv1884
Rv2031
Rv2428
Rv2450
Rv2558
Rv2624
Rv2801
Rv3129
Rv3130
Rv3478
Rv3541
Rv3619
Rv3620
33
⫺58
⫺18
0
⫺38
⫺132
⫺106
⫺59
1
⫺78
1
⫺18
0
⫺72
1
⫺34
⫺36
⫺41
⫺58
⫺34
⫺82
1
⫺49
⫺24
⫺38
⫺122
⫺41
0
⫺54
2
1
1
2
⫺48
⫺21
⫺78
⫺233
29
⫺39
20
⫺248
⫺186
⫺213
⫺39
11
⫺256
37
⫺163
9
102
⫺65
⫺174
⫺84
⫺76
⫺127
⫺125
⫺203
32
⫺74
⫺91
31
⫺83
32
21
⫺51
⫺134
11
8
⫺23
⫺110
⫺128
⫺271
⫺3643
⫺50
⫺126
10
64
21
⫺21
89
5
⫺55
3
31
15
21
69
⫺173
52
12
40
26
⫺164
26
75
⫺54
⫺56
⫺72
65
4
⫺99
69
10
18
⫺73
15
⫺58
26
⫺6254
130
⫺213
59
30
1114
343
91
35
⫺81
18
⫺5
71
122
129
625
197
54
90
⫺7
404
31
39
220
⫺28
51
⫺81
25
48
113
70
96
146
64
108
⫺123
⫺358
⫺305
⫺250
1
11
⫺338
⫺466
3
1
⫺151
0
1
0
12
3
⫺553
53
5
14
12
⫺238
2
15
36
9
⫺47
8
0
15
24
4
12
13
9
10
13
111
257
⫺234
30
0
730
264
⫺41
39
⫺199
17
14
30
⫺66
15
⫺133
439
126
207
⫺80
⫺108
45
12
116
⫺28
⫺282
8
59
⫺94
20
4
12
⫺2
104
⫺131
⫺276
1226
⫺729
76
0
⫺348
⫺799
⫺1284
⫺148
0
⫺713
0
⫺377
⫺31
⫺423
⫺123
⫺1130
⫺317
⫺84
⫺140
⫺301
⫺924
⫺148
⫺1119
⫺848
⫺382
⫺440
⫺262
0
⫺462
⫺457
⫺186
⫺676
⫺303
⫺199
⫺338
⫺171
1274
⫺1007
⫺1045
⫺60
⫺332
⫺444
⫺879
⫺32
31
⫺594
⫺118
⫺150
14
⫺218
⫺56
⫺471
⫺201
⫺202
⫺179
⫺156
⫺779
⫺11
⫺573
⫺179
⫺345
⫺482
⫺88
⫺233
⫺621
⫺234
⫺365
⫺171
⫺36
⫺244
⫺733
⫺825
⫺889
⫺606
⫺295
⫺24
⫺58
35
⫺2
20
⫺3
⫺45
18
⫺35
⫺8
48
14
⫺236
98
⫺22
⫺24
⫺43
⫺293
⫺24
136
⫺7
3
32
⫺3
39
⫺68
⫺52
23
110
11
2
27
⫺174
⫺1074
⫺1190
⫺1064
⫺49
⫺201
⫺539
⫺696
⫺4
⫺49
⫺310
⫺115
⫺73
⫺22
0
⫺55
⫺80
3
⫺140
⫺167
⫺139
⫺922
⫺68
⫺25
⫺227
⫺81
⫺180
⫺144
⫺87
⫺200
⫺159
⫺67
⫺26
⫺24
⫺114
⫺212
⫺583
⫺376
1199
2989
0
564
862
⫺463
0
0
31
0
0
0
1463
0
⫺528
⫺40
0
0
0
⫺613
0
103
0
597
⫺226
0
0
⫺132
0
0
0
0
0
0
⫺209
⫺2192
⫺1373
⫺883
⫺161
⫺382
⫺761
⫺1238
⫺249
⫺77
⫺639
⫺155
⫺169
⫺194
29
⫺295
⫺1280
⫺869
⫺217
⫺312
⫺569
⫺1283
⫺140
⫺376
⫺259
⫺385
⫺739
⫺520
⫺140
⫺682
⫺206
⫺140
⫺217
⫺315
⫺304
⫺667
⫺856
TBC-01
TBC-04
TBC-06
TBC-07
TBC-08
TBC-09
a
Whole blood was obtained from six TB patients at time zero (T0) and 6 months (T6) of antibiotic treatment and stimulated with individual recombinant
M. tuberculosis protein for 24 h at 37°C. Plasma levels of IFN-␥ or TNF and IL-10 were measured using multiplexing ELISA. A negative value indicates a higher level
of IL-10 than of IFN-␥ or TNF.
1382
BERTHOLET ET AL.
CLIN. VACCINE IMMUNOL.
FIG. 2. Kinetics of whole-blood cytokine responses to a subset of M. tuberculosis antigens. Whole blood was obtained from six TB patients at
time 0 (T0), 2 weeks (T0.5), and 1 (T1), 3 (T3), 6 (T6), 9 (T9), and 12 months (T12) of antibiotic treatment and from seven PPD-negative healthy
controls (N) and stimulated with Tris buffer, PHA, PPD, MtbL, or an individual recombinant M. tuberculosis protein for 24 h at 37°C. Plasma levels
of IFN-␥, TNF, and IL-10 were measured using multiplexing ELISA. At each time point, data from the six subjects were averaged for a given in
vitro stimulation and color coded depending on cytokine concentration.
was a trend for a greater IL-10 than IFN-␥ response detected
at T6 compared to their levels at T0 (T6 value ⬍ T0 value).
Kinetics of cytokine responses to M. tuberculosis antigens
during chemotherapy. Whole-blood samples were obtained at
T0, T0.5 (2 weeks), and T1, T3, T6, T9, and T12 (months) after
the initiation of therapy and stimulated for 24 h with the subset
of stimulating M. tuberculosis antigens identified in the experiments discussed above (Fig. 1). Plasma levels of IFN-␥, TNF,
and IL-10 were measured and averaged for the six patients at
each time point. For IFN-␥, the cytokine level associated with
each single M. tuberculosis antigen was either maintained or
increased at all the other time points except T0 (Fig. 2A). This
observation is of particular interest as it suggests that blood
cells from TB subjects already become more responsive to
antigen-specific stimulation within 2 weeks after starting on
antibiotics. In comparison, IFN-␥ levels measured from blood
cells of PPD-negative healthy controls were low (⬍50 pg/ml,
except for Rv0287 and Rv0455). The TNF levels in response to
M. tuberculosis antigen stimulation seemed generally higher at
the later time points, T6 through T12 (Fig. 2B), with the exception of Rv0287, Rv0455, Rv1174, Rv1253, Rv1288, Rv1738,
and Rv2428, which induced strong TNF responses at all time
points, as well as in healthy controls. Responses to these M. tuberculosis antigens in healthy controls are probably due to
homology with and exposure to environmental nontuberculous
mycobacteria. Likewise, the highest IL-10 responses were generally observed at T6 and T9, with Rv0287, Rv0455, Rv1253,
and Rv1738 inducing IL-10 levels of ⬎500 pg/ml (Fig. 2C). At
T12, however, the IL-10 responses resemble those of healthy
controls. It appears that secretions of TNF and IL-10 have
both more similar kinetics and more reactive M. tuberculosis
antigen subsets than those of IFN-␥ and IL-10.
Quantitative changes in cytokine responses to M. tuberculosis antigens during chemotherapy. In the experiments discussed above, we identified a subset of 36 M. tuberculosis antigens inducing IFN-␥, TNF, and/or IL-10 responses in the
VOL. 18, 2011
T-CELL CYTOKINE RESPONSES IN TUBERCULOSIS PATIENTS
1383
and presented as a group response at the different time points
tested. IFN-␥ responses to Rv0467, Rv1738, and Rv3619 were
already upregulated 5- to 25-fold at T0.5, while responses to
ESAT-6, Ag85B, Rv0655, and Rv1174 increased at T3 or later
(Fig. 4A). TNF responses to all but three antigens were upregulated at T6 and further increased at T9 and/or T12 (Fig.
4B). In addition, 2- to 10-fold changes in TNF responses to
Rv0164, Rv1818, and Rv2425 were already occurring at T0.5.
Finally, IL-10 responses to most antigens were downregulated,
especially at T12 (Fig. 4C).
DISCUSSION
FIG. 3. Fold changes in cytokine responses to a subset of M. tuberculosis antigens 6 months into antibiotic therapy. Whole blood was
obtained from six TB patients at time 0 (T0) and 6 months (T6) of
antibiotic treatment and stimulated with Tris buffer, PHA, PPD, MtbL,
or an individual recombinant M. tuberculosis protein for 24 h at 37°C.
Plasma levels of IFN-␥, TNF, and IL-10 were measured using multiplexing ELISA. Increases (positive fold change) or decreases (negative
fold change) in cytokine responses at T6 compared to the data at T0
are shown and color coded based on intensity.
blood of two-thirds of the TB subjects. We further examined
whether cytokine response(s) to some of these M. tuberculosis
antigens changed during the course of therapy (Fig. 3 and
Fig. S2).
The fold changes in IFN-␥, TNF, and IL-10 responses to the
subset of 36 M. tuberculosis antigens over the period from T0 to
T6 were first calculated for each subject. Among the 19 M.
tuberculosis antigens selected, IFN-␥ responses to latency antigens Rv467, Rv655, and Rv1738 were upregulated (2- to
25-fold) in 50% of the TB subjects (Fig. 3A). Similarly, at T6,
25 of the 33 M. tuberculosis antigens initially selected induced
increased TNF levels (2- to 150-fold) (Fig. 3B), and 8 of 31
increased IL-10 responses (2- to 25-fold) (Fig. 3C) in 50% of
the TB subjects. Finally, a subset of 8 M. tuberculosis antigens
did induce positive changes at T6 for at least two cytokines in
ⱖ50% of the patients (Table 3) and might be pursued further
as a test of cure. While cytokine responses to some antigens
were downregulated at T6 compared to their levels at T0, the
occurrence was rare and never occurred in more than onethird of the subjects. Finally, fold changes in cytokine levels
during the course of therapy were averaged for the six subjects
The identification of surrogate markers of bacterial clearance in pulmonary TB patients receiving chemotherapy is a
critical step toward early assurance of treatment success and
identification of patients at risk of relapse and subsequent
transmission of TB to others following short-course TB therapy. These biomarkers may also serve as a surrogate endpoint
in clinical trials evaluating new TB medications. In this longitudinal study, we demonstrated that whole-blood cytokine production patterns from pulmonary TB patients vary during standard TB treatment. Blood culture stimulation with a set of 71
recombinant M. tuberculosis proteins and complex antigen mixtures (PPD and MtbL) resulted predominantly in Th1 (IFN-␥
and TNF) and regulatory (IL-10) cytokine responses; the levels
of Th2 (IL-5)-, Th17 (IL-17)-, and memory (IL-2)-associated
cytokines were low to undetectable for most antigens in all
subjects. IFN-␥, TNF, and/or IL-10 changes in expression profiles during chemotherapy were associated with a subset of the
M. tuberculosis antigens tested.
Diagnosis of active TB relies on clinical criteria, patient
TABLE 3. Increase in IFN-␥, TNF, and IL-10 responses to a subset
of M. tuberculosis antigens
Antigen
Rv287
Rv455
Rv467
Rv523
Rv655
Rv1174
Rv1253
Rv1288
Rv1397
Rv1511
Rv1626
Rv1738
Rv1818
Rv1884
Rv2428
Rv2624
Rv3478
Rv3619
Rv3620
% of patients with positive cytokine responsea
IFN-␥
TNF
IL-10
33
33
50
17
50
33
33
0
17
0
17
50
33
17
17
17
17
33
33
50
67
50
33
33
33
50
50
50
50
50
50
67
50
50
50
67
50
33
33
50
17
17
17
17
33
50
50
50
50
67
17
17
50
33
17
33
50
a
Percentage of patients with positive fold-change cytokine responses to a
subset of M. tuberculosis antigens 6 months into antibiotic therapy. Whole blood
was obtained from six TB patients at time zero (T0) and 6 months (T6) of
antibiotic treatment and stimulated with individual recombinant M. tuberculosis
protein for 24 h at 37°C. Plasma levels of IFN-␥, TNF, and IL-10 were measured
using multiplexing ELISA. Antigens marked in boldface induced positive
changes at T6 for at least two cytokines in ⱖ50% of the patients.
1384
BERTHOLET ET AL.
FIG. 4. Fold changes in cytokine responses to a subset of M. tuberculosis antigens at different times during antibiotic treatment. Whole
blood was obtained from six TB patients at time 0 (T0), 2 weeks (T0.5),
and 1 (T1), 3 (T3), 6 (T6), 9 (T9), and 12 months (T12) of antibiotic
treatment and stimulated with Tris buffer, PHA, PPD, MtbL, or individual recombinant M. tuberculosis protein for 24 h at 37°C. Plasma
levels of IFN-␥, TNF, and IL-10 were measured using multiplexing
ELISA. Increases (positive fold change) or decreases (negative fold
change) in cytokine responses at the different time points compared to
the data at T0 were calculated. For each time point and in vitro
stimulation, data from the six subjects were averaged and color coded
based on intensity.
history, chest radiography, TST, and confirmation by sputum
smear or AFB culture positivity. However, an unexpectedly
high number of individuals can be sputum positive (and therefore potentially infectious) without any apparent symptoms,
whereas many patients who have significant symptoms remain
sputum negative, thus calling this simple definition into question. The recent development of short standardized IFN-␥
release assays (QuantiFERON-TB Gold and T-SPOT.TB)
based on recall responses to ESAT-6 and/or CFP-10 M. tuberculosis antigens offers new possibilities for the early diagnosis
of active TB in adults (11, 32). The use of IFN-␥ release assays
in assessing treatment efficacy in adults and children, however,
has yielded conflicting results, with some studies reporting
good correlations between bacterial loads and IFN-␥ responses
(mainly to CFP-10) (9, 14, 20, 26, 27, 36), while other studies
did not (3, 16, 31, 38, 41, 43). Our study confirms the lack of
reliability of ESAT-6 as a single marker because only two
patients (TBC-04 and TBC-08) of the six tested showed a
moderate IFN-␥ response to this antigen at T0, while all pa-
CLIN. VACCINE IMMUNOL.
tients had moderate to strong cytokine responses to M. tuberculosis-specific (PPD and MtbL) and nonspecific (PHA) stimulations. TBC-04 showed an initial increase in IFN-␥ 2 weeks
into therapy, consistent with previous observations (20), and
both TBC-04 and TBC-08 showed further-reduced IFN-␥ responses to ESAT-6 from T1 to T6 but returned to T0 levels at
T12. A similar kinetic was reported by Dominguez et al. (14).
Among the four other patients, one never responded to
ESAT-6, and the three others showed mild increases at T3 and
onward. It is unclear why these four patients had poor correlations between the presence of bacteria and IFN-␥ responses
to ESAT-6, but this observation is consistent with the considerable interindividual variation reported across studies in
terms of percentage of responders and rate of decline of the
response (14). When taken collectively, our data indicate that
there are no differences in IFN-␥ responses to ESAT-6 during
the first 3 months of treatment, followed by a moderate increase thereafter. Furthermore, our screening of 70 additional
M. tuberculosis antigens revealed that for a majority of patients,
none of these antigens was associated with IFN-␥ declines
during the course of treatment, while a small subset of antigens
(the 15 antigens Ag85B, Rv0287, Rv0455, Rv467, Rv523,
Rv0655, Rv1174, Rv1253, Rv1626, Rv1738, Rv1818, Rv1884,
Rv2428, Rv2624, and Rv3619) induced higher IFN-␥ recall
responses at T6 in ⱖ67% of TB patients. Patient responses to
latency antigens were generally low, as previously observed by
Leyten et al. (29), except for Rv1738. The relative discrepancy
between our Rv1738 data and Leyten’s might be explained by
the use of whole blood instead of PBMC or by a more restricted definition of TB patients. Antigen stimulation of whole
blood has the advantage of capturing the totality of the cellular
response compared to that seen in PBMC, where loss of lowfrequency cells during the purification process cannot be ruled
out. Altogether, these observations suggest that increased
rather than decreased IFN-␥ responses to a selected subset of
M. tuberculosis antigens might better correlate with bacterial
load and treatment efficacy. Furthermore, antigen stimulation
of whole-blood cultures is a simple method that does not require labor-intensive PBMC purification and that might be
more suitable in resource-limited laboratory settings. Nevertheless, further characterization of the T-cell response to
these 15 M. tuberculosis antigens and phenotypic analyses at
the single-cell level might provide additional information on
whether higher IFN-␥ responses are associated with increased
frequencies of blood antigen-specific circulating effector T
cells.
In this study, we hypothesized that short (⬍24 h) in vitro
blood stimulations will target circulating effector rather than
memory cells. Consistent with this hypothesis, the levels of
IL-2 measured in response to all single M. tuberculosis antigens
were low (⬍100 pg/ml). Positive-control cultures incubated
with the mitogen PHA showed elevated levels of IL-2 (500 to
1,500 pg/ml) at all time points, indicating that the lack of IL-2
responses to M. tuberculosis antigens were not due to an overall
low T-cell response or cytokine detection problem. The IL-5
and IL-17 levels, as a measure of Th2 and Th17 cell responses,
respectively, were also low (⬍100 pg/ml) for all M. tuberculosis
antigens, including PPD and MtbL, in agreement with observations from Hussain et al. (23). As for IL-2, we found IL-5 and
IL-17 responses to PHA stimulation (250 to 1,500 pg/ml and
VOL. 18, 2011
T-CELL CYTOKINE RESPONSES IN TUBERCULOSIS PATIENTS
100 to 1,000 pg/ml, respectively). However, PHA-driven IL-2,
IL-5, and IL-17 responses were not consistently modulated
during the course of therapy. Therefore, the measure of IL-2,
IL-5, and IL-17 expression patterns in response to M. tuberculosis antigens or PHA stimulation was not predictive of sputum
conversion and therapy outcome.
The ratios of IFN-␥/IL-2 (6, 30) and IFN-␥/IL-10 (24) have
also been used with some success in the diagnosis of active TB
and/or assessment of therapy outcome. In addition, Eum et al.
recently demonstrated that whole-blood TNF levels in response to culture filtrate proteins were uniquely correlated
with sputum conversion in patients treated for multidrug-resistant TB (15). However, they reported a decrease in TNF in
sputum-negative patients at 6 months, while we observed an
increase. Similarly, Harari et al. reported that subjects with
active TB had elevated frequencies of antigen-specific (ESAT-6
and CFP-10) TNF-positive CD4 T cells that decreased upon
completion of antibiotic treatment (19). The reasons for this
difference are still unclear, but it might be related to the M.
tuberculosis antigens tested in their and our study. Nevertheless, TNF, IL-10, and IFN-␥ responses to M. tuberculosis antigens were the most dynamic and likely to better predict therapy outcomes.
In summary, our results suggest that a subset of defined
M. tuberculosis antigens can be used to monitor IFN-␥, TNF,
and IL-10 expression patterns associated with sputum conversion in pulmonary TB patients during chemotherapy. However, the sample size in the present study is too small to make
a definitive conclusion, and this hypothesis will have to be
validated in larger patient cohorts. These studies (i) confirm
the relevance of IFN-␥, TNF, and IL-10 expression profiles as
an immune signature associated with reduction in bacterial
load and (ii) define novel M. tuberculosis antigens with diagnostic potential. Early detection of treatment failure has significant implications for TB control programs, as it may allow
the identification of TB patients whose clinical courses need to
be reviewed for nonadherence, malabsorption of medications,
poor penetration of TB drugs to the affected sites, or drugresistant TB. Conversely, accurate diagnostics may allow for
shortening treatment in early responders and a focusing of
resources on patients with a higher likelihood of poor outcome
in order to limit the chance of relapses. Accurate biomarkers
are urgently needed as surrogate endpoints in clinical trials for
evaluating tuberculosis medications, especially phase 2 trials.
23.
ACKNOWLEDGMENTS
24.
This work was supported by National Institutes of Health grant
AI-044373 (S.G.R.).
We are thankful to Gregory C. Ireton for discussion and selection of
M. tuberculosis antigens, to Jeffrey Guderian, Raodoh Mohamath,
Garrett Poshusta, and Jackie Whittle for protein expression and purification, to Qiong Pan and the clinical staff of the TB Control Program,
Public Health—Seattle & King County, to Anna Marie Beckmann for
regulatory affairs, to Randall F. Howard for reviewing the manuscript,
and to Rick Sorensen for heat map display programming.
REFERENCES
1. Aiken, A. M., et al. 2006. Reversion of the ELISPOT test after treatment in
Gambian tuberculosis cases. BMC Infect. Dis. 6:66.
2. Al-Attiyah, R., et al. 2006. Cytokine profiles in tuberculosis patients and
healthy subjects in response to complex and single antigens of Mycobacterium tuberculosis. FEMS Immunol. Med. Microbiol. 47:254–261.
3. Al-Attiyah, R., A. S. Mustafa, A. T. Abal, N. M. Madi, and P. Andersen. 2003.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
25.
26.
27.
28.
29.
30.
1385
Restoration of mycobacterial antigen-induced proliferation and interferongamma responses in peripheral blood mononuclear cells of tuberculosis
patients upon effective chemotherapy. FEMS Immunol. Med. Microbiol.
38:249–256.
Barry, S. M., M. C. Lipman, B. Bannister, M. A. Johnson, and G. Janossy.
2003. Purified protein derivative-activated type 1 cytokine-producing CD4⫹
T lymphocytes in the lung: a characteristic feature of active pulmonary and
nonpulmonary tuberculosis. J. Infect. Dis. 187:243–250.
Bertholet, S., et al. 2008. Identification of human T cell antigens for the
development of vaccines against Mycobacterium tuberculosis. J. Immunol.
181:7948–7957.
Biselli, R., et al. 2010. Detection of interleukin-2 in addition to interferongamma discriminates active tuberculosis patients, latently infected individuals, and controls. Clin. Microbiol. Infect. 16:1282–1284.
Blumberg, H. M., et al. 2003. American Thoracic Society/Centers for Disease
Control and Prevention/Infectious Diseases Society of America: treatment
of tuberculosis. Am. J. Respir. Crit. Care Med. 167:603–662.
Carrara, S., et al. 2004. Use of a T cell-based assay for monitoring efficacy
of antituberculosis therapy. Clin. Infect. Dis. 38:754–756.
Chee, C. B., et al. 2007. Latent tuberculosis infection treatment and T-cell
responses to Mycobacterium tuberculosis-specific antigens. Am. J. Respir.
Crit. Care Med. 175:282–287.
Dheda, K., et al. 2007. Interpretation of Mycobacterium tuberculosis antigen-specific IFN-gamma release assays (T-SPOT.TB) and factors that may
modulate test results. J. Infect. 55:169–173.
Dheda, K., Z. F. Udwadia, J. F. Huggett, M. A. Johnson, and G. A. Rook.
2005. Utility of the antigen-specific interferon-gamma assay for the management of tuberculosis. Curr. Opin. Pulm. Med. 11:195–202.
Diel, R., R. Loddenkemper, and A. Nienhaus. 2010. Evidence-based comparison of commercial interferon-gamma release assays for detecting active
TB: a metaanalysis. Chest 137:952–968.
Doherty, T. M., et al. 2005. Effect of sample handling on analysis of cytokine
responses to Mycobacterium tuberculosis in clinical samples using ELISA,
ELISPOT and quantitative PCR. J. Immunol. Methods 298:129–141.
Dominguez, J., et al. 2009. T-cell responses to the Mycobacterium tuberculosis-specific antigens in active tuberculosis patients at the beginning, during,
and after antituberculosis treatment. Diagn. Microbiol. Infect. Dis. 63:43–51.
Eum, S. Y., et al. 2010. Association of antigen-stimulated release of tumor
necrosis factor-alpha in whole blood with response to chemotherapy in
patients with pulmonary multidrug-resistant tuberculosis. Respiration 80:
275–284.
Ferrand, R. A., G. H. Bothamley, A. Whelan, and H. M. Dockrell. 2005.
Interferon-gamma responses to ESAT-6 in tuberculosis patients early into
and after anti-tuberculosis treatment. Int. J. Tuberc. Lung Dis. 9:1034–1039.
Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev.
Immunol. 19:93–129.
Handzel, Z. T., et al. 2007. Increased Th1 and Th2 type cytokine production
in patients with active tuberculosis. Isr. Med. Assoc. J. 9:479–483.
Harari, A., et al. 2011. Dominant TNF-alpha⫹ Mycobacterium tuberculosisspecific CD4⫹ T cell responses discriminate between latent infection and
active disease. Nat. Med. 17:372–376.
Herrmann, J. L., et al. 2009. Temporal dynamics of interferon gamma
responses in children evaluated for tuberculosis. PLoS One 4:e4130.
Hirsch, C. S., et al. 2001. Augmentation of apoptosis and interferon-gamma
production at sites of active Mycobacterium tuberculosis infection in human
tuberculosis. J. Infect. Dis. 183:779–788.
Horne, D. J., et al. 2010. Sputum monitoring during tuberculosis treatment
for predicting outcome: systematic review and meta-analysis. Lancet Infect.
Dis. 10:387–394.
Hussain, R., et al. 2002. Cytokine profiles using whole-blood assays can
discriminate between tuberculosis patients and healthy endemic controls in
a BCG-vaccinated population. J. Immunol. Methods 264:95–108.
Hussain, R., N. Talat, F. Shahid, and G. Dawood. 2007. Longitudinal tracking of cytokines after acute exposure to tuberculosis: association of distinct
cytokine patterns with protection and disease development. Clin. Vaccine
Immunol. 14:1578–1586.
Jalapathy, K. V., C. Prabha, and S. D. Das. 2004. Correlates of protective
immune response in tuberculous pleuritis. FEMS Immunol. Med. Microbiol.
40:139–145.
Katiyar, S. K., A. Sampath, S. Bihari, M. Mamtani, and H. Kulkarni. 2008. Use
of the QuantiFERON-TB Gold In-Tube test to monitor treatment efficacy in
active pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 12:1146–1152.
Kobashi, Y., et al. 2009. Transitional changes in T-cell responses to Mycobacterium tuberculosis-specific antigens during treatment. J. Infect. 58:197–
204.
Lalvani, A., et al. 2001. 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. 183:469–477.
Leyten, E. M., et al. 2006. Human T-cell responses to 25 novel antigens
encoded by genes of the dormancy regulon of Mycobacterium tuberculosis.
Microbes Infect. 8:2052–2060.
Millington, K. A., et al. 2007. Dynamic relationship between IFN-gamma
1386
31.
32.
33.
34.
35.
36.
BERTHOLET ET AL.
and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen
load. J. Immunol. 178:5217–5226.
Pai, M., et al. 2007. Sensitivity of a whole-blood interferon-gamma assay
among patients with pulmonary tuberculosis and variations in T-cell responses during anti-tuberculosis treatment. Infection 35:98–103.
Pai, M., L. W. Riley, and J. M. Colford, Jr. 2004. Interferon-gamma assays
in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect.
Dis. 4:761–776.
Pathan, A. A., et al. 2001. Direct ex vivo analysis of antigen-specific IFNgamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J. Immunol. 167:5217–5225.
Ribeiro, S., et al. 2009. T-SPOT.TB responses during treatment of pulmonary tuberculosis. BMC Infect. Dis. 9:23.
Ribeiro-Rodrigues, R., et al. 2002. Sputum cytokine levels in patients with
pulmonary tuberculosis as early markers of mycobacterial clearance. Clin.
Diagn. Lab. Immunol. 9:818–823.
Sauzullo, I., et al. 2009. In vivo and in vitro effects of antituberculosis
treatment on mycobacterial interferon-gamma T cell response. PLoS One
4:e5187.
CLIN. VACCINE IMMUNOL.
37. Snider, D. E., Jr. 1985. Bacille Calmette-Guerin vaccinations and tuberculin
skin tests. JAMA 253:3438–3439.
38. Ulrichs, T., R. Anding, S. H. Kaufmann, and M. E. Munk. 2000. Numbers of
IFN-gamma-producing cells against ESAT-6 increase in tuberculosis patients during chemotherapy. Int. J. Tuberc. Lung Dis. 4:1181–1183.
39. Vankayalapati, R., et al. 2003. Serum cytokine concentrations do not parallel
Mycobacterium tuberculosis-induced cytokine production in patients with
tuberculosis. Clin. Infect. Dis. 36:24–28.
40. Veenstra, H., et al. 2007. Changes in the kinetics of intracellular IFNgamma production in TB patients during treatment. Clin. Immunol. 124:
336–344.
41. Vekemans, J., et al. 2001. Tuberculosis contacts but not patients have higher
gamma interferon responses to ESAT-6 than do community controls in The
Gambia. Infect. Immun. 69:6554–6557.
42. WHO. 2007. Global tuberculosis control: surveillance, planning, financing,
vol. 376. World Health Organization, Geneva, Switzerland.
43. Wu-Hsieh, B. A., et al. 2001. Long-lived immune response to early secretory
antigenic target 6 in individuals who had recovered from tuberculosis. Clin.
Infect. Dis. 33:1336–1340.