Tuberculosis 90 (2010) 252e261

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Tuberculosis
journal homepage: http://intl.elsevierhealth.com/journals/tube

IMMUNOLOGICAL ASPECTS

Regulatory T cell frequency and modulation of IFN-gamma and IL-17 in active and
latent tuberculosis
Nancy D. Marin a, c, Sara C. París a, Viviana M. Vélez a, d, Carlos A. Rojas b, Mauricio Rojas a, Luis F. García a, *
a
Grupo de Inmunología Celular e Inmunogenética, Centro de Investigaciones Médicas, Universidad de Antioquia, Medellín, Colombia
b
Grupo de Epidemiología, Facultad de Salud Pública, Universidad de Antioquia, Medellín, Colombia
c
NDM is recipient of a predoctoral scholarship from Colciencias, Bogotá, Colombia
d
VMV is recipient of a “Joven Investigador” award from Vicerrectoría de Investigaciones, Universidad de Antioquia, Medellín, Colombia

a r t i c l e i n f o s u m m a r y

Article history: Regulatory T cells (Tregs) play an essential role in immune homeostasis. In infectious diseases Tregs may
Received 24 February 2010 inhibit protective responses facilitating pathogen multiplication and dissemination, but they may also
Received in revised form limit the inflammatory response diminishing tissue damage. Although there is experimental and clinical
5 May 2010
evidence that Tregs are induced during Mycobacterium tuberculosis infection, their role in the immu-
Accepted 20 May 2010
nopathogenesis of tuberculosis is still not completely understood. In this study, the phenotype, frequency
and activity of circulating Tregs in active and latent tuberculosis were evaluated. Phenotypic analysis
Keywords:
showed that Tregs were CD4þCD25highFOXP3þCD45ROþCD127-. High levels of circulating Tregs were
Tuberculosis
Latent infection
found in patients with active pulmonary tuberculosis, compared to individuals with latent infection. Treg
Regulatory T cells activity was evaluated by ELISPOT by determining the effect of CD25þ cell depletion on the frequency of
Interferon gamma IFN-g and IL-17 producing cells after in vitro stimulation with ESAT-6, CFP-10 and PPD. Treg depletion
IL-17 increased the frequency of IFN-g producing cells, without affecting the frequency of IL-17 producing cells,
in both active and latent tuberculosis, irrespective of the antigen used. Neutralization of IL-10 did not
have any effect on the frequency of IFN-g and IL-17 producing cells. Altogether, these results suggest that
during active tuberculosis Tregs inhibit protective Th1 responses, but not the proinflammatory Th17
responses, facilitating mycobacterial replication and tissue damage.
Ó 2010 Published by Elsevier Ltd.

1. Introduction the development or reactivation of ATB in absence of an immu-

According to the World Health Organization, one third of the
word population is infected with Mycobacterium tuberculosis (Mtb),
but only 10% of the infected individuals would develop active TB
during their lifetime.1 It is well known that bacterial, host and
environmental factors influence the development of active TB.2e4
In most of cases, the host immune response controls the Mtb
replication and a latent infection (LTBi) is established, but when the
host immune response fails to control the tubercle bacilli replica-
tion, active TB (ATB) is developed.5 Latency is maintained by a fine
balance between the pathogen persistence and the immune
response, therefore perpetuating the risk of reactivation. Thus, the
immune response against Mtb infection is associated with
the establishment of latency, but the phenomena responsible for
* Corresponding author. LFG. Grupo de Inmunología Celular e Inmunogenética,
Sede de Investigación Universitaria, Cra 53 N 61-30, Lab. 410, Medellín, Colombia. .
Tel.: þ57 4 219 6446; fax: þ57 4 219 6450
E-mail address: lfgarcia@udea.edu.co (L.F. García).
nosuppressive event are not well understood.
Tuberculosis has many clinical manifestations, but the most
common form is pulmonary tuberculosis. Individuals with
pulmonary tuberculosis spread bacilli in aerosol, transmitting the
infection to other persons. The extent and the strength of the
exposure affect the transmission rate, thus a higher exposure to M.
tuberculosis results in a higher risk of infection and active disease.6,7
Household contacts (HHC) in close contact to patients with ATB are
exposed to high bacterial loads and therefore, they have a higher
probability to be infected and develop active disease.6
T cell responses are critical components of the protective
immunity against M. tuberculosis. IFN-g producing Th1 cells are
essential to control the mycobacterial replication by inducing
macrophages antimycobacterial mechanisms and activating CD8þ
cytotoxic cells,8e10 but Th1 cells alone do not explain the resistance/
susceptibility to infection and disease.11,12 Th1 cells are important
for protection, but they are also involved in the inflammation and
tissue damage that occurs during active TB.13 The more recently
described Th17 cells have also been associated with Mtb infec-
tion.12,14 IL-17 is produced early during immune response against

1472-9792/$ e see front matter Ó 2010 Published by Elsevier Ltd.

doi:10.1016/j.tube.2010.05.003
 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261
Mtb and it has been proposed to be associated with reactivation in
latent TB infected individuals.15,16 Importantly, the kinetics of IFN-g
and IL-17 production and the phenotypic and functional charac-
teristics of Th1 and Th17 cells are different,15,17,18 as well the
susceptibility to Treg suppression, which is diminished in Th17
cells.19 Knowing the role of IFN-g in the defense against Mtb and its
ability to inhibit IL-17 production,20 in addition to the proposed role
for IL-17 in tuberculosis, it is important to understand their regu-
lation and function during latent and active TB. There is evidence
that many ATB patients present suppression of Mtb specific T cell
responses, including decreased production of IL-2 and IFN-g,[21e23]
suggesting that T cell responses during infection are subject to
regulatory mechanisms; however, little is known about IL-17
producing cells during Mtb infection and the development of active
disease.
The suppressive mechanisms described in the immune response
against Mtb include increased activity of regulatory T cells.24e34
Tregs are recruited to infected organs down-regulating the
immune response against Mtb infection and preventing the clear-
ance of M. tuberculosis by suppressing antigen specific CD4þ cells
and interfering with antigen presenting cells.28,30,33 Thus Tregs
have the capacity to control the tissue damage while dampening
the adequate control of mycobacterial replication,29 allowing the
persistence and the establishment of a chronic infection, but they
may also be involved in the reactivation and dissemination of Mtb.
Regulatory T cells with the CD4þCD25þFOXP3þ phenotype
(Tregs) represent 5e10% of circulating CD4þ cells,25,27,35 but in
humans only the subset expressing higher levels of CD25 (a chain of
IL-2R) exhibit a strong suppressive capacity.36 Tregs are a key
component of peripheral tolerance suppressing auto-reactive T
cells and preventing autoimmune diseases. However, there is
strong evidence that Tregs are involved in the immune response
against Mtb and have been detected in a higher frequency in TB
patients peripheral blood mononuclear cells (PBMC) associated
with decreased effectors responses.25,27,30e32
The immunological and physiological events triggered after the
establishment of active TB have been extensively studied, but those
events responsible for maintaining the latency and causing reac-
tivation in immunocompetent individuals are not yet well defined.
Therefore, in this study the frequency of Tregs and their effect on
IFN-g and IL-17 production in response to mycobacterial antigens
were studied in individuals with ATB, LTBi individuals with a high
level of exposure (HHC) and LTBi individuals with a low level of
exposure (no HHC) to Mtb. Results show an increased frequency of
circulating CD4þCD25high and CD4þCD25highFOXP3þ cells in ATB
patients compared to LTBi individuals. The functional evaluation of
these cells showed a higher capacity of CD4þCD25high cells to
inhibit the IFN-g production and a lesser capacity to inhibit IL-17
producing cells in both ATB and LTBi individuals. These results
suggest an important role of Tregs in the reactivation of the latent
infection and the development of active tuberculosis by decreasing
IFN-g responses, while IL-17 may continue facilitating the accu-
mulation of cells in the inflamed tissues.
2. Materials and methods
2.1. Study population
Thirty-one newly diagnosed, active TB (ATB) patients were
recruited at the Tuberculosis Control Program in Medellín
(Colombia) and its metropolitan area. ATB patients had acid fast
smear or culture positive for Mtb. ATB patients were studied before
or within the first 2 weeks of anti-TB treatment. Thirty-eight
subjects with latent TB infection (LTBi) were selected according to
an IFN-g positive response to CFP-10, as evaluated by ELISA in
253
seven-days whole blood culture supernatants, as previously
reported by our Group.5 LTBi individuals included 26 HHC of
pulmonary TB patients who were followed for 3 years between
2005 and 2008 in our cohort study, remaining healthy without
clinical evidence of active TB.6 A household contact was considered
to be someone who had spent time regularly (weekly) in the same
household as the index case (active TB) for at least one month prior
to the time when the index case’s diagnosis was confirmed. Twelve
LTBi individuals who were not household contacts (no HHC) and
who did not have a recent documented exposure to active TB were
selected among laboratory personal according a positive response
to CFP-10 as described by del Corral H et al.6 All subjects studied
were HIV negative, as tested by DoubleCheckGoldÔ HIV 1&2 kit
(Orgenics, Courbevole, France), following the manufacturer
instructions. The study was approved by the Ethical Committee of
the Instituto de Investigaciones Médicas of the Universidad de
Antioquia and a written informed consent was obtained from all
participants. Individuals infected with HIV, using immunosup-
pressive drugs, with diabetes, or younger than 15 years old were
excluded.
2.2. Sample preparation
Ten to 20 mL of blood were obtained using heparin as antico-
agulant, and the PBMC were obtained by Ficoll-Hypaque density
gradient centrifugation (Biowittaker, Walkersville, MD). PBMC
were washed twice in PBS (Invitrogen, Carlsbad, CA) and counted in
a hemocytometer. Viability, as tested by trypan blue staining, was
always 95%.
2.3. Mycobacterial antigens
Recombinant ESAT-6 and CFP-10 were provided by the
Department of Microbiology and Immunology at Colorado State
University, Fort Collins, CO through the “Tuberculosis Vaccine
Testing and Research Material Contract No. HHSN26266400091C
NIH, NIAID (N01-AI-40091). PPD (RT50) was obtained from Statens
Serum Institute (Copenhagen, Denmark).
2.4. Phenotypic analyses
The expression of CD4, CD25 and FOXP3 were determined in
freshly isolated PBMC. One million PBMCs were incubated at
room temperature for 30 min with anti-CD4-FITC (clone RPA-T4)
plus anti-CD25-PeCy5 (clone M-A251) (BD Biosciences, San Diego,
CA). Mouse IgG1k-PeCy5 (clone MOPC-21) was used as isotype
control. Thereafter, cells were washed with PBS and non-per-
meabilized cells were fixed with 2% paraformaldehyde (J.T.Baker.
Phillipsburg, NJ). For FOXP3 detection, cells stained for CD4 and
CD25 were fixed and permeabilized using anti-human FOXP3
staining buffer (eBioscience). Thereafter, anti-FOXP3-PE (clone
PCH101) or rat IgG2a-PE isotype control (Clone eBR2a) was added
for 30 min 4  C. One hundred cells were acquired and the analysis
included identification of CD25þFOXP3þ cells and
CD25 high
FOXP3 cells within the CD4þ gate. The CD25þ pop-
þ
ulation was defined by isotype control and the CD25h population
was defined as the population expressing higher CD25 MFI on
a dotplot. To further evaluated the memory phenotype of Tregs as
previously reported,37 CD45RO expression on CD4þCD25þ/high-
þ
FOXP3 cells was evaluated in some representative samples, using
anti-CD45RO-APC (clone UCHL-1) plus anti-CD4-FITC (clone RPA-
T4) and anti-CD25-PeCy5 (clone M-A251) (BD Biosciences. San
Diego, CA), followed by intracellular staining with anti-FOXP3-PE.
Mouse IgG1k-PeCy5 (clone MOPC-21), mouse IgG2ak-APC (Clone
eBM2a) and rat IgG2a-PE (Clone eBR2a) were used as isotype
254 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261
controls. One hundred thousand cells were acquired in FACS
Canto II Flow Cytometer (San Jose, CA) and analyzed using
Cytomation Summit (Fort Collins, CO) and BD FACSDiva Software
v6.1.2. (San Jose, CA). Furthermore, for a better characterization of
Tregs, the CD127 expression that had been associated with cells
with effector phenotype, and lacking in Tregs,26,38 was evaluated
in 1  106 PBMC cells using anti-CD127-FITC (clone eBioRDR5)
(eBioscience) plus anti-CD4-ECD (clone SFCl12T4D11) (T4)
(Beckman Coulter. San Diego, CA) and anti-CD25-PeCy5 (clone M-
A251) (BD Biosciences), followed by intracellular staining with
anti-FOXP3-PE. Mouse IgG1k-FITC (clone MOPC-21) and mouse
IgG1k-PeCy5 (clone MOPC-21) were used as isotype controls. To
determine CD127 expression, 1  105 cells were acquired in
a CoulterÒ EPICSÒ XLÔ Flow Cytometer (Hialeah, FL).
2.5. ELISPOT
The frequency of IFN-g and IL-17 producing cells was evaluated
by ELISPOT using human IFN-g and IL-17 ELISPOT kit (eBioscience),
according to the manufacturer’s instructions. Briefly, each well in
MultiScreenHTS 96-well filter plates (Millipore. Billerica, MA) was
covered overnight with either anti-IFN-g or anti-IL-17 capture
antibody at room temperature, followed by blockade with RPMI-
1640 (Invitrogen) supplemented with 10% Fetal Bovine Serum
(Invitrogen) plus Penicillin/streptomycin (Biowittaker) (complete
medium) at room temperature for 1e2 h. Then, 1.5  105 PBMC per
well were cultured in duplicate wells with complete medium in the
presence of ESAT-6 (1 mg/ml), CFP-10 (5 mg/ml), and PPD (10 mg/ml)
for 48 h at 37  C, 5% CO2. Non-stimulated wells were used as
controls. After incubation, supernatants were discarded and the
plates washed, followed by incubation with anti-IFN-g or anti-IL-17
detection antibodies for 2 h at room temperature. The wells were
washed again and HRP-streptavidin was added for 45 min, light
protected, and after washing, the AEC substrate (BD Pharmingen)
was added. The reaction was stopped with distilled water. When
the plates were dried, the spot forming units were determined in an
ImmnunoSpot ReaderÒ (CTL, Shaker Heights, OH). Readings
obtained in the nil control were subtracted from samples stimu-
lated with antigens. The spot forming units (SFU) are reported as
SFU  106 cells.
2.6. Evaluation of the suppressive function of Tregs
To evaluate Treg activity two strategies were used. First, the
frequency of IFN-g and IL-17 producing cells in non-depleted and
CD25high-depleted PBMC cultures stimulated with ESAT-6, CFP-10
and PPD was compared. Second, CD4þCD25high cells, obtained by
sorting, were added back into CD25high-depleted PBMC cultures,
and the IFN-g and IL-17 production in response to CFP-10 and PPD
was compared with CD25high-depleted, non-reconstituted PBMC
cultures, and non-depleted PBMC cultures.
2.6.1. Depletion of regulatory T cells
The depletion of Tregs was performed using the Human
Regulatory T Cell Isolation kit (R&D systems, Minneapolis, USA)
following manufacturer instructions. Briefly, 6e7106 PBMC were
washed in MagCellet buffer 1 and incubated for 15 min at 4  C
with suboptimal amounts of anti-CD25 ferrous beads (8 ml
compared to 15 ml recommended by manufacturers), ensuring the
depletion of the CD25high population. Thereafter, 1 ml of Mag-
Cellet buffer 1 was added and the mix incubated for 6 min on
the MagCellet magnet, allowing the CD25þ cells to attach to the
magnet. CD25-depleted PBMC were collected and washed with
complete medium. Cells were counted and the efficiency of
CD4þCD25high depletion was confirmed by flow cytometry using
anti-CD4 plus anti-CD25 followed by intracellular staining with
anti-FOXP3 antibodies, as described above.
To determine the effect of CD25high depletion, 1.5  105 non-
depleted PBMC or Treg-depleted PBMC were cultured in ELISPOT
plates in duplicate wells in absence or presence of ESAT-6 (5 mg/ml),
CFP-10 (5 mg/ml) and PPD (10 mg/ml) for 48 h at 37  C, 5% CO2. Non-
stimulated plates were used as controls. After incubation, the spot
forming units were determined as described above.
2.6.2. CD4þCD25high Treg reconstitution assay
Ten million PBMCs were stained with anti-CD4-FITC and
anti-CD25-PECy5 antibodies and sorted in a MoFloÔ XDP Cell
Sorter (Beckman Coulter. Brea, CA). CD4þ cells were gated on
a dotplot allowing the selection of CD4þCD25high positive cells
to be sorted. Sorted cells were collected in RPMI-1640 plus
penicillin/streptomycin, and added to Treg-depleted PBMC
cultures at the same proportion of Tregs that were present
before depletion of Tregs with magnetic beads. The effect of
CD4þCD25high reconstitution on IFN-g and IL-17 producing cells
was compared with the response of non-depleted PBMC
cultures and CD4þCD25high-depleted PBMC in response to CFP-
10 and PPD and evaluated by ELISPOT as described above. The
purity of CD4þCD25high cells sorted was 80% with a FOXP3
expression 85%.
2.7. Neutralization of IL-10
For IL-10 neutralization, 1.5  105 PBMCs were preincubated
in duplicate wells in ELISPOT plates in absence or presence of
0.125 mg/mle2 mg/ml of neutralizing anti-human IL-10 antibody
(R&D systems), as suggested by the manufacturer, or 0.25 mg/
mle2 mg/ml of goat IgG Isotype control (R&D systems) for
30 min at 37  C. Thereafter, CFP-10 (5 mg/ml) and PPD (10 mg/ml)
were added for 48 h at 37  C, 5% CO2. The ELISPOT was per-
formed as described above and the SFU are reported as
SFU  106 cells.
2.8. Statistical analysis
The frequency of CD4þCD25þ/highFOXP3þ Tregs in LTBi no
HHC, LTBi HHC and ATB individuals were compared by Krus-
kalleWallis and Dunn’s post test. Wilcoxon test was used for
evaluate differences between non-depleted and Treg-depleted
PBMCs. Statistical differences and significance are shown in each
graph. Statistical significance was considered when p  0.05. All
analyses were carried out using the Prism 5 software (GraphPad,
San Diego, CA).
3. Results
3.1. Clinical characteristics of studied groups
Twelve individuals with LTBi no HHC, 26 individuals with LTBi
HHC and 31 smear or culture positive ATB patients were studied.
Their median (range) ages were: 31(27e61) years for LTBi no HHC,
38(15e68) years for LTBi HHC and 45 (16e70) years for ATB
patients. The male/female ratio for each group was: 4/8 for LTBi no
HHC, 9/17 for LTBi HHC and 22/9 for ATB (Table 1). Twenty-eight
ATB patients had pulmonary tuberculosis, 2 patients had military
tuberculosis and another one had laryngeal tuberculosis. Most
pulmonary TB individuals had a high bacterial load as detected by
acid fast staining of sputum smear.
 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261
Table 1
Demographic and clinical characteristics of the populations studied.
Latent TB no Latent TB
HHC HHC
Median age (range) 31 (27e63) 36 (15e68)
Gender Male 4 9
Female 8 17
AFB sputum þ 3
þþ
þþþ
Without
data
Type of clinical
disease
3.2. Regulatory T cells CD4þCD25þFOXP3þ are CD127 and
CD45ROþ
For the characterization of regulatory T cells, different surface
and intracellular markers associated to Tregs were evaluated.
Although CD25 is a marker of regulatory T cells, it is also expressed
by effector cells after activation, albeit at lower expression.39
Additionally, the transcription factor FOXP3 is considered the best
marker for regulatory T cells40 and therefore the phenotypic anal-
ysis of Tregs was done within the CD4þ population according to the
total and high CD25 expression, in addition to FOXP3 (Figure 1A).
Previous reports have shown the differential expression of CD127
(a chain of IL-7R) between effector and regulatory T cells. Regula-
tory T cells are CD127 negative whereas effector cells are positive
for this marker.26,38 Thus, CD127 expression on
CD4þCD25þ/highFOXP3þ Tregs was evaluated and 97% of them
were negative for CD127 (Figure 1B). Additionally, Tregs have been
reported to exhibit a memory phenotype,37 thus the expression of
CD45RO was also evaluated and more than 95% of
CD4þCD25þ/highFOXP3þ cells were found CD45ROþ (Figure 1C).
Therefore, the complete phenotype of Tregs studied under our
experimental conditions was CD4þCD25highFOXP3þ CD127
CD45ROþ.
3.3. Active TB patients have a higher frequency of Tregs and HHC
have lower levels
To determine whether the frequency of circulating CD4þ cells
expressing low or high CD25 is different in LTBi and ATB, the
percentage of CD4þCD25þ and CD4þCD25high in LTBi no HHC, LTBi
HHC and ATB individuals were compared. There were not differ-
ences in the frequency of CD4þCD25þ cells among the groups (data
not shown). ATB patients had a higher frequency of CD4þCD25high
cells (5.35% [Interquartile range, IQR 2.6e6.3]), compared to LTBi
HHC individuals who had the lowest frequency of CD4þCD25high
cells (median 1.7 [IQR 1.2e2.8]) (p < 0.001) (Figure 2A). The
frequency of CD4þCD25þFOXP3þ cells was similar among LTBi no
HHC, LTBi HHC and ATB individuals (Figure 2B). Active TB patients
had a higher frequency of CD4þCD25highFOXP3þ (median 2.0 [IQR
1.3e2.7]) compared to LTBi HHC (median 0.95 [IQR 0.6e1.8])
(p < 0.001). No differences were observed between LTBi no HHC
compared to LTBi HHC and ATB patients (Figure 2C). When ATB
patients were compared with the two groups of LTBi individuals
considered together, the percentages of CD4þCD25high and
CD4þCD25highFOXP3þ (data not shown) were still increased in the
ATB group (p ¼ 0.0025 and p ¼ 0.004, respectively). Thus in the
remaining sections of results, the 2 groups of HHCs will be shown
together. These results indicate that during active TB there is
255
a higher frequency of circulating CD4þCD25highFOXP3þ Tregs
compared with latent TB infection.
Active TB
3.4. Active TB patients have more IFN-g producing cells in response
42 (16e70) to mycobacterial antigens than LTBi individuals
22
9
It has been reported that during ATB there is a reduced
6 production of IFN-g in response to different stimuli.21,41,42 There-
15 fore, the frequency of IFN-g and IL-17 producing cells in response to
4 ESAT-6, CFP-10 and PPD was evaluated by ELISPOT in 48 h cultures.
ATB patients, compared to LTBi individuals, showed higher
28
pulmonary
frequency of IFN-g producing cells in response to CFP-10
1 laryngeal (p ¼ 0.0071) and PPD (p ¼ 0.0009). No differences were found in
2 miliary response to ESAT-6 between the studied groups (Figure 3A), nor in
the frequency of IL-17 producing cells in response to ESAT-6, CFP-10
and PPD. In addition the IFN-g/IL-17 ratio in response to ESAT-6,
CFP-10 and PPD was evaluated and the IFN-g/IL-17 ratio in response
to CFP-10 was 14.7 [IQR 9.9e32.9] for ATB patients and 8.7 [IQR
2.9e14.6] for LTBi individuals (p ¼ 0.0097). No differences were
found in the IFN-g/IL-17 ratio in cultures stimulated with ESAT-6 or
PPD (Figure 3B).
3.5. Tregs suppress IFN-g producing cells but not IL-17 producing
cells
There is evidence that Tregs can suppress both Th1 and Th17
responses,34,43 but IL-17 producing cells seem to be less susceptible
to suppression by Tregs.19 To investigate the effect of Tregs on IFN-g
and IL-17 production in response to mycobacterial antigens in LTBi
individuals and ATB patients, non-depleted PBMC and Treg-
depleted-PBMC cultures were stimulated with ESAT-6, CFP-10 and
PPD and the frequency of the IFN-g and IL-17 producing cells was
evaluated by ELISPOT. Depletion reduced the number of
CD4þCD25high cells by 94  5.6% in LTBi individuals and 91  11.8%
in ATB patients (Figure 4 and data not shown). The depletion of
CD25high cells resulted in a significant increase in the frequency of
IFN-g producing cells responding to ESAT-6, CFP-10 and PPD in LTBi
and ATB subjects (Figure 5A). On the contrary, depletion of
CD4þCD25high cells did not affect the frequency of IL-17 producing
cells, with the exception of the cultures of LTBi individuals stimu-
lated with PPD that showed a lower frequency of IL-17 in CD25high-
depleted cultures (p ¼ 0.034) (Figure 5B). These findings suggest
that Tregs have a less suppressive capacity on IL-17 production, and
a higher susceptibility of IFN-g producing cells to the suppression
by regulatory T cells.
To further confirm that Tregs are responsible for the suppression
observed, reconstitution of Tregs in Treg-depleted PBMC cultures
was performed. CD4þCD25high Tregs were purified by sorting and
added back into Treg-depleted PBMC cultures maintaining the
initial proportion of Tregs observed before depletion. The results
were not conclusive (data not shown) because there was a high
variability among LTBi and ATB individuals studied.
3.6. Tregs suppress IFN-g and IL-17 production is not IL-10-
dependent
Regulatory T cells suppress effector responses by different
mechanisms.44,45 One of these mechanisms is controlled by IL-10.
To evaluate whether the effect of CD4þCD25highFOXP3þ cells on
IFN-g producing cells observed under our experimental conditions
is IL-10-dependent, different concentrations of neutralizing anti-IL-
10 or isotype control antibody (0.125 mg/ml to 2 mg/ml) were added
to PBMC cultures stimulated with CFP-10 and PPD. However, the
addition of IL-10 did not affect the response to CFP-10 and PPD in
either LTBi or ATB individuals (Figure 6). These results suggest that
256 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261

Figure 1. Phenotypic characterization of Tregs according to CD4, CD25, FOXP3, CD127 and CD45RO expression. Cells were stained with anti-CD4, anti-CD25, anti-CD27 and anti-
CD45RO followed by intracellular staining with anti-FOXP3. One hundred thousand cells were analyzed for CD25/FOXP3 expression and the CD127, CD45RO expression were
evaluated among CD4þ cells. (A) Top CD4þCD25þFOXP3þ T cells and bottom CD4þCD25highFOXP3þ T cells. (B) More than 97% of CD4þCD25hFOXP3þ cells were CD127 negative and
(c) more than 95% were CD45RO positive. A representative experiment is shown.

IL-10 is not involved in the suppression of IFN-g and IL-17
responses to mycobacterial antigens under our experimental
conditions.
4. Discussion
The events responsible for reactivation of tuberculosis in indi-
viduals latently infected with M. tuberculosis are poorly understood.
Patients with active tuberculosis frequently have decreased levels
of IFN-g and IL-2, and high levels of immunomodulatory cytokines
IL-10 and TGF-b in response to mycobacterial antigens.21,25,46,47
Tregs, which are increased during active TB, have been associated
with the regulation of immune functions such as self-tolerance,
autoimmunity and anti-tumor response,48,49 but they also have
been associated with the regulation of the immune response in
infectious disease.50,51 In some conditions Tregs may regulate
effector cells during long-persistent diseases protecting them from
the tissue damage caused by effector cells,52,53 but during a chronic
infection, like M. tuberculosis infection, they may be deleterious
because they may down regulate antigen specific T cells, damp-
ening the effective macrophage activation and therefore the Mtb
replication control.29,30,33,54 However, the role of Tregs in TB is not
well understood; nor it is clear whether their expansion is a cause
or a consequence of the disease. Probably they are expanded as an
adaptive host response to limit the inflammatory reaction and
tissue damage induced during the immune reaction against the
mycobacteria. But it is also possible that they are expanded in
response to M. tuberculosis infection by recognition of particular
bacterial products, such as ManLAM that promotes Treg expansion
in a PGE2-dependent manner33 or through the induction of IL-10
 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261
Figure 2. Frequency of circulating (A) CD4þCD25high, (B) CD4þCD25þFOXP3þ, and (C)
CD4þCD25highFOXP3þ cells in individuals with latent and active TB. PBMC were stained
with anti-CD4-FITC plus anti-CD25-PeCy5, followed by intracellular staining with anti-
FOXP3-PE. One hundred thousand cells were analyzed and the total and high CD25
expression was evaluated among CD4þ cells. (A) ATB patients had higher frequency of
CD4þCD25high T cells, whereas LTBi HHC had the lowest frequency. No differences were
257
and TGF-b produced during the infection, as supported by previous
reports showing increased levels of IL-10 and TGF-b in patients
with active TB.46,47,55,56
In this study, we compared the frequency of Tregs and their
suppressive capacity on IFN-g and IL-17 production in individuals
with active TB and two groups of latently infected individuals.
Latent TB infection in our study was confirmed by a positive IFN-g
response to CFP-10 as previously defined.6 They were classified as
HHC or not-HHC according to whether or not they had a recent and
prolonged exposure to a TB index case. HHCs were exposed to high
bacterial loads and probably this exposure had conditioned their
specific immune response and the ability of establish an effective
response against M. tuberculosis infection.
The phenotypic characterization of human Tregs is difficult
because they lack specific markers. In this study we used the most
accurate available markers: CD25high and FOXP3 expression,57 and
additionally, the expression of CD45RO and the lack of CD127
expression confirmed their phenotype as regulatory T cells. The
expression of CD45RO and the lack of CD127 suggest an activated
memory phenotype of these cells and it is in concordance with the
high expression of CD25.26,37,38 As previously reported by other
authors, we found a higher frequency of CD4þCD25highFOXP3þ cells
in patients with active TB, compared to individuals latently infected
with M. tuberculosis,25,30,32 but no differences were observed
between no HHC and HHC LTBi individuals. This finding might be
explained by the time elapsed (about 2 years) between the initia-
tion of anti-TB treatment of the index cases and the recruitment of
their HHCs for this study. The low levels of CD4þCD25highFOXP3þ
cells in HHC LTBi support their ability to control the mycobacterial
replication, despite their high exposure to the mycobacteria, pre-
venting reactivation of latent TB and the development of active
disease.
In agreement with previous reports using the same procedure,58
we found a higher frequency of IFN-g producing cells in ATB
patients in response to CFP-10 and PPD, compared to LTBi indi-
viduals. However, these results are not in agreement with other
reports that show decreased IFN-g production during ATB.23,41,42
The explanation for such discrepancy could be the culture time
and the type of T cell involved. Whereas in short-term cultures
(24e48 h), as used herein, IFN-g is produced mainly by effector T
cells that do not require proliferation to initiate cytokine produc-
tion, in long-term cultures (120e144 h) IFN-g is produced mainly
by central memory T cells that require IL-2-dependent prolifera-
tion.59,60 It is also known that TB patients have decreased IL-2
production in response to different mycobacterial antigens.21,41
The hallmark of the regulatory T cells is their capacity of control
effector T cell responses, like cytokine production and cell prolif-
eration. Thus to assess Treg activity in ATB patients and LTBi indi-
viduals, the frequency of IFN-g and IL-17 producing cells in
non-depleted and Treg-depleted PBMC cultures stimulated with
ESAT-6, CFP-10 and PPD was evaluated by ELISPOT. The depletion of
Tregs in both ATB patients and LTBi individuals PBMC cultures led
to an increased frequency of IFN-g producing cells, indicating that
Tregs were actively functioning. However, the depletion of Tregs in
PBMC cultures did not affect the frequency of IL-17 producing cells
in response to the antigens used, except in response to PPD in
latently infected individuals, indicating a differential susceptibility
of Th1 and Th17 cells to the suppression exerted by Tregs. The
reason why IL-17 producing cells are less susceptible to the
observed in LTBi no HHC compared to LTBi HHC and ATB individuals. (B) The
proportion of CD4þCD25þFOXP3þ Tregs was not different among studied groups. (C)
Active TB group had a higher frequency of CD4þCD25highFOXP3þ compared to LTBi
HHC individuals. KruskalleWallis test and Dunn’s multiple comparison post test
***p < 0.001.
258 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261

Figure 3. Comparison of the frequency of IFN-g and IL-17 producing cells in ATB patients and LTBi individuals in response to ESAT-6, CFP-10 and PPD. (A) PBMC from ATB patients
and LTBi individuals were stimulated with ESAT-6, CFP-10 and PPD and the frequency of IFN-g and IL-17 producing cells was evaluated by ELISPOT as described in Materials and
Methods. ATB patients compared to LTBi individuals had a higher frequency of IFN-g producing cells in response to CFP-10 and PPD. (B) The IFN-g/IL-17 ratio was compared between
LTBi individuals and ATB patients in response to ESAT-6, CFP-10 and PPD. ATB patients had a higher IFN-g/IL-17 ratio, compared to LTBi individuals, in response to CFP-10
(p ¼ 0.0097). Mann Whitney test was used and p values are shown in the graphs.

Figure 4. Effectiveness of CD4þCD25high depletion. Seven million PBMC were depleted of CD25high using anti-CD25 ferrous beads as described in Materials and Methods. The
effectiveness of depletion was evaluated in the cell fraction that was not attached to the magnet. (A). The figure shows a representative example of LTBi (n ¼ 22) and ATB (n ¼ 15) .
 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261 259

Figure 5. Effect of Treg depletion on the frequency of IFN-g and IL-17 producing cells in response to mycobacterial antigens. One hundred and fifty thousand non-depleted and
Tregs-depleted PBMC, as described in Materials and Methods, were stimulated with ESAT-6, CFP-10 and PPD for 48 h at 37  C, thereafter, the SFU were determined by ELISPOT. (A)
Frequency of IFN-g producing cells in response to ESAT-6, CFP-10 and PPD. The depletion of CD25high cells increased the frequency of IFN-g producing cells in LTBi individuals and
ATB patients in response to ESAT-6, CFP-10 and PPD. (B) Frequency of IL-17 producing cells in response to ESAT-6, CFP-10 and PPD. The frequency of IL-17 producing cells was lower
in Treg-depleted cultures from LTBi individuals in response to PPD, but no differences were observed in response to CFP-10 and PPD in LTBi and ATB individuals. Wilcoxon test was
used and p values are shown for each graph.

suppressive effect of Tregs is not yet clear; however, it is possible
that Th17 cells are dependent on TGF-b, produced by Tregs for their
expansion or differentiation.61 In fact, in LTBi individuals, PBMC
stimulation with PPD down regulated IL-17 producing cells in Treg-
depleted PBMC, compared to non-depleted PBMC. This finding is
not in agreement with a recent report34 showing in TST þ individ-
uals that both IFN-g and IL-17 are susceptible to the suppressive
effect of Tregs. One possible explanation may be the differences in
the methodology used to evaluate this phenomenon. Also, it must
be noted that our ATB patients were studied before or within the
first 2 weeks of anti-TB treatment. Although it is unlikely that such
a short time under treatment would decrease the number or the
activity of Tregs, we cannot rule out this possibility, since in the
guinea pig model of TB it has been demonstrated that the standard
anti-TB treatment eliminates Tregs.62 Unfortunately, our experi-
ments of Treg reconstitution did not provide consistent results. It is
possible that manipulation of the Tregs during the sorting proce-
dure affected their suppressive capacity, but more probably that the
260 N.D. Marin et al. / Tuberculosis 90 (2010) 252e261
Figure 6. Effect of anti-IL-10 on the frequency of IFN-g producing cells in response to
PPD and CFP-10. PBMC (1.5  105) were preincubated in duplicate wells in ELISPOT
plates with either 0.25 mg/mle2 mg/ml of neutralizing anti-human IL-10 antibody or
goat IgG Isotype control for 30 min at 37  C. Thereafter, CFP-10 (5 mg/ml) and PPD
(10 mg/ml) were added during 48 h and the frequency of IFN-g producing cells was
determined by ELISPOT as described in Materials and Methods. The figure shows the
results of one ATB patient of 4 experiments done in ATB (2) and LTBi (2) subjects.
amount of Treg cells added back to the depleted cells, which in our
experiments was equivalent to the pre-sorting percentage, was not
enough to suppress the effector cell response, since other authors
have used a higher proportion of Treg/effector cells.32,63
Despite the high levels of Tregs in inflamed tissues during
tuberculosis disease,25 the persistence of the inflammatory process
may indicate a selective suppressive function of different compo-
nents of the adaptive immune response. It is possible to suggest
that the high suppressive capacity of Tregs on IFN-g producing cells
and the low suppressive capacity over IL-17 producing cells
observed in our experiments, allow the persistence of IL-17
producing cells in inflamed tissues and thus the perpetuation or
recrudescence of the inflammatory reaction.15,64
IL-17 is considered a proinflammatory cytokine and it has been
proposed to participate early in the defense against M. tuberculosis,
due to its capacity to induce cytokine and chemokine production,
favoring the recruitment of other cells, including neutrophils, to the
sites infected with M. tuberculosis.65 The role of neutrophils in
tuberculosis is controversial.66e68 Neutrophils are important
components of the innate immune system and are considered the
first line of defense against many invading microorganisms.69 On
the other hand, previous studies considered the neutrophils to be
deleterious in the defense against M. tuberculosis. High levels of
these cells have been observed in patients with active disease70 and
TB susceptible animals were found to accumulate neutrophils in TB
lesions compared to resistant animals.67,71
Regulatory T cells have an arsenal of suppressor mechanisms
including IL-10 production,44 high level of this cytokine have been
detected in patients with active TB.72 In tuberculosis, IL-10 is
produced as a result of the chronic stimulation by mycobacterial
antigens and produced by Tregs and Tr1 regulatory cells.73,74 IL-10
production in patients with active disease is associated with anergy
to the stimulation with mycobacterial antigens,47 dampening
proliferation and cytokine production by effector cells. However, in
agreement with previous reports,33 the neutralization of IL-10 in
cultures stimulated with CFP-10 and PPD showed no association of
IL-10 with the inhibition of IFN-g and IL-17 production, suggesting
that Tregs use another suppressive mechanism, different from IL-10
production, to inhibit effector T cell responses. Future studies
should focus on the elucidation of the suppressive mechanisms
used by Tregs to inhibit proliferation and cytokine production in
response to mycobacterial antigens by TB patients, since the
increased frequency of circulating Tregs and their suppressive
activity on IFN-g production during active disease and their low
suppressive capacity on IL-17 producing cells support their
involvement in the development of tuberculosis disease. Currently
the role of Tregs in the latency phenomenon is not clear and more
studies are necessary to clarify it, and to possibly used them as
predictive biomarkers for the development of active TB in people
with a high risk of infection or disease by M. tuberculosis.
Acknowledgements
The authors thank the patients and the healthy volunteers for
their acceptance to participate in this study. We also thank the
Tuberculosis Control Programs of the Servicio Seccional de Salud de
Antioquia and the Secretaria de Salud de Medellín for allowing us to
have access to the clinical records of patients. This work was sup-
ported by Colciencias, (Bogotá, Colombia) grant 1115-408-20488
Funding: None
Competing interests: None declared.
Ethical approval: Not required.
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