ORIGINAL ARTICLE—LIVER, PANCREAS, AND BILIARY TRACT
Elevated frequency and function of regulatory T cells in patients
with active chronic hepatitis C
Kuo-Chih Tseng•Yun-Che Ho•Yu-Hsi Hsieh•
Ning-Sheng Lai•Zhi-Hong Wen•Chin Li•
Received: 20 June 2011/Accepted: 11 January 2012/Published online: 28 February 2012
? Springer 2012
role in the persistence of hepatitis C virus infection. The
aim of this study was to evaluate the frequency and func-
tion of Tregs in patients with chronic hepatitis C (CHC).
We enrolled 44 CHC patients with elevated
alanine aminotransferase (ALT) levels (CH group), 13
CHC patients with persistent normal ALT levels (PNALT
group), and 14 age-matched healthy subjects (HS group;
controls). Tregs were identified as CD4?, CD25?, and
forkhead box P3 (Foxp3)? T lymphocytes, using three-
color fluorescence-activated cell sorting (FACS). The
frequency of Tregs was determined by calculating the
percentage of CD4?CD25highT cells among CD4 T cells.
CD127 and CD45RA were also analyzed for subsets of
Tregs. The levels of serum transforming growth factor
(TGF)-b and interleukin (IL)-10 in immunosuppressive
assays were detected by enzyme-linked immunosorbent
assay (ELISA). The immunosuppressive abilities of Tregs
Regulatory T cells (Tregs) play a pivotal
were evaluated by measuring their ability to inhibit the
proliferation of effector cells.
Higher proportions of Tregs were found in the
CH and PNALT groups compared with the HS group. The
populations of CD127 low/negative and CD45RA negative
cells were higher in the CH group than in the PNALT
group. The expressions of IL-10 and TGF-b in the CH and
PNALT groups were significantly higher than those in the
HS group. In addition, the immunosuppressive ability of
Tregs from the CH group was increased relative to that in
the PNALT and the HS group.
CHC patients, irrespective of liver function,
had higher frequencies of Tregs than healthy subjects;
however, only CHC patients with inflammation showed
enhanced immunosuppressive function of Tregs.
Regulatory T cells
Hepatitis C ? Immunosuppression ?
Hepatitis C virus
Chronic hepatitis C
Regulatory T cell
Forkhead box P3
Transforming growth factor
Hepatitis C virus (HCV) is a small, enveloped, single-
stranded, positive-sense RNA virus that belongs to the
K.-C. Tseng ? Y.-H. Hsieh ? N.-S. Lai
Department of Internal Medicine, Buddhist Dalin Tzu Chi
General Hospital, Chia-Yi, Taiwan
K.-C. Tseng ? Y.-H. Hsieh ? N.-S. Lai
School of Medicine, Tzuchi University, Hualien, Taiwan
Y.-C. Ho ? C. Li ? S.-F. Wu (&)
Department of Life Science, Institute of Molecular Biology,
National Chung-Cheng University, No. 168, University Rd.,
Min-Hsiung, Chia-Yi 62102, Taiwan
Department of Marine Biotechnology and Resources,
National Sun Yat-sen University, Kaohsiung, Taiwan
J Gastroenterol (2012) 47:823–833
family flaviviridae and genus hepacivirus . After HCV
infection, the immune systems of 55–85% of infected
patients cannot eliminate the virus, and in these cases,
chronic hepatitis C (CHC) may develop .
Chronic hepatitis C is often asymptomatic, with persis-
tently normal serum alanine aminotransferase (ALT) levels
in 20–30% of patients, although it is usually associated
with fluctuating or persistently elevated ALT levels [2, 3].
Major long-term complications of CHC include cirrhosis,
end-stage liver disease, and hepatocellular carcinoma
(HCC) . Retrospective and prospective studies on the
long-term natural history of HCV infection have shown
that 15–20% of CHC patients develop cirrhosis within
30 years . Once cirrhosis occurs, the annual risk of
HCC, hepatic decompensation, and liver-related death is
approximately 1–4, 5, and 2–4%, respectively [4–7].
After HCV infection, interactions between innate and
adaptive immune responses play pivotal roles in the per-
petuation or clearance of HCV. Natural killer (NK) cells
are specialized lymphocytes that provide one of the first
lines of defense during HCV infection. An overall reduc-
tion in NK cell activity may cause a predisposition to
chronic HCV infection or, alternatively, HCV gene prod-
ucts may downregulate NK cell activity . Evidence also
suggests that the clearance and control of HCV infection is
dependent on vigorous, multispecific immune responses by
both CD8 and CD4 T lymphocytes . An early and per-
sistent Th1-dominant CD4 response appears to be critical
for preventing chronic infection, whereas a weak or absent
Th1 response with a more pronounced Th2 response is
associated with the development and maintenance of
chronic infection [10, 11].
Recently, regulatory T cells (Tregs) have been identified
as a specialized subset of T cells that can suppress auto-
reactive immune responses to maintain immunological
tolerance and inhibit autoimmunity . There are two
general categories of Tregs. One Treg subset develops
during the process of T-cell maturation in the thymus,
resulting in the generation of a naturally occurring popu-
lation of regulatory T cells. A second subset develops from
naı ¨ve CD4?CD25- T cells during immune responses in
the periphery . Although these Tregs are CD4? cells
with high expression of CD25?, the most reliable marker
for this subset is the transcription factor forkhead box P3
(Foxp3) . The virus-specific induction of Tregs may
have two different consequences. First, it may be an
important process that occurs to prevent excessive
immuno-pathological damage. Second, it may enable the
virus to establish viral persistence .
Many studies have investigated the relationship between
Tregs and the outcome of CHC infection [16–23]. The
majority of these studies showed that Tregs were present at
higher frequencies in patients with persistent infections and
elevated liver function than in patients who recovered and
had normal liver function and in normal subjects. These
findings resulted from the Treg-related suppression of
HCV-specific T-cell responses [16–22]. However, Bolac-
chi et al.  demonstrated that CHC patients with normal
ALT levels had higher levels of transforming growth factor
(TGF)-b production by Tregs than CHC patients with
elevated ALT levels. Of note, the majority of these studies
only focused on the frequency but not the function of
Our aim in this study was to investigate the frequency
and function of immune cells, including Tregs, in CHC
patients with elevated liver function and to compare these
patients with CHC patients who had persistently normal
liver function and healthy subjects.
Patients and methods
A total of 71 subjects who had been followed at the Dalin
Tzu Chi General Hospital in southern Taiwan during the
period between November 2008 and June 2010 were
recruited for this study. Among them, 44 were CHC
patients with elevated ALT levels (CH group), 13 were
CHC patients with persistent normal ALT levels (PNALT
group), and 14 were healthy subjects (HS group; con-
trols). The clinical backgrounds of these 3 groups were
similar, except for their serum AST and ALT levels
The criteria for inclusion in the CH group were a posi-
tive anti-HCV antibody test for at least 6 months, a serum
ALT level greater than the upper limit of the normal level,
and detectable serum HCV RNA. The criteria for inclusion
in the PNALT group were a positive anti-HCV antibody
test for at least 6 months, but with a normal ALT level for
more than 12 months. Exclusion criteria included malig-
nant neoplasms, decompensated liver disease, acute hepa-
titis, autoimmune diseases, alcohol abuse, positive hepatitis
B surface antigen, and HIV infection. The study was
approved by the Ethics Committee of the Dalin TzuChi
General Hospital (approval number: B09604008-1). All
patients signed written informed consents.
HCV quantification and genotyping
Serum HCV RNA levels were measured using the COBAS
TaqMan HCV assay (Roche Molecular Diagnostics, Basel,
Switzerland), with a lower limit of quantification of 25 IU
per ml. HCV genotyping was performed using the LIN-
EAR ARRAY Hepatitis C Virus Genotyping Test (Roche
824 J Gastroenterol (2012) 47:823–833
Preparation of peripheral blood mononuclear cells
Heparinized venous blood was obtained (10 ml), and cell
surface markers were analyzed immediately after collec-
tion. PBMCs were isolated by Ficoll/sodium isophthala-
mide density-gradient centrifugation (relative density =
1.077 g/ml) at 400g for 30 min at room temperature. Cells
at the interphase were collected, washed, and resuspended
in RPMI-1640 medium supplemented with 10% fetal calf
serum (FCS), 5 mg/ml L-glutamine, 100 U/ml penicillin,
and 100 lg/ml streptomycin .
Surface and intracellular antibody staining
The monoclonal antibodies used to detect cell surface
markers in direct immunofluorescence assays were anti-CD3
(clone HIT31, or UCHT1), anti-CD4 (clone RPA-T4,), anti-
CD25 (clone M-A251s), anti-CD45RA (clone HI100), anti-
CD56 (clone MEM188, or B159), and anti-CD127 (clone
HIL-7R-M21); all the antibodies were obtained from BD
Pharmingen (Franklin Lakes, NJ, USA). Negative control
samples were stained with isotype-matched control mono-
clonal antibodies (mAbs). After staining, the cells were
washed extensively and analyzed by flow cytometry. To
analyze the intracellular expression levels of Foxp3, freshly
isolated PBMCs were fixed with 1% paraformaldehyde and
permeabilized with 0.5% Triton X-100, followed by detec-
tion using anti-Foxp3 (eBioscience, San Diego, CA, USA).
Flow cytometry was performed with a Becton Dickinson
(Franklin Lakes, NJ, USA) FACScalibur cytometer, and the
data were analyzed using CellQuest software (BD Biosci-
ences, San Jose, CA, USA).
Isolation of different cell populations
PBMC single-cell suspensions were prepared from whole
blood and depleted of erythrocytes by ammonium chloride
lysis. To purify Tregs, the cell suspensions were incubated
with a human Treg separation cocktail containing biotin-
conjugated anti-CD8, CD11b, CD16, CD19, CD36, CD41a,
CD56, CD123, CD235a, and cd TCR, and APC-conjugated
anti-CD25 mAbs (BD Biosciences, San Jose, CA, USA).
Streptavidin-conjugated beads (BD Biosciences) were
added, and the bound cells were isolated using a BD IMag-
net. Bead-bound cells were used as supporting cells. Sus-
pensions ofthese cells were incubatedwith anti-APCcoated
beads (BD Biosciences), and then purified again using a BD
IMagnet. After this second round of cell purification, the
suspended cells were used as responder cells and the bead-
3 types of cells from the same donor were used.
Table 1 Characteristics of the study subjects
CharacteristicsCH group (n = 44)PNALT group (n = 13) HS group (n = 14)
56.0 ± 10.757.9 ± 7.054.9 ± 11.6 0.749c
CH vs. PNALT\0.001d
CH vs. HS\0.001d
PNALT vs. HS 0.02d
CH vs. PNALT\0.001d
CH vs. HS\0.001d
PNALT vs. HS 0.319d
CH vs. PNALT 0.077e
93.6 ± 54.423.5 ± 5.1 19.4 ± 3.3
137.8 ± 82.822.4 ± 7.220.1 ± 4.3
HCV RNA (106IU/ml)a,f
2.84 ± 3.712.64 ± 4.02NA
1 275 NA CH vs. PNALT: 0.699b
AST aspartate aminotransferase, ALT alanine aminotransferase, HCV hepatitis C virus, CH group chronic hepatitis C patients with elevated
alanine aminotransferase, PNALT chronic hepatitis C patients with persistent normal alanine aminotransferase, HS healthy subjects, NA not
aValues are expressed as means ± SD
bv2test or Fisher’s exact test
cAnalysis of variance (ANOVA)
fHCV RNA was undetectable for six subjects in the PNALT group
J Gastroenterol (2012) 47:823–833825
Detection of cytokines by enzyme-linked
immunosorbent assay (ELISA)
To measure plasma cytokine concentrations, samples were
obtained from each individual’s peripheral blood. The
resulting plasma or the supernatant of cocultured medium
was transferred to a microtube and stored at -80?C. The
sample was thawed at room temperature at the time of
cytokine measurement. Each cytokine was measured
according to the manufacturer’s instructions for each rele-
vant assay kit. The concentration of IL-10 was measured
detection limit was 2 pg/ml. The concentration of TGF-b
was measured with a commercial ELISA kit (Quantikine
MN, USA). The total TGF-b1 concentration was measured
according to the manufacturer’s instructions: 0.1 ml of 1 N
activate TGF-b1 and then incubated for 1 h at room tem-
of 1 N NaOH. The detection limit was 4.61 pg/ml.
Measurement of the immunosuppressive function
To evaluate the immunosuppressive function of Tregs,
supporting cells were resuspended in T-cell medium at a
concentration of 5 9 107cells/ml and incubated with
20 lg/ml mitomycin C (Sigma-Aldrich, Saint Louis, MO,
USA) at 37?C for 40 min. The cells were then washed
twice and resuspended in T-cell medium. Responder cells
were resuspended in T-cell medium at 106cells/ml and
incubated with 10 lM carboxyfluorescein diacetate succ-
inimidyl ester (CFSE) (Invitrogen, Grand Island, NY,
USA) at 37?C for 10 min. After a fourfold volume of pre-
chilled T-cell medium was added, the cells were washed
twice and resuspended in T-cell medium. Supporting cells
and responder T cells were cocultured at a ratio of 5:1 in
the presence or absence of activating anti-CD3 antibodies
(BD Biosciences) at 1 lg/ml. Immunosuppressive ability
was assessed by adding additional Tregs at a ratio of 1/5:1
or 1/2:1 to the effector T cells. After 4.5 days of culture,
the cells were evaluated by flow cytometry. The percent
suppression of proliferation by Tregs was calculated as
proliferation with Tregs
proliferation without Tregs
follows :1 ?
For the baseline variables, analysis of variance (ANOVA),
Student’s t-test, and the Mann–Whitney U-test were used
to compare the means of continuous variables. The v2test
was used for the comparisons between categorical
variables. Fisher’s exact test was used instead of the v2test
when the numbers were small. The percentages of cell pop-
ulations and cytokine expression were compared using Stu-
dent’s t-test. A P value of\0.05 was considered significant.
Lymphocyte populations were altered in the peripheral
blood of the PNALT and CH groups
To investigate whether CHC patients had significant
alterations in lymphocyte subpopulations in their periphe-
ral blood, we primarily focused on NK cells, CD4? T cells,
and CD8? T cells. The PBMCs of 44 CHC patients with
elevated liver function (CH group), 13 CHC patients with
normal liver function (PNALT group), and 14 age-matched
healthy subjects (HS group) were stained with anti-CD3,
anti-CD4, anti-CD8, anti-CD25, and anti-CD56 antibodies
and analyzed by flow cytometry. CD4? T cells were
identified as the CD3?CD4? population (Fig. 1a). The
percentage of CD4? T cells was increased in the CH and
PNALT groups compared with that in the HS group
(Fig. 1c; CH vs. HS: 34.158 ± 10.174 vs. 26.815 ±
8.362%, P = 0.014; PNALT vs. HS: 35.822 ± 10.400 vs.
26.815 ± 8.362%, P = 0.015). However, the percentage
of CD4? T cells did not significantly differ between the
CH and PNALT groups. NK cells were identified as the
CD3-CD56? population (Fig. 1b). The percentage of NK
cells was significantly decreased in the CH and PNALT
groups compared to that in the HS group (Fig. 1c; CH vs.
HS: 14.843 ± 9.120 vs. 30.430 ± 13.242%, P\0.001;
PNALT vs. HS: 18.774 ± 11.543 vs. 30.430 ± 13.242%,
P = 0.020). However, the percentage of NK cells did not
significantly differ between the CH and PNALT groups.
The CD3?CD8? population, which consisted of cytotoxic
anti-virus immune cells other than NK cells, also showed
no differences between these groups (Fig. 1c).
Regulatory T-cell populations were increased
in the PNALT and CH groups
As described above, the population of CD4? T cells was
increased in the CHC patients. CD4 T-helper cells include
a variety of subtypes, including Tregs. Human peripheral
blood contains heterogeneous populations of CD4?
CD25? cells with either high expression of CD25, which is
associated with regulatory functions, or moderate expres-
sion of CD25 [16, 26]. We defined a threshold for CD25
expression such that those cells that showed greater
expression than CD4-CD25? T cells were considered to
be CD4?CD25highTreg cells (Fig. 2b). As shown in
Fig. 2, Tregs were identified by CD3?CD4?CD25high
826J Gastroenterol (2012) 47:823–833
staining (Fig. 2a, b). Tregs were significantly increased in
both the CH and PNALT groups compared to the HS group
(Fig. 2c; CH vs. HS: 2.860 ± 1.459 vs. 1.521 ± 0.363%,
P = 0.001; PNALT vs. HS: 2.832 ± 1.416 vs. 1.521 ±
0.363%, P = 0.002).
CD127 and CD45RA expression in the PNALT
and CH groups
CD4?CD25highwere significantly increased in both the
PNALT and CH groups (Fig. 2c). It has been indicated that
low expression levels of CD127, the receptor alpha chain
of IL-7, identify CD4?CD25? cells that express a high
level of Foxp3 and have suppressive activities. To char-
acterize the CD cell populations in CHC patients, CD4? T
cells were examined for the expression of CD25 and
CD127. As shown in Fig. 3a, the population was defined by
CD127 low/- and CD25?. We compared the percentages
of CD4?CD25?CD127 low/- cells between the PNALT
and CH groups. The percentage was significantly higher in
the CH group compared to the PNALT group (Fig. 3b; CH
vs. PNALT: 18.016 ± 5.200
P = 0.018). We also detected the expression of CD45RA
in the PNALT and CH groups. As shown in Fig. 3c, the
expression of CD25? populations was mostly distributed
in the CD45RA- cells. We compared the percentages of
CD4?CD25?CD45RA- cells between the PNALT and
abovedata showed thatthe percentagesof
vs. 9.230 ± 2.789%,
CH groups. The percentage was significantly higher in the
CH group compared to the PNALT group (Fig. 3d; CH vs.
PNALT: 12.512 ± 3.999 vs 3.976 ± 1.107%, P = 0.003).
Increased Treg Foxp3 expression in the PNALT
and CH groups
Foxp3 is the most important transcription factor expressed
by Tregs that influences their survival, differentiation, and
function. To investigate the expression of Foxp3 in Tregs
from the three studied subject groups, freshly isolated
PBMCs were evaluated after intracellular staining with anti-
Foxp3 (Fig. 4a). The percentage of Foxp3-expressing Tregs
HS group (Fig. 4b; CH vs. HS: 2.263 ± 1.122 vs. 0.787 ±
0.751%, P = 0.007; PNALT vs. HS: 2.350 ± 1.292 vs.
0.787 ± 0.751%, P = 0.018); however, there was no dif-
ference between the CH and PNALT groups.
Increased IL-10 expression in regulatory T-cell
inhibition assay and TGF-b expression in PBMCs
in the PNALT and CH groups
To estimate the cytokine expression in the immunosup-
pressive assay of Tregs in the three studied subject groups,
we detected the IL-10 and TGF-b expression levels. The
supernatants of Tregs cocultured with effector T cells (1:2)
were collected from the HS, PNALT, and CH groups.
Fig. 1 Expression of CD4, CD8 and CD56 on peripheral blood
mononuclear cells (PBMCs) in the HS, PNALT, and CH groups.
PBMCs were isolated from the whole blood of healthy subjects (HS,
n = 14), chronic hepatitis C patients with persistent normal alanine
aminotransferase (PNALT, n = 13), and chronic hepatitis C patients
with elevated alanine aminotransferase (CH, n = 44). a CD4? T-cell
analysis of PBMCs was performed by anti-CD3 andanti-CD4 staining.
CD3?CD8? cells, CD3?CD4? cells, and CD3-CD56? cells in the
PBMCs were determined. CD3?CD4? cells were statistically signif-
icantly increased in the PNALT (P = 0.015) and CH (P = 0.014)
groups compared with the HS group (HS 26.815 ± 8.362%, PNALT
35.822 ± 10.400%,CH34.158 ± 10.174%); however, CD3-CD56?
cells were statistically significantly decreased in the PNALT (P =
0.020) and CH (P\0.001) groups compared with the HS group
(HS 30.430 ± 13.242%, PNALT 18.774 ± 11.543%, CH 14.843 ±
J Gastroenterol (2012) 47:823–833 827
ELISA was performed. The expression of IL-10 in the CH
and PNALT groups was significantly higher than that in the
HS group (Fig. 5a; CH vs. HS: 64.812 pg/ml ± 9.724 vs.
29.657 pg/ml ± 5.933, P\0.001; PNALT vs. HS: 72.818
pg/ml ± 11.989 vs. 29.657 pg/ml ± 5.933, P\0.001). The
IL-10 production was dependent on the ratio of cocultured
effector T cells and Tregs (E:T ratio). As shown in Fig. 5b,
the IL-10 concentration was highest at an E:T ratio of 1:1,
then it gradually decreased. TGF-b is important in the sup-
pressivefunctionofTregs. However, we could not detectany
differences between the three subject groups (Fig. 5c), prob-
ably due to the culture medium containing a high level of
TGF-b in serum. But we also evaluated the TGF-b concen-
tration in the serum of the three studied groups. The TGF-b
than that in the HS group (Fig. 5d; CH vs. HS: 898.213 pg/
ml ± 113.897vs.5.005 pg/ml ± 0.114,P\0.001;PNALT
vs. HS: 837.413 pg/ml ± 192.182 vs. 5.005 pg/ml ± 0.114,
Enhanced immunosuppressive ability of circulating
Tregs in CH patients
We next evaluated the immunosuppressive abilities of
Tregs. The ability of Tregs to mediate immunosuppression
was analyzed by coculturing Tregs with CFSE-labeled,
autologous CD4?CD25- responder T cells at different
ratios. After 4.5 days of culture, the cells were evaluated by
As a result of this stimulation, the mean fluorescence
intensity (MFI) of the dividing cells containing CFSE was
decreased, as shown in Fig. 6a. The decrease in the MFI of
the positive control sample without Tregs was the most
apparent. When Tregs were added to the coculture system,
the proliferation of the effector cells was inhibited to
varying extents. The histogram of one of the CH patients is
shown in Fig. 6a. The immunosuppressive ability of the
Tregs was determined by calculating the ratio of the MFI
from the effector cells cocultured with Tregs to the MFI of
Fig. 2 AnalysisofregulatoryTcellsamongthegroups.aPBMCswere
CD25 withintheT-cell gate.bCD4?CD25highTcells areshowninthe
rectangle drawn in the upper right region of the density plot illustrating
CD4?CD25highTreg cells. The threshold for CD25highexpression was
line); CD4?CD25lowand CD4?CD25- populations are also shown.
c The mean percentages and standard deviations of CD4?CD25high
cells in the CD4 T cells were calculated (HS 1.521 ± 0.363%, PNALT
2.832 ± 1.416%, CH 2.860 ± 1.459%), and these results were com-
pared between the HS andCH groups (P = 0.001) and between the HS
and PNALT groups (P = 0.002)
828 J Gastroenterol (2012) 47:823–833
the positive controls (effector cells only), as described in
the ‘‘Patients and methods’’ section.
When cocultured at an effector:Treg (E:T) ratio of 1:1/2
or 1:1/5, the suppressive percentages of Treg were calcu-
lated to be, for 1:1/2: HS 14.73%, PNALT 13.386%, CH
45.02%; for 1:1/5: HS 3.208%, PNALT 4.686%, CH
26.82%. The immunosuppressive abilities of the Tregs
were significantly increased in the CH group compared
with the PNALT and HS groups (CH vs. HS: 1:1/2,
P = 0.010; 1:1/5, P = 0.033; CH vs. PNALT: 1:1/2,
P = 0.013; 1:1/5, P = 0.055), but the immunosuppressive
ability was not significantly different in the PNALT and
HS groups (Fig. 6b). This indicated that the Tregs derived
from the CH group exhibited a stronger ability to suppress
effector T-cell functions than the Tregs from the PNALT
and the HS groups.
Fig. 3 CD127 and CD45RA
expression in the PNALT and
CH groups. a Expression of
CD127 and CD25 in CD4? T
cells in the PNALT and CH
groups. Plots were gated for
CD4? T cells. CD127 low/-
CD25? cells were gated in the
box R15. b The percentages of
CD127 low/- CD25? cells in
the PNALT and CH groups
were calculated (PNALT
9.230 ± 2.789%, CH
18.016 ± 5.200%, P = 0.018).
c Expression of CD45RA and
CD25 in CD4? T cells in the
PNALT and CH groups.
CD45RA and CD25 were
analyzed in CD4? T cells.
The major population
expressing CD25? was
CD45RA-. d The percentages
of CD45RA-CD25? cells in
the PNALT and CH groups
were calculated (PNALT
3.976 ± 1.107%, CH
12.512 ± 3.999%,
P = 0.003)
Fig. 4 Forkhead box P3 (Foxp3) expression of PBMCs in the HS,
PNALT, and CH groups. a Foxp3 expression in CD4? T cells was
detected by intracellular staining. b The mean percentages and
standard deviations of the Foxp3? cells were determined for the HS,
PNALT, and CH groups (HS 0.787 ± 0.751%, PNALT 2.350 ±
1.292%, CH 2.263 ± 1.122%). Foxp3? cells were statistically
significantly increased in the PNALT (P = 0.018) and CH (P = 0.007)
groups compared with the HS group
J Gastroenterol (2012) 47:823–833 829
In this study, we evaluated the frequency and function of
Tregs and the associated T-cell responses in PBMCs from
CHC patients with elevated ALT levels and compared
these data with the data for both CHC patients with per-
sistent normal ALT levels and healthy subjects. We dem-
onstrated that the frequencies of Tregs in the CH and
PNALT groups were higher than that in the HS group.
Furthermore, we also demonstrated that the functional
immunosuppression mediated by Tregs from the CH group
was stronger than that mediated by Tregs from the PNALT
and HS groups.
There are two plausible mechanisms for cytotoxic T
lymphocyte (CTL)-mediated HCV clearance from the liver
after HCV infection: (1) the induction of apoptosis in
infected hepatocytes (cytolytic mechanism) or (2) the
release of interferon (IFN)-c to suppress HCV replication
(non-cytolytic mechanism) . Only the former mecha-
nism leads to an elevation of ALT levels, due to hepatocyte
Chronic infections are usually marked by reduced
frequencies of virus-specific CD4 and CD8 T cells, sug-
gesting that the appropriate immune response required for
spontaneous recovery either fails to develop or is sup-
pressed . Approximately 15–30% of patients achieve
spontaneous viral clearance after the initial HCV-specific
CD4 and CD8 cell responses . Tregs appear to play an
important role in determining whether resolution or per-
sistence occurs during acute HCV infection . Smyk-
Pearson et al.  demonstrated that patients in whom an
acute hepatitis C infection had resolved showed a decrease
in Treg functional suppression compared with the level of
suppression seen with persistence. This phenomenon is
similar with regard to the status of CHC infection.
During chronic infection with HCV, HCV-specific CD4
and CD8 T cells respond to the HCV viremia, which causes
hepatocyte destruction. Our hypothesis was that the more
rigorous the T-cell response, the stronger the Treg response
would be to compensate for the destructive immune
responses. However, this stronger Treg response may also
cause viral persistence. Our study showed that the fre-
quencies of Tregs in the CH and PNALT groups were
higher than that in the HS group, a finding which was also
demonstrated in previous studies [16–22].
In addition to the frequency of Tregs, we also investi-
gated the immunosuppressive function of Tregs. Although
there was no statistically significant difference in the
Fig. 5 Cytokine expressions of regulatory T cells (Tregs) in the HS,
PNALT, and CH groups. a The supernatants of effector T cells
cocultured with Tregs (Teff/Treg = 2:1) were collected from the HS,
PNALT, and CH groups. Enzyme-linked immunosorbent assay
(ELISA) was performed to detect interleukin (IL)-10 (CH vs. HS,
P\0.001; PNALT vs. HS, P\0.001). b The Teff:Treg ratios from
one of the patients were 1:1, 2:1, and 5:1, and IL-10 was detected.
c The supernatants of effector T cells cocultured with Tregs (Teff/
Treg = 2:1) were collected from the HS, PNALT, and CH groups.
ELISA was performed to detect transforming growth factor (TGF)-b.
d Serum was collected from the HS, PNALT, and CH groups. ELISA
was performed to detect TGF-b (CH vs. HS, P\0.001; PNALT vs.
830J Gastroenterol (2012) 47:823–833
frequency of Tregs between the CH and PNALT groups,
there was a statistically significant difference in the func-
tional suppressive abilities of Tregs between these two
groups. That is, Tregs had more potent suppressive abilities
in CHC patients with inflammation. This result was con-
sistent with a previous study . Furthermore, our study
also demonstrated that there was a significant difference
between the frequency of Tregs in the PNALT and HS
groups, but there was no statistically significant difference
in the immunosuppressive function of Tregs between these
two groups. Combined with the baseline characteristics, we
concluded that CHC patients, irrespective of liver function,
had higher frequencies of Tregs than the healthy subjects;
however, the immunosuppressive function of Tregs was
enhanced only in CHC patients with inflammation (CH
group). There have also been a few studies that investi-
gated the functional suppression of Tregs in CHC patients
with and without abnormal liver function [16, 18, 19].
However, these studies focused on changes in Th1 cytokine
expression by CD4 T cells with or without Treg depletion.
IL-10 and TGF-b are two important cytokines for the
development and function of Tregs. The release of TGF-b
and IL-10 by Tregs suppresses effector T-cell functions and
immune responses. In our data, the IL-10 secretion deter-
mined from the Treg suppression assay was significantly
higher in CHC patients than in the healthy subjects;
furthermore, the TGF-b concentration in the serum of these
CHC patients was also significantly elevated compared to
that in the HS group. These findings indicated that the
immune condition in CHC patients is suppressed compared
with that in healthy subjects, and Tregs are more easily
induced in these patients .
Tregs appear to play an important role in determining
whether resolution or persistence occurs during acute HCV
infection. A better marker than the CD25 marker in CD4 T
cells is needed to discriminate Foxp3 Tregs in patients.
IL-7 receptor (CD127) appears to be a specific marker to
discriminate Foxp3 Tregs. Our present data showed that
the expression of CD127 low/- was more significantly
enhanced in the CH group compared to the PNALT group.
Whether these Tregs belong to the memory or naı ¨ve T-cell
compartment is still debatable. A previous study indicated
that thymus-derived Tregs that expressed CD45RA? were
naturally occurring Tregs with naı ¨ve phenotype . Our
present data showed that the CD4?CD25? populations in
the CH group mostly expressed CD45RA-, indicating that
these cells were not naı ¨ve, but inducible Treg cells. On
comparing the expression of CD45RA- populations in
CD4?CD25? cells in the CH and PNALT groups, we
found that the CH group had a significantly higher
expression level than the PNALT group, indicating that the
CHC patients with inflammation (CH group) had a higher
Fig. 6 Immunosuppressive ability of Tregs in the HS, PNALT, and
CH groups. Supporting cells, effector T cells, and Tregs were purified
from PBMCs with isolation kits using whole blood from the HS
(n = 5), PNALT (n = 5), and CH (n = 11) groups. a Immunosup-
pression was estimated using carboxyfluorescein diacetate succinim-
idyl ester (CFSE) dilution; the histogram from one sample from the
CH group is shown here. Supporting cells and effector T cells were
cocultured at a ratio of 5:1 and stimulated with anti-CD3 (1 lg/ml).
The immunosuppressive ability of Tregs was assessed using different
ratios of effector T cells to Tregs (1:1/5 or 1:1/2) and estimated by
determining the mean fluorescence intensity (MFI). b The suppressive
percentages were calculated (1:1/2: HS 14.73%, PNALT 13.386%,
CH 45.02%; 1:1/5: HS 3.208%, PNALT 4.686%, CH 26.82%), and
these results were compared between the HS and CH groups (1:1/2,
P = 0.010; 1:1/5, P = 0.033) and between the PNALT and CH
groups (1:1/2, P = 0.013; 1:1/5, P = 0.055)
J Gastroenterol (2012) 47:823–833831
level of inducible Tregs than the CHC patients with a
persistent normal ALT level (PNALT group), probably due
to the response to liver inflammation.
NK cells use a complex set of receptor interactions to
differentiate between ‘‘self’’ and ‘‘non-self’’  and these
cells constitute an important component of innate immune
responses to invading pathogens. NK cells recognize
infected cells in an antigen-independent manner, destroy
them using their cytotoxic activity, and rapidly produce
large amounts of IFN-c to activate cellular adaptive
immune responses . Reduced NK cell activity may
predispose individuals to CHC infection. In addition, HCV
gene products can also downregulate NK cell function .
In our study, there was a statistically significant decrease in
the percentage of NK cells in the CH and PNALT groups
compared with the HS group (Fig. 1b, c), a finding which
was consistent with a previous study . Golden-Mason
et al.  demonstrated that altered NK cell functions may
contribute to impaired cellular immune responses and the
chronicity of disease following HCV infection.
It remains difficult to determine whether Tregs play a
protective or detrimental role in chronic HCV infection. On
one hand, a weak Treg response accompanied by an
effective T-cell response against pathogens may induce
severe inflammation and tissue damage. On the other hand,
a strong Treg response accompanied by a weak T-cell
response can minimize the damage to tissues but may lead
to chronic infection. It remains unclear which mechanism
is responsible for the precise balance between inflamma-
tory and anti-inflammatory reactions.
The increased frequency and function of Tregs in the
CH group found in our study is reflective of the normal
physiological responses of CHC patients with viremia and
inflammation. However, increased Treg function cannot
restore liver function to a normal level. It has been sug-
gested that CHC patients’ viremia is the key factor that
induces sequential innate and adaptive immune responses.
Therefore, it is important to clear the HCV via the use of
interferon and ribavirin.
In conclusion, our study demonstrated that the frequency
of Tregs in the CH and the PNALT groups was higher than
that in the HS group; and the immunosuppressive function
of Tregs in the CH group was stronger than that in the
PNALT group and the HS group. Further sequential pro-
spective examinations of the frequencies and functions of
Tregs in CHC patients receiving antiviral therapy are
Tzuchi General Hospital through grant number DTCRD97-11.
This study was funded in full by the Dalin
Conflict of interest
The authors declare no conflicts of interest.
1. Hoofnagle JH. Course and outcome of hepatitis C. Hepatology.
2. MarcellinP,Le ´vyS,ErlingerS.TherapyofhepatitisC:patientswith
normal aminotransferase levels. Hepatology. 1997;26:S133–6.
3. Tassopoulos NC. Treatment of patients with chronic hepatitis C
and normal ALT levels. J Hepatol. 1999;31(Suppl 1):193–6.
4. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J
5. Fattovich G, Giustina G, Degos F, Tremolada F, Diodati G,
Almasio P, et al. Morbidity and mortality in compensated cir-
rhosis type C: a retrospective follow-up study of 384 patients.
6. Fattovich G, Stroffolini T, Zagni I, Donato F. Hepatocellular
carcinoma in cirrhosis: incidence and risk factors. Gastroenter-
7. Serfaty L, Aumaı ˆtre H, Chazouille `res O, Bonnand AM, Ro-
smorduc O, Poupon RE, et al. Determinants of outcome of
compensated hepatitis C virus-related cirrhosis. Hepatology.
8. Golden-Mason L, Rosen HR. Natural killer cells: primary target
for hepatitis C virus immune evasion strategies? Liver Transpl.
9. Diepolder HM. New insights into the immunopathogenesis of
chronic hepatitis C. Antiviral Res. 2009;82:103–9.
10. Cramp ME, Carucci P, Rossol S, Chokshi S, Maertens G, Wil-
liams R, et al. Hepatitis C virus (HCV) specific immune
responses in anti-HCV positive patients without hepatitis C vi-
raemia. Gut. 1999;44:424–9.
11. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC. Detection of
type 2-like T-helper cells in hepatitis C virus infection: impli-
cations for hepatitis C virus chronicity. Hepatology. 1997;25:
12. Sakaguchi S. Naturally arising Foxp3-expressing CD25? CD4?
regulatory T cells in immunological tolerance to self and non-
self. Nat Immunol. 2005;6:345–52.
13. Belkaid Y. Regulatory T cells and infection: a dangerous
necessity. Nat Rev Immunol. 2007;7:875–88.
14. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the
development and function of CD4? CD25? regulatory T cells.
Nat Immunol. 2003;4:330–6.
15. Alatrakchi N, Koziel M. Regulatory T cells and viral liver dis-
ease. J Viral Hepat. 2009;16:223–9.
16. Itose I, Kanto T, Kakita N, Takebe S, Inoue M, Higashitani K,
et al. Enhanced ability of regulatory T cells in chronic hepatitis C
patients with persistently normal alanine aminotransferase levels
than those with active hepatitis. J Viral Hepat. 2009;16:844–52.
17. Sugimoto K, Ikeda F, Stadanlick J, Nunes FA, Alter HJ, Chang
KM. Suppression of HCV-specific T cells without differential
hierarchy demonstrated ex vivo in persistent HCV infection.
18. Cabrera R, Tu Z, Xu Y, Firpi RJ, Rosen HR, Liu C, et al. An
immunomodulatory role for CD4(?)CD25(?) regulatory T
lymphocytes in hepatitis C virus infection. Hepatology. 2004;40:
19. Boettler T, Spangenberg HC, Neumann-Haefelin C, Panther E,
Urbani S, Ferrari C, et al. T cells with a CD4? CD25? regulatory
phenotype suppress in vitro proliferation of virus-specific CD8?
T cells during chronic hepatitis C virus infection. J Virol. 2005;
20. Rushbrook SM, Ward SM, Unitt E, Vowler SL, Lucas M,
Klenerman P, et al. Regulatory T cells suppress in vitro
832 J Gastroenterol (2012) 47:823–833
proliferation of virus-specific CD8? T cells during persistent Download full-text
hepatitis C virus infection. J Virol. 2005;79:7852–9.
21. Ebinuma H, Nakamoto N, Li Y, Price DA, Gostick E, Levine BL,
et al. Identification and in vitro expansion of functional antigen-
specific CD25? FoxP3? regulatory T cells in hepatitis C virus
infection. J Virol. 2008;82:5043–53.
22. Yoshizawa K, Abe H, Kubo Y, Kitahara T, Aizawa R, Matsuoka
M, et al. Expansion of CD4(?)CD25(?)FoxP3(?) regulatory T
cells in hepatitis C virus-related chronic hepatitis, cirrhosis and
hepatocellular carcinoma. Hepatol Res. 2010;40:179–87.
23. Bolacchi F, Sinistro A, Ciaprini C, Demin F, Capozzi M, Card-
ucci FC, et al. Increased hepatitis C virus (HCV)-specific CD4?
CD25? regulatory T lymphocytes and reduced HCV-specific
CD4? T cell response in HCV-infected patients with normal
versus abnormal alanine aminotransferase levels. Clin Exp
24. Lin CT, Yu MT, Li C, Ho YC, Shen CH, Liu DW, et al. Dys-
function of natural killer cells in patients with transitional cell
carcinoma. Cancer Lett. 2010;291:39–45.
25. Lutsiak ME, Semnani RT, De Pascalis R, Kashmiri SV, Schlom
J, Sabzevari H. Inhibition of CD4(?)25? T regulatory cell
function implicated in enhanced immune response by low-dose
cyclophosphamide. Blood. 2005;105:2862–8.
26. Smyk-Pearson S, Golden-Mason L, Klarquist J, Burton JR Jr,
Tester IA, Wang CC, et al. Functional suppression by FoxP3?
CD4? CD25(high) regulatory T cells during acute hepatitis C
virus infection. J Infect Dis. 2008;197:46–57.
27. Kanto T, Hayashi N. Immunopathogenesis of hepatitis C virus
infection: multifaceted strategies subverting innate and adaptive
immunity. Intern Med. 2006;45:183–91.
28. Bowen DG, Walker CM. Adaptive immune responses in acute
and chronic hepatitis C virus infection. Nature. 2005;436:946–52.
29. Valmori D, Merlo A, Souleimanian NE, Hesdorffer CS, Ayyoub
M. A peripheral circulating compartment of natural naive CD4
Tregs. J Clin Invest. 2005;115:1953–62.
30. Herman RB, Koziel MJ. Natural killer cells and hepatitis C: is
losing inhibition the key to clearance? Clin Gastroenterol Hepa-
31. Thimme R, Lohmann V, Weber F. A target on the move: innate
and adaptive immune escape strategies of hepatitis C virus.
Antiviral Res. 2006;69:129–41.
32. Golden-Mason L, Madrigal-Estebas L, McGrath E, Conroy MJ,
Ryan EJ, Hegarty JE, et al. Altered natural killer cell subset
distributions in resolved and persistent hepatitis C virus infection
following single source exposure. Gut. 2008;57:1121–8.
J Gastroenterol (2012) 47:823–833833