Scand J Med Sci Sports 2011 & 2011 John Wiley & Sons A/S

doi: 10.1111/j.1600-0838.2010.01288.x

Effect of exercise training intensity on murine T-regulatory cells and

vaccination response
J. Wang1,2,*,w, H. Song3,*, X. Tang3, Y. Yang2, V. J. Vieira4, Y. Niu3, Y. Ma1,5
1
Key Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng, China, 2Institute of Molecular Medicine, Henan
University, Kaifeng, China, 3College of Physical Education, Henan University, Kaifeng, China, 4Obesity and Metabolism Laboratory,
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Universit, Boston, Massachusetts, USA, 5Institute of
Immunology, College of Medicine, Henan University, Kaifeng, China
Corresponding author: Junpeng Wang, PhD, Institute of Molecular Medicine, College of Medicine, Henan University, Kaifeng
475004, China. Tel/fax:186 378 388 0398. E-mail: jpwangchina@henu.edu.cn
Accepted for publication 13 December 2010

To understand the underlying mechanism(s) for the effect of
exercise at different intensities on T cell and DNA vaccina-
tion responses, we treated mice in a training protocol with
regular moderate-intensity exercise (MIE) or prolonged,
exhaustive high-intensity exercise (HIE). After 6 weeks of
training, splenocytes were isolated to evaluate cytokine
expression and T-regulatory (Treg) cell proportion by RT-
PCR and FACS, respectively. Another set of mice that
completed the same training protocol were used to deter-
mine DNA vaccination responses. These mice were immu-
nized three times with HBV DNA vaccine at 2-week
intervals and euthanized on day 14 after the last immuniza-
tion. Serum and splenocytes were isolated to determine
humoral and cell-mediated immunity (CMI). Results
showed that HIE increased anti-inflammatory cytokine
expression and CD41CD251 Treg cell proportion. Further,
HIE decreased IFN-c expression, T-lymphocyte proli-
feration, and antigen-specic cytotoxic response in HBV
DNA vaccine-immunized mice. MIE did not change
anti-inflammatory cytokine expression or CD41CD251
Treg cell proportion but increased pro-inflammatory cyto-
kine expression and augmented antigen-specific CMI.
Thus, MIE lower the risk of cancer and infectious illness
through enhancing the pro-inflammatory responses. By
contrast, HIE might increase the risk of common infections,
such as upper respiratory tract infection, due to an up-
regulation of CD41CD251 Treg cells and anti-inflamma-
tory responses.

Exercise, depending on its intensity, can have either
positive or negative effects on immune function and
general health (Pedersen & Hoffman-Goetz, 2000).
Regular moderate-intensity exercise (MIE) enhances
immune functions compared with the typical seden-
tary individuals in terms of potentiation of T cell-
mediated immunity (CMI), NK cell cytotoxicity, and
Th1 response in human or animal models (Sugiura et
al., 2001; Davis et al., 2004; Murphy et al., 2004).
However, prolonged, exhaustive high-intensity exer-
cise (HIE) and intensive periods of endurance exercise
training may impair immune function, increasing
susceptibility to infections and decreasing NK cell
activity, pro-inflammatory cytokine (Interleukin [IL-
2]) expression, and IFN-g production (Davis et al.,
1997; Davis et al., 1998; Steensberg et al., 2001). The
*Contributed equally.
w
Current address: Nutrition Immunology Laboratory, Jean Mayer
USDA Human Nutrition Research Center on Aging at Tufts
University, 711 Washington Street, Boston, Massachusetts 02111,
USA.
J. W. and Y. M. share senior authorship.
negative effects of HIE and positive effects of MIE,
however, do not extend equally to all aspects of
immune function. Interestingly, a recent study of the
patients with type 2 diabetes mellitus (DM) showed
that regular Tai Chi Chuan (TCC), a type of tradi-
tional Chinese martial art exercise, alters the Th1/Th2/
T-regulatory (Treg) balance by increasing the gene
encoding forkhead/winged helix transcription factor
(Foxp3) but not TGF-b expression in patients with
type 2 DM or health middle-aged volunteers (Yeh
et al., 2006; Yeh et al., 2009).
CD41CD251 Treg cells, a CD41 T cell subtype,
specifically express Foxp3, a master specific and
functional marker of Treg cells (Sakaguchi, 2005).
In addition, these cells constitutively express IL-2R
a-chain (CD25) and suppress immune responses via
cell contact-dependent mechanisms (i.e., natural
Treg cells). Studies show that the depletion of these
cells causes autoimmune disease and enhances the
immune response to foreign antigens (Furuichi et al.,
2005; Sakaguchi, 2005; Fernandez et al., 2008).
However, whether prolonged, exhaustive HIE may
change Treg cell number and suppressive function,

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Wang et al.
which is regarded as a factor that increases the risk
for upper respiratory tract infection (URTI) (Cur-
otto de Lafaille & Lafaille, 2009), is still unclear.
The purpose of the present study was to examine
whether regular MIE and prolonged, exhaustive HIE
affect CD41CD251 Treg cell proportion and DNA
vaccination responses and if so, through what me-
chanism(s), utilizing a mouse model of exercise. Our
objectives were to (1) measure pro- and anti-inflam-
matory cytokines and CD41CD251 Treg cells in
exercise-trained mice; (2) determine whether regular
MIE and prolonged, exhaustive HIE affect humoral
and cellular immune responses in exercised mice
vaccinated with the HBV DNA vaccine encoding
hepatitis B surface antigen (HBsAg, pVAX-S2), as a
model DNA vaccine.

Materials and methods

Experimental animals and reagents
Six- to 8-week-old syngeneic, female C57BL/6 mice were
purchased from the Animal Institute of the Chinese Medical
Academy (Beijing, China) and housed under a 12 h light cycle.
The mice were provided with pathogen-free water and food,
and were randomly distributed among the experimental groups.
All management procedures were approved by the Institutional
Animal Care and Use Committee of Henan University. Chinese
hamster ovary cells, mammalian cells for transfection, expres-
sion, and large-scale recombinant protein production, expres-
sing HBsAg (rHBsAg) were purchased from China North
Pharmaceutical Group Corp. (NCPC, Hebei, China). The
HBsAg-derived peptide S208-215 (ILSPFLPL; H-2Kb-re-
stricted) and ovalbumin peptide 257–264 (SIINFEKL; H-
2Kb-restricted) were synthesized by GL Biochem Co. Ltd.
(Shanghai, China). Plasmid pVAX-S2, encoding the HBV
surface antigens preS2 and S, was constructed using pVAX1
as described previously (Wang et al., 2008). The plasmid was
maxi-prepared, purified by PEG8000 precipitation, and then
dissolved in saline at a concentration of 1 mg/mL.
Exercise program
Seventy-two mice were divided into four groups: naı̈ve, MIE,
HIE, and treadmill control (TC). The exercise regimen was 5
days a week for 6 weeks. It has been shown that the percentage
of maximal oxygen consumption (%VO2max) of small rodents
running on the treadmill can be predicted by their speed or
relative intensity of exercise (Fernando et al., 1993). Based on
this study, the following exercise protocols were used for mice
to achieve MIE and HIE (about 70% and 491% of
%VO2max, respectively). Two different experiments were per-
formed, shown schematically in Fig. 1. The mice in the MIE
group were subjected to treadmill exercise on a level belt
without any slope for 30 min at 8–18 m/min during the first
week and for 60 min at 18 m/min during the next 5 weeks. The
mice in the HIE group trained five times for 30 min each on a
treadmill set at 15 m/min (5% gradient) during the first week,
five times for 60 min each at 15–26.8 m/min (5% gradient)
during the second week, and five times for 60 min each at
26.8 m/min (10% gradient) during the next 4 weeks. To
control for any non-exercise effects of treadmill running
such as handling, novel environment, noise, and vibration,
animals of the TC group were kept in the same room and
placed on a slow-moving treadmill (5 m/min, 5 min/day,
Fig. 1. Experimental design for evaluation of the effects of
MIE and HIE on inflammatory cytokine production, T-
regulatory (Treg) cell proportion, and HBV DNA vaccine
responses. Thirty-two mice were randomized to four groups
(8/group, high-intensity exercise [HIE], moderate-intensity
exercise [MIE], treadmill control [TC], and naı̈ve) as de-
scribed in the ‘‘Materials and methods.’’ Following 6 weeks
of prescribed exercise, mice were sacrificed and splenocytes
were collected to determine the effects of MIE and HIE on
cytokine production and Treg cell proportion (a). Forty
mice in four groups (n 5 10/group) were given the same
exercise-training as mentioned above and then immunized
with HBV DNA vaccine three times to assess the effects of
MIE and HIE on HBV DNA vaccine responses (b). Serum
was collected at time points as indicated for Ab analysis and
splenocytes were collected to determine T cell proliferation,
intracellular cytokines, and cytotoxicity.
5 days/week). Within 16–20 h after the completion of 6 weeks
of exercise according to their respective program, 32 mice were
euthanized by the inhalation of carbon dioxide. Splenocytes
were isolated as required for the evaluation of cellular surface
markers, intracellular staining, and RT-PCR. In addition,
after the completion of 6 weeks of exercise according to their
respective program, 40 mice were immunized with HBV DNA
vaccine pVAX-S2 on days 0, 14, and 28 as described in our
previous study (Wang et al., 2008), during which time they
continued running. Serum samples were collected from the
retro-orbital sinus before and after immunization at 2 week
intervals. The mice were euthanized by the inhalation of
carbon dioxide on day 14 after the third immunization, and
splenocytes were isolated to evaluate T cell proliferation,
cytotoxic activity, and intracellular staining. Twenty mice
were used to evaluate T cell proliferation and cytokine
production and the remaining 20 mice were used to determine
in vivo cytotoxic activity.

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Analysis of cell surface markers and intracellular cytokine
expression
Fluorescent-conjugated monoclonal antibodies recognizing
CD4, CD25, IFN-g, IL-10, Foxp3, and isotype controls
were purchased from Biolegend (San Diego, California,
USA). Purified antibodies to CD16/CD32 were purchased
from BD Biosciences Pharmingen (San Jose, California,
USA). Within 16–20 h after the completion of 6 weeks of
exercise, single-cell splenocyte suspensions were harvested and
2  106 cells were restimulated for 4 h with 50 ng/mL PMA
and 500 ng/mL ionomycin (both from Sigma, St. Louis,
Missouri, USA) in the presence of monensin (GolgiStop, BD
Pharmingen, San Jose, California, USA), and then 1  106
cells were stained as described previously (Tai et al., 2008).
Cellular fluorescence was detected and 2  104 cells were
acquired using the FACSCalibur system. The data were
analyzed using FlowJo software.
Detection of antigen-specific cytokine production
Fourteen days after the third immunization, single-cell sple-
nocyte suspensions were harvested as described previously
(Wang et al., 2008) and these cells at 2  106 cells in a 24 well
plate were re-stimulated with HBsAg-derived peptide S208–
215 (10 mg/mL) and anti-CD28 (5 mg/mL) mAb for 6 h at 37 1C
and 5% CO2. For intracellular staining of IFN- g and IL-4,
cells were re-stimulated for the last 4 h with 50 ng/mL PMA
and 500 ng/mL ionomycin in the presence of monensin. One
million cells were harvested and stained as described in the
above-mentioned methods. Cellular fluorescence was detected
and 2  104 cells were acquired using the FACSCalibur
system. The data were analyzed using FlowJo software.
Exercises, Treg cells and vaccination
T cell proliferation
Fourteen days after the third immunization, single-cell sple-
nocyte suspensions were harvested and the cells stained with
1 mM CFSE as described previously (Wang et al., 2008). CFSE
is an ester that can diffuse into cells, where it reacts with amine
groups to become fluorescent. The fluorescence is stably
retained in cells and equally distributed between daughter
cell populations with every cell division. Cell cultures were
prepared by inoculating triplicate 96-well round-bottom plates
with 5  105 cells/mL prepared in RPMI 1640 medium con-
taining 5% FCS. The plates were incubated at 37 1C in a 5%
CO2 incubator. T cell proliferation was evaluated by a FACS
assay in cells re-stimulated with rHBsAg. The data were
analyzed using FlowJo software.
In vivo cytotoxic assay
In vivo cytotoxic assays were carried out as described pre-
viously (Wang et al., 2008). In brief, single-cell splenocyte
suspensions from naı̈ve mice were pulsed with HBsAg CTL
peptide S208–215 and labeled with a high concentration
(5 mM) of CFSE (CFSEhigh); these cells served as the target
cells. An equal fraction of splenocytes was pulsed with OVA
peptide (negative control) and labeled with a low concentra-
tion (0.5 mM) of CFSE (CFSElow); these cells served as the
non-target control. The target and control cells were mixed
together and injected into immunized mice at 2  107 total
cells in 200 mL PBS via tail vein on day 14 after the third
immunization. Four hours after injection, the mice were
euthanized by CO2 inhalation and their splenocytes isolated.
The labeled cells were analyzed based on their differential
CFSE fluorescence intensities using the FACSCalibur system.
Specic lysis was calculated according to the following formula:
% specic lysis 5 [1  (% specic peptide  loaded target cells/%
control peptide  loaded target cells)]  100%.

RT-PCR
Total RNA was extracted from the splenocytes and reverse-
transcribed. The resulting cDNAs were PCR-amplified with
the primers listed in Table 1, separated on a 1.5% agarose gel,
and visualized by staining with ethidium bromide.
Statistical analysis
Results are expressed as means  SEM. Statistical analysis
was conducted using Sigmastat software. Signicant differences
were determined using ANOVA for different exercises effect
and was followed by Fisher’s least signicance difference post
hoc test for individual comparisons. Po0.05 indicated statis-
tical significance.

Antibody ELISA Results

Serum samples were analyzed by ELISA on day 14 after the
third immunization. Total IgG binding to HBsAg was mea-
sured with an ELISA kit according to the manufacturer’s
instructions (SIIC Kinghaw Biotech Co. Ltd., Beijing, China)
and expressed in international units. The amount of total anti-
HBsAg antibody was calculated as described previously
(Wang et al., 2008).
Effect of MIE and HIE on anti- and pro-inflammatory
cytokine expression
Cytokines play important roles in the polarization of
immune responses. To determine the cytokine pro-
files of C57BL/6 mice subjected to an MIE/HIE
program, the genes, IL-12p40 (for IL-12), TGF-b,

Table 1. The Primers of target genes

Target gene Primers sequences Annealing temperatures (1C)

b-actin Sense: 5 0 -GGGACCTGACAGACTACCTCAT-3 0 60

Anti-sense: 5 0 -CAAGAAGGAAGGCTGGAAA-3 0
IL-12p40 Sense: 5 0 -CTGGCCAGTACACCTGCCAC-3 0 59
Anti-sense: 5 0 -GTGCTTCCAACGCCAGTTCA-3 0
IL-2 Sense: 5 0 -TCCACTTCAAGCTCTACAG-3 0 55
Anti-sense: 5 0 -GAGTCAAATC CAGAACATGC C-3 0
TGF-b Sense: 5 0 -CTCCCACTCCCGTGGCTTCTAG-3 0 60
Anti-sense: 5 0 -GTTCCACATGTTGCTCCACACTT-3 0
Ebi3 Sense: 5 0 -CTTTGTGGCTGAGCGAAT-3 0 58
Anti-sense: 5 0 -CAGTCACTTGGTTTCCCATA-3 0

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Wang et al.
Fig. 2. Effects of exercise on pro- and anti-inflammatory
cytokine expression, as determined by RT-PCR. Total RNA
was collected from the splenocytes of mice subjected to
moderate- or high-intensity exercise (MIE/HIE) or from
naı̈ve or TC mice after 6 weeks. The mRNA levels of IL-2,
IL-12p40, EBi3, and TGF-b were analyzed by RT-PCR and
electrophoresis (a) and were normalized according to b-actin
to evaluate the relative expression of the target (b). Results
means  SEM, n 5 8. *Po0.05 compared with the naı̈ve
and TC group.
and Ebi3 (for IL-35) were detected by RT-PCR,
while IL-10 expression was examined by FACS
analysis. As shown in Figs 2 and 3, HIE up-regulated
TGF-b, Ebi3, and IL-10 expression and down-regu-
lated IL-12p40 expression compared with the naı̈ve
and TC groups. By contrast, MIE increased the
expression of IL-12p40 but had no effect on the
expression of TGF-b, Ebi3, and IL-10.
HIE increases IL-2R but reduces IL-2 and IFN-g expression
IL-2 is a key cytokine promoting T cell proliferation,
and changes in its production are often associated
with T cell activation and proliferation. We therefore
examined whether exercise of varying intensities
alters IL-2 production. Our results showed that,
compared with the two control groups, IL-2
mRNA expression was lower in the HIE group and
higher in the MIE group (Fig. 2). By contrast,
production of the T cell cytokine IFN-g was inhib-
ited by HIE (Fig. 3a and c), consistent with finding in
the expression of pro-inflammatory cytokine IL-12
(Fig. 2).
Because IL-2 up-regulates IL-2R expression and
exerts its effects on T cells through the high-affinity
IL-2R, the effects of HIE and MIE on IL-2Ra
4
(CD25) expression in splenocytes were examined.
FACS analysis showed that both HIE and MIE
increased CD25 expression in total splenocytes
(Fig. 4a and b).
Effect of exercise on CD41CD251 Treg cells
To further investigate whether HIE/MIE affects the
CD41CD251 Treg cells, splenocytes from exercise-
trained mice were isolated and T cell surface makers
were co-stained and detected by FACS. Our results
indicated that the proportion of CD41CD251 T cells
was significantly higher in the HIE group than in
either of the control groups, whereas there was no
significant difference between the MIE group and the
controls (Fig. 4c). In addition, HIE but not MIE
markedly increased Foxp3 expression, a CD41
CD251 Treg cell-specific marker (Fig. 4d). These
results suggest that HIE, but not MIE, may impair
immune response in mice by increasing Treg cells.
IL-10 is a key immunomodulatory factor that
inhibits the release of pro-inflammatory cytokines
by innate immune cells (Maynard et al., 2007).
Because IL-10 is produced by CD41 T cells, we
asked whether exercise of varying intensities alters
the production of IL-10. Our results showed that IL-
10 expression by CD41 T cells was significantly
higher in HIE compared with control groups (Fig.
4e), whereas no effect was seen for MIE (Fig. 4e).
These results also suggest that HIE may impair
immune responses possibly through an increase in
IL-10 production by CD41 T cells.
Effect of exercise on HBV DNA vaccination
DNA vaccines have a number of advantages over
conventional vaccines, including the ability to induce
strong humoral and cellular immune responses. We
therefore examined whether exercise of varying in-
tensities differentially affects humoral and cellular
immunity, specifically, in terms of the immune re-
sponse to HBV DNA vaccination, as a model DNA
vaccine. We first analyzed the ability of HIE/MIE to
elicit a humoral and cellular immune response to
HBV DNA vaccine. The running C57BL/6 mice were
vaccinated intramuscularly with 100 mg of pVAX-S2.
Total serum IgG against HBsAg was determined by
quantitative ELISA. As shown in Fig. 5, significant
differences in total anti-HBsAg IgG were not de-
tected among the four groups.
To further determine the effects of HIE/MIE on
CMI responses, T cell proliferation was evaluated in
vitro. Figure 6 shows that the MIE group exhibited a
higher level of T cell proliferation, whereas the HIE
group exhibited a lower T cell proliferation com-
pared with the two control groups.
 Exercises, Treg cells and vaccination

Fig. 3. Effects of exercise on IL-10 and IFN-g expression, as determined by FACS analysis. Splenocytes isolated from C57BL/
6 mice on week 6 after moderate- or high-intensity exercise were stained for IL-10 and IFN-g and analyzed by FACS.
Representative results are shown. The percent expression of IL-10 and IFN-g in splenocytes from each group (a and b) and the
per cent expression of IL-10 and IFN-g, summarized as the means of three independent experiments (c), are indicated. Results
are means  SEM, n 5 8. Means without a common letter differ, Po0.05.

Next, we wanted to determine the effects of HIE/
MIE on HBsAg-specific Th cell responses. Cytokine
expression of CD41 and CD81 T cells were exam-
ined by intracellular staining. The data showed that
MIE increased the IFN-g expression for antigen-
specific CD41 and CD81 T cells (Fig. 7). By con-
trast, HIE reduced the IFN-g expression in antigen-
specific CD41 and CD81 T cells (Fig. 7). In addition,
neither HIE nor MIE had an effect on IL-4 expres-
sion in total CD41 T cells, indicating an unaltered
Th2 response (Fig. 7).
Specific cytotoxic responses have been demon-
strated as another key effector required to clear
HBV-infected cells at the carrier stage (Davis et al.,
1996). To confirm the effect of HIE/MIE on CMI,
the specific cytotoxic response was tested in vivo on
day 14 after the third immunization. As shown in
Fig. 8, the MIE groups had a significantly augmented
effect on HBsAg-specific cytotoxic responses com-
pared with the other groups. By contrast, HIE
impaired antigen-specific cytotoxic activities. Over-
all, the magnitude of impairment of the HBsAg-
specific cytotoxic response corresponded with the
amounts of HBsAg-specific IFN-g-positive CD81 T
cells observed in Fig. 6 in the same group.
Discussion
Most studies on the effects of exercise on immunity
have shown that habitual, moderate physical activity
augments immune responses by increasing the ex-
pression of pro-inflammatory cytokines, including
IL-2, IL-1b, and TNF-a (Haahr et al., 1991; Zaldivar
et al., 2006) while exhaustive exercise tends to be
immunosuppressive. In the current study, MIE was
shown to increase both IL-2 production and the
expression of pro-inflammatory cytokine IL-12 (IL-
12p40), which promotes Th1-type immunity and
decreases the risk of URTI. The increased expression
of IL-12 was consistent with that of IFN-g. More-
over, regular MIE did not affect the anti-inflamma-
tory cytokines TGF-b and IL-10 but increased IL-
2Ra (CD25) expression. In our study, we did not
determine IL-2/IL-2R activity. However, we specu-
late that IL-2/IL-2R signaling is involved in inducing

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Wang et al.

Fig. 4. Effect of exercise on CD41CD251 T-regulatory cells and IL-10 expression. Splenocytes isolated from C57BL/6 mice on
week 6 after exercise of moderate or high intensity were stained either for surface markers or intracellularly with anti-CD4
jointly with anti-CD25, anti-Foxp3, or anti-IL-10 antibodies and then analyzed by FACS. Representative results showing the
per cent expression of CD25 in splenocytes from each group (a) and the per cent expression of CD25 are included. The results
in (b) and the abundance of CD251 (c), Foxp3 (d), and IL-10 (e) in CD41 T cells means  SEM of eight independent
experiments. Means without a common letter differ, Po0.05.

IFN-g production by regular MIE. In addition, IL-2/
IL-2R signaling is essential for maintaining self-
tolerance, as mice deficient in either IL-2 or IL-2R
exhibit lethal autoimmunity (Nelson, 2004; Antony
et al., 2006). These in vivo studies strongly favored a
model whereby IL-2 controls autoimmunity through
the production of CD41CD251 Treg cells. In the
current study, MIE did not alter the proportion of
CD41CD251 Treg cells, thus supporting recent
studies in which regular moderate exercise (TCC)

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had no effect on the CD41CD251 Treg cells of
healthy humans (Yeh et al., 2009). In a recent human
study, regular TCC exercise increased the T-bet
expression, a specific transcription factor for Th1
cells, and Foxp3 expression in patients with type 2
DM, but not in normal age-matched adults, suggest-
ing that the immunological effects of MIE may be
modulated by health status.
Unlike MIE, prolonged, exhaustive HIE has been
shown to suppress immune responses and increase
the risk of URTI (Nieman, 1997; Nieman et al.,
2006). Although the plasma level of IL-6, an inflam-
mation-responsive cytokine, increases following an
acute bout of prolonged, exhaustive exercise, anti-
Exercises, Treg cells and vaccination
inflammation cytokines (e.g., IL-1 receptor antago-
nist [IL-1ra], IL-4, IL-10, and cortisol) also increase,
which may minimize the inflammatory process (Hoff-
man-Goetz., 1996; Steensberg et al., 2003). Further-
more, IL-6 is regarded as anti- and pro-inflammatory
depending on the tissues where it is secreted (Peder-
sen & Bruunsgaard, 2003; Calle & Fernandez, 2010).
Lastly, studies demonstrate that an acute bout of
intensity exercise can increase the production of pro-
inflammatory cytokines in response to exercise, but
these cytokines decline post-exercise (Pedersen &
Hoffman-Goetz, 2000; Petersen & Pedersen,
2005).Our results suggest that the mechanism by
which HIE increases the risk of URTI may be

Fig. 5. Effect of exercise on humoral responses in immu-

nized running mice. Mice subjected to moderate- or high-
intensity exercise were subsequently immunized with HBV
DNA vaccine on days 0, 14, and 28. Serum samples from
C57BL/6 mice were collected for ELISA on day 14 after the
final boost. Total anti-HBsAg antibodies were quantitated
with a commercial kit, and values were determined relative
to the standard concentration of anti-HBsAg antibody.
Mean titers are expressed in milli-international units per
milliliter. Total anti-HBsAg IgG in serum was detected as
described in the ‘‘Materials and methods.’’
Fig. 7. Effect of exercise on antigen-specific cytokine pro-
ductions in T cells. T cells isolated from the spleen of
C57BL/6 mice on day 14 after the final boost were stimu-
lated with HBsAg-derived peptide S208-215 for 6 h in
culture. Intracellular staining for IL-4 or IFN-g in CD41
T cells, and IFN-g in CD81 T cells was performed by FACS.
The summaries of percentage of IL-4 in CD41, IFN-g in
CD41 and CD81 T cells are shown. Results are means
 SEM, n 5 5. *Po0.05 compared with the naı̈ve and TC
group.

Fig. 6. Effect of exercise on T cell proliferation in immunized running mice. To confirm whether HIE/MIE impaired/enhanced
cell-mediated immune responses to the HBV DNA vaccine, the T cells were isolated from animals of all groups on day 14 after
the final immunization and then the T cell proliferation was performed as described in ‘‘Materials and methods.’’ The
histograms show a presentative result (a) and the percentage of divided cells was summarized in the means of five independent
experiments (b). Means without a common letter differ, Po0.05.

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Wang et al.

Fig. 8. Effect of exercise on antigen-specific cytotoxic responses in vivo. To analyze effect of exercise on HBsAg-specific
cytotoxicity in vivo, a 1:1 mixture of the CSFEhigh labeled specific target and CSFElow labeled non-specific target cells were
transferred into each group via i.v. After 4 h, these mice were killed and the specific lysis was analyzed by FACS and calculated
as described in ‘‘Materials and methods.’’ The histograms show a presentative result (a) and the percentage of specific lysis was
summarized in the means of five independent experiments (b). Means without a common letter differ, Po0.05.

through a down-regulation of pro-inflammatory cy-
tokines (mainly IL-2, IFN-g, and IL-12) together
with an increase in anti-inflammatory cytokines
(TGF-b, IL-10, and IL-35 [Ebi3]). In addition, HIE
increased IL-2R expression relative to the control
groups. Thus, the HIE-mediated impairment of im-
mune responses may be due to a defective IL-2/IL-
2R signaling; however, this hypothesis needs to be
validated. Whether an acute bout of high-intensity
exercise could affect IL-12, IL-2, IL-2R, and IFN-
g expression is also yet to be determined.
It has been demonstrated that the presence of
higher numbers of Treg cells in the lungs of mice
and humans during infection may lead to failure to
clear the infection. This may contribute to the estab-
lishment of a chronic phase in which Treg cells are
involved in limiting immune-mediated tissue damage
(Curotto de Lafaille & Lafaille, 2009). We found that
prolonged, exhaustive HIE increased the proportion of
CD41CD251 T cells and the expression of Foxp3, a
CD41CD251 Treg cell-specific marker, in spleen. It is
imperative to further investigate whether prolonged or
exhaustive HIE increases the risk of URTI through
elevated number of Treg cells in infection models.
It is well known that hormonal changes occur in
response to exercise, including increases in the
plasma concentration of epinephrine (adrenaline),
cortisol, growth hormone, and prolactin, all of which
have immunomodulatory effects (Pool & Axford,
2001; Venkatraman et al., 2001). Moreover, recent
studies have shown that a novel anti-inflammatory
cytokine, IL-35, expands Treg cells and inhibits IFN-
g production. Thus, it is possible that HIE expands
CD41CD251 Treg cells by increasing IL-35 produc-
tion (Niedbala et al., 2007). Furthermore, cortisol
and epinephrine suppress Th1 cell cytokine produc-
tion and Th1 response promotes CMI, which affords
the host protection against viruses (Franchimont et
al., 2000). In addition, cortisol, and anti-inflamma-
tory cytokines such as TGF-b, can also induce the
expansion of CD41CD251 Treg cells (Horwitz et al.,
2008; Kang et al., 2008). However, whether hor-
mones or other immune-modulating factors are in-
volved in expanded CD41CD251 Treg cells seen
after HIE is worth further investigation.
Compared with what we know about HIE, we
know even less about how regular MIE augments
immune responses. Studies have suggested that the
density of b-adrenergic receptor, which is expressed
on Th1, not Th2 cells, is involved in mediating the
endurance exercise-training associated increase in
Th1 response (Nieto et al., 1997; Kohut et al.,
2004). Another mechanism that deserves further
validation is that MIE may enhance T cell function
by increasing CD28 expression, because CD28 has
been shown to induce IL-2 production and IL-2R
expression leading to T cell proliferation (Jenkins et
al., 1991; Shimizu et al., 2008).
DNA vaccines specific for HBV hold particular
promise in the treatment of chronic HBV infections
and have been shown to induce strong humoral
immunity and CMI in animal models and healthy
volunteers (Mancini-Bourgine et al., 2004). In addi-
tion, several studies have shown that MIE increases
secondary antibody responses to antigens especially
certain protein antigens (Sugiura et al., 2001). How-
ever, whether exercise of varying intensities has
differential effects on the immune response to DNA-
based vaccinations had not been examined. In this
study, we showed that MIE augmented CMI by
increasing the production of Th1 cell cytokines and
causing the proliferation of HBsAg-specific T cells,

8

thereby stimulating HBsAg-specific cytotoxic activ-
ities, which provide protection against viruses. In
contrast, HIE led to impaired CMI in mice vaccinated
with the HBV DNA vaccine. However, whether this
effect is mediated by the HIE-specific expansion of
CD41CD251 Treg cells or an increase in IL-10
expression by CD41 T cells remains to be investigated.
Perspectives
We report here that regular/habitual physical activity
(i.e., MIE) enhances the pro-inflammatory cytokine
and CMI response in mice, which have an implica-
tion in lowering the risk of cancer and infectious
illness in humans. However, as chronic low grade
inflammation is being increasingly associated with
the risk of developing chronic cardiovascular and
metabolic diseases, regular exercise that includes
some high-intensity work can improve health benefit
probably via an altered Th1/Treg balance (Balducci
et al., 2010). HIE training appears to suppress
immune function by increasing Treg cells and results
in a reduced pro-inflammatory and an increased anti-
Exercises, Treg cells and vaccination
inflammatory cytokine expression, which might in-
crease risk of common infections such as URTI
(Curotto de Lafaille & Lafaille, 2009). A recent study
showed that the combination of high-intensity aero-
bic plus resistance exercise training, in addition to
daily physical activity, is required to achieve a
significant anti-inflammatory effect in type 2 diabetic
patients (Balducci et al., 2010). However, it would be
interesting to determine whether HIE, or its combi-
nation with resistance exercise training is more ben-
eficial than MIE in reducing risk of chronic
cardiovascular and metabolic diseases via its anti-
inflammatory effects, especially CD41CD251 Treg
cells.
Key words: cell-mediated immunity, cytokines, lym-
phocytes.
Acknowledgements
This work was supported in part by the National Natural
Science Foundation of China (30972687) (to Y. M) and a
research initiation fund from Henan University to J. W.

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