Stress, March 2009; 12(2): 134143

q Informa Healthcare USA, Inc.

ISSN 1025-3890 print/ISSN 1607-8888 online
DOI: 10.1080/10253890802137437

Chronic restraint stress impairs T-cell immunity and promotes tumor

progression in mice

L. R. FRICK1, M. L. BARREIRO ARCOS1, M. RAPANELLI2, M. P. ZAPPIA3, M. BROCCO3,

C. MONGINI1, A. M. GENARO1, & G. A. CREMASCHI1
1
Centro de Estudios Farmacologicos y Botanicos, Consejo Nacional de Investigaciones Cientficas y Tecnicas, 18 Catedra de
Farmacologa, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 Piso 15, Buenos Aires 1121, Argentina,
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2
Instituto de Biologa y Medicina Experimental., Consejo Nacional de Investigaciones Cientficas y Tecnicas, Vuelta de Obligado
2490, Buenos Aires, 1428, Argentina, and 3Instituto de Investigaciones Biotecnologicas, Instituto Tecnologico de Chascomus.
Consejo Nacional de Investigaciones Cientficas y Tecnicas, Universidad Nacional de General San Martn. Av. Gral. Paz 5445,
INTI, Edificio 24, Buenos Aires 1650, Argentina

(Received 2 February 2008; revised 24 March 2008; accepted 17 April 2008)

Abstract
For personal use only.

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of
anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter
the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress
affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a
reduction in CD4T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-a) and Interferon-gamma
(IFN-g) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of
tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival.
Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic
exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated
immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the
therapeutic management of stress to improve the prognosis of cancer patients.

Keywords: Cancer, CD4/CD8, chronic stress, cytokines, T-cell immunity, tumor growth

Introduction
In the last decades, stress has become an important
aspect of daily life. A growing body of evidence
indicates that stress is a key factor in the development
of several pathologies, including psychiatric diseases
such as anxiety and major depression, disruption of
neuroendocrine systems, alterations of the immune
response and even cancer (Reiche et al. 2004). Several
animal models in rodents and primates using physical
and/or psychological stressors are useful tools for the
study of deleterious effects induced by chronic stress
exposure (Willner and Mitchell 2002, Van Kampen
et al. 2002). These models are validated in accordance
with typical behavioral alterations, i.e. altered per-
formance in tasks such as the open field, forced
swimming and tail suspension tests (Palumbo et al.
2007; Morgulis et al. 2004). Using such models, it has
been reported that chronic stress alters the immune
system. This is evidenced by a reduction of T-
cytotoxic and natural killer (NK) activities (Nunez
et al. 2006), alterations of T- and B-lymphocyte
proliferative responses to mitogens (Edgar et al. 2003;
Silberman et al. 2002), impairment of antibody
production (Silberman et al. 2003) and changes in
cytokine secretion (Merlot et al. 2004). T cells and
NK cells are the major components of anti-tumoral

Correspondence: Luciana Romina Frick. Tel/Fax: 5411 49624435. E-mail: lfrick@fmed.uba.ar


immunity. Both CD8T-cytotoxic and CD4T-helper
lymphocytes are crucial regulators of tumor growth
(Foss 2002; Ostrand-Rosenberg 2005). NK cells are
potent effectors against tumor cells by inducing
cytotoxicity (OHanlon 2004; Abbas et al. 2000).
Cellular cytokines, such as tumor necrosis factor-
alpha (TNF-a) and interferon-gamma (IFN-g) are
key mediators in these processes (Knutson and Disis
2005; Dredge et al. 2002). Taken together, the
evidence strongly suggests that chronic stress could
enhance tumor growth by suppressing anti-tumoral
immunity (Calcagni and Elenkov 2006).
This proposal was first made by Vernon Riley
(1975, 1981) and since then considerable progress in
this field has been achieved (Cohen, Rabin 1998;
Freire-Garabal et al. 2004; Saul et al. 2005; Thaker
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et al. 2006). However, the influence of chronic stress
exposure on the immune response and hence on
tumor progression and the mechanisms underlying
these processes are still far from being completely
understood. In this scenario, the aim of the present
work was to evaluate the effects of chronic stress
exposure on cellular immunity and on tumor
evolution. With this purpose, we first validated our
repeated restraint model by performing behavioral
measurements in order to confirm that this paradigm
For personal use only.
induced a state of chronic stress. Next, we evaluated
the lymphoproliferative response to T- and B- selective
mitogens, as well as the NK cytotoxic activity against
YAC-1 cells of splenocytes from normal and stressed
mice. In addition, we evaluated balance of the
CD4/CD8 subsets by dual fluorescence flow
cytometry in stressed and normal mice. We also
studied the effects of chronic stress on TNF-a and
IFN-g mRNA expression in these animals using real
time RT-PCR. Finally, we analyzed the influence of
chronic stress on the biological behavior of a T-cell
lymphoma (LBC cells) growing in vivo in syngeneic
mice, and the specific cytotoxicity against these tumor
cells in stressed mice bearing tumors.
Materials and methods
Animals
Inbred female BALB/c (H-2d) mice between 60 and
100 days old were purchased from the Instituto
Nacional de Tecnologia Agropecuaria (INTA, Caste-
lar, Buenos Aires, Argentina). Animals were cared for
in accordance with the Guide for the Care and Use of
Laboratory Animals of the National Institute of
Health (NIH, USA); experimental protocols were
approved by the Internal Ethics Committee of the
School of Medicine of the University of Buenos Aires,
i.e. Comite Institucional para Cuidado y Uso de los
Animales de Laboratorio (CICUAL).
All the mice were housed in groups of 3 6,
depending on the experiment, per cage and main-
Stress alters T-immunity and tumor growth 135
tained on a 12/12 h light/dark cycle with the lights on
from 8 AM to 8 PM under controlled temperatures
between 188C and 228C. Food and water were
available ad libitum.
Experimental procedures
Mice were randomly assigned to the stress or control
groups (n 47 mice for each condition in total;
numbers per experimental group are in Tables and
Figures legends), according to our previous protocol
(Alfonso et al. 2006). All experiments were repeated at
least three times. For the chronic restraint stress
model, mice were restrained daily for 6 h (from 11 AM
to 5 PM) in well-ventilated polypropylene tubes
(2.8 cm diameter 11.5 cm length) without access to
food and water. The mice were not physically
compressed and did not experience pain. This
protocol was performed for at least three weeks. For
immunity studies mice were killed at the third week by
cervical dislocation between 9 and 11 AM. For
lymphoma evaluation, tumor cells were injected at the
third week, and the stress protocol continued until the
mice died or were killed; mice were monitored every
day, and were killed if a series of observations
indicated the need for humane euthanasia following
the guidelines for animal care, including hypothermia
and slow locomotion among others. The survival time
was counted as if euthanased mice had died naturally.
For LBC cytolytic activity assays and for flow
cytometric analysis of LBC proliferation assays mice
were stressed for three weeks, injected with LBC cells
and stressed for two more weeks and then killed. Mice
from control groups were housed in normal con-
ditions. Each experiment used control mice of the
same weight and age.
Behavioral tests
Open field test. The open field test was performed using
a rectangular plastic chamber (42 35 15 cm),
with its floor divided into 30 equal squares. The mice
were placed in the center of the open field and allowed
to explore freely for 10 min. The parameters registered
were: rearing (vertical exploration), crossed lines
(horizontal exploration) and immobility time.
Forced swimming test. Mice were placed in a
transparent cylinder (17 cm diameter 25 cm high)
half-filled with warm water (308C) for 6 minutes.
Latency to begin floating was scored as the time
between introduction of a mouse into the cylinder and
the first moment of complete immobility of the entire
body, irrespective of the duration of the first floating
episode. The total time spent floating was scored
during the last 4 min of the test.
 136 L. R. Frick et al.
Tail suspension test. Mice were suspended by the tail to
a plastic string positioned horizontally 75 cm above
the table top, using hypoallergenic adhesive tape
placed at 1 cm from the tip of the tail. Total duration of
immobility was measured over 6 min.
Syngeneic lymphoma model
The LBC cell line is an aggressive T-cell lymphoma
derived from an early T-cell lymphocyte precursor in
BALB/c mice (H-2d), as described by Mongini et al.
(2001). LBC T lymphoma cells were cultured in RPMI
1640 (Invitrogen, Carlsbad, CA, USA) supplemented
with 10% fetal bovine serum (Invitrogen), 2 mM
glutamine (Invitrogen), 100 mg/ml streptomycin (Invi-
trogen), and 50 mM 2-b-mercaptoethanol (Sigma
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Aldrich, St. Louis, MI, USA). Before implantation,
LBC cells were washed with phosphate buffered saline
(PBS), counted according to Trypan blue staining
(Sigma Aldrich) and resuspended in sterile PBS.
Syngeneic mice from the indicated experimental
groups were subcutaneously inoculated with 1 106
LBC cells in 100 ml PBS to generate a solid tumor.
Tumor length and width were measured every two days
using calipers (resolution: 1 mm), and tumor volume
was calculated as V p/6 length width2.
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Lymph node cell suspensions, culture conditions,
and proliferation assays
Lymphocytes from spleens (which are enriched in B-
cells and NK cells) and lymph nodes (which are
enriched in T-cells) were obtained as previously
described (Edgar et al. 2003). Briefly, mice were
killed by cervical dislocation and spleens and lymph
nodes were quickly removed and disrupted through a
1-mm metal mesh. Cell suspensions were centrifuged
and washed twice with RPMI 1640 supplemented
with 10% fetal bovine serum, 2 mM glutamine and
100 mg/ml streptomycin. Cell viability was estimated
according to Trypan blue exclusion criteria and was
greater than 90%. Proliferation was determined by
culturing 2 105 cells per well in 96-well plates in a
final volume of 0.2 ml in triplicate aliquots of
supplemented medium. The T-cell mitogen Conca-
navalin A (Con A, Sigma Aldrich) or the B-cell
mitogen lipopolysaccharides from Escherichia coli
(LPS, Sigma Aldrich) were added to the microcul-
tures to yield the optimal mitogen concentrations of 1
and 30 mg/ml, respectively (Mamchak and Hodgkin
2000). Cells were cultured at 378C in a 5% CO2
atmosphere. Mitogenic activity was measured by
adding 0.75 mCi [3H]thymidine (Amersham Bios-
ciences, Little Chalfont, Buckinghamshire, UK) per
well for the last 24 h of culture. [3H]Thymidine
incorporation was measured by scintillation counting
after retention over GF/C glass-fiber filters (What-
man, Brentford, Middlesex, UK) of the acid-insoluble
macromolecular fraction. Mitogen-stimulated cells
displayed the expected proliferation kinetic, with a
peak of proliferation at the third day of culture.
Natural killer activity assay
YAC-1 cells were purchased from ATCC (Manassas,
VA, USA). Cells were cultured in supplemented
medium as described for LBC cells. Specific cytotoxic
activity against tumor cells was evaluated according to
the JAM method (Ayres et al 2003; Wunderlich et al.
1997). Briefly, YAC-1 cells were labeled with 5 mCi
[3H]thymidine 3 h before the cytotoxicity assays were
carried out. Cell suspensions from spleens from
normal and stressed mice were obtained as described
above. Different ratios of effector and target cells
(1:100 to 1:1) were added to microtiter plates (100 ml
each) at final volumes of 200 ml, and incubated for
3.5 h at 378C in a 5% CO2 atmosphere. Plates were
harvested as described above. Percentages of cytotoxic
activity were calculated as NK 100 (SR 2
ER)/SR, where SR is the spontaneous release and
ER is the experimental release.
Lymphocyte subsets determination by flow cytometry
CD4T-helper/inducer and CD8T-cytotoxic/suppres-
sor lymphocytes were determined in lymph node cell
suspensions by flow cytometry (Silberman et al. 2003).
Briefly, 1 106 cells in 100 ml cytometry buffer (2%
bovine fetal serum and 0.01% NaN3 in PBS) were
stained with 0.50 ml of fluorescein-conjugated anti-
mouse CD4 (CD4-FITC, clone GK1.5) and 0.50 ml of
phycoerythrin-conjugated anti-mouse CD8 (CD8-PE,
clone 536.7) monoclonal antibodies (eBioscience,
San Diego, CA, USA), washed twice and fixed with 2%
formaldehyde in PBS. Lymphocytes were identified by
FACS analysis using a BD FACSCalibur flow
cytometer (BD Biosciences, San Jose, CA, USA). Dot
plots of two color fluorescence analysis as well as
percentages of lymphocytes expressing CD4 and CD8
were determined. Isotype controls (clones MOPC-141
and UPC-10, respectively, SigmaAldrich) were used
for each assay to determine non-specific staining.
Total RNA and poly(A) mRNA isolation
and cDNA synthesis
Lymph nodes were stored in liquid nitrogen until use.
As we described previously (Alfonso et al. 2006),
tissues were homogenized in Trizol Reagent (Invitro-
gen) and total RNA was isolated following the
manufacturers instructions. Poly(A) mRNA was
isolated from total RNA using the PolyATract mRNA
isolation System (Promega, Madison, WI, USA).
Complementary DNA was synthesized by retro-
transcription using oligo(dT) and SuperScript II
Reverse Transcriptase enzyme (Invitrogen).

Quantitative real time reverse transcription polymerase
chain reaction
Real time RT-PCRs were carried out in a
GeneAmp 5700 Sequence Detection System (Applied
Biosystems, Foster City, CA, USA), as we previously
described (Alfonso et al. 2006). cDNA amounts
present in each sample were determined in duplicates
using SYBR Green PCR Core Reagents kit (Applied
Biosystems). Primer sequences were designed using
Primer Express software (Applied Biosystems). To
verify that the SYBR Green dye detected only one
PCR product, all the reactions were subjected to a
heat dissociation protocol following the final cycle of
the PCR. Oligonucleotide sequences used were:
TNF-a forward 50 -GCA CCA CCA TCA AGG
ACT CAA-30 , TNF-a reverse 50 -TTG CAG AAC
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TCA GGA ATG GAC A-30 , IFN-g forward 50 -TGC
TGA TGG GAG GAG ATG TCT AC-30 , IFN-g
reverse 50 -ACC TGA CAC ATT CGA GTG CTG T-
30 , b-actin forward 50 -CAA CTT GAT GTA TGA
AGG CTT TGG T-30 , b-actin reverse 50 -ACT TTT
ATT GGT CTC AAG TCA GTG TAC AG-30 . For
data normalization, values were referred to b-actin as a
housekeeping gene.
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Flow cytometric analysis of LBC proliferation assays
LBC cells were cultured as described above. Before
tumor implantation, tumor cells were stained with
carboxyfluorescein diacetate succinimidyl ester
(CFSE) using the Vybrant CFDA SE Cell Tracer
Kit (Invitrogen) following the manufacturers instruc-
tions. Briefly, cell suspensions were incubated with
20 mM CFSE for 15 min at 378C, washed and re-
incubated for 30 min. Traced cells were subcu-
taneously injected as described above. Two weeks
later, mice were killed and tumors were quickly
removed and disrupted through a 1 mm metal mesh.
Cell suspensions were centrifuged and washed twice
with PBS and then fixed with 3.7% formaldehyde in
PBS. LBC cells were identified by fluorescence
analysis at 492 nm using a BD FACSCalibur flow
cytometer (Lyons 1999).
LBC cytolytic activity assay
Specific cytotoxic activity against tumor cells was
evaluated according to the JAM method as described
for NK activity. Briefly, LBC were labeled over night
with 5 mCi [3H]thymidine and co-cultured with cell
suspensions from spleens from normal and stressed
mice that were injected with the lymphoma cells two
weeks before and hence were bearing solid tumors.
Different concentrations of effector and target cells
were added to microtiter plates (100 ml each) at final
volumes of 200 ml, and incubated for 3.5 h at 378C in
a 5% CO2 atmosphere. Plates were harvested as
Stress alters T-immunity and tumor growth 137
described above. Percentages of cytotoxic activity were
calculated as the relation CTL 100 (SR
ER)/SR, where SR is the spontaneous release and
ER is the experimental release.
Statistical analysis
Group means were analyzed for statistical significance
with the software XLSTAT 2007.8.01 Version
(Addinsoft, New York, NY, USA), using unpaired
two-tailed Student t tests. For survival analysis,
Kaplan Meier curves were constructed and com-
pared using Log Rank tests. Differences between
means were considered significant if p , 0.05. Results
are expressed as groups mean ^ standard error of the
mean, except in the case of animal survival data, which
are expressed as mean ^ standard deviation.
Results
To investigate the effects of chronic stress on immunity
and cancer prognosis, we used the well known model of
repeated restraint stress in mice. Stressed mice
displayed a significant reduction in body weight gain
after three weeks of repeated restraint (as a percentage
of initial body weight, Control 108.44 ^ 2.44% vs.
Stress 95.84 ^ 1.78%, p , 0.01), as we have
previously shown (Alfonso et al. 2006). Additionally,
chronically restrained mice displayed altered loco-
motor activity in the open field test, i.e. an increase of
crossed lines and rearings and a reduction of the
immobility time (Table I; p , 0.05 and p , 0.01). As
expected, these mice also showed a reduction in the
latency to escape as well as an increase of the
immobility time in the forced swimming test and in
the tail suspension test (Table I; p , 0.05).
Chronic stress impairs the T-cell lymphoproliferative
response
In order to evaluate the status of the cellular and
humoral immune response in normal and stressed
mice, lymphocyte proliferation to both T- and B-
selective mitogens was evaluated by [3H]thymidine
incorporation. Cell suspensions from lymph nodes
and spleens were stimulated with Con A (T-cell
specific) and LPS, respectively (B-cell specific). A
significant reduction in T-cell proliferation was
observed in cells from stressed mice (Figure 1A; p ,
0.01), whereas B-cell proliferation was unchanged
(Figure 1B).
Chronic stress does not alter natural killer activity
We also studied whether chronic stress affects NK cell
activity. Cytotoxicity was evaluated using [3H]thymi-
dine, previously incorporated into the DNA (Ayres
et al 2003; Wunderlich et al. 1997). DNA fragments
 138 L. R. Frick et al.
Table I. Behavioral alterations induced by chronic restraint stress.
Behavioral paradigm Parameter
Open field test Lines crossed
Rearings
Immobility (s)
Forced swimming test Latency to floating (s)
Immobility (s)
Tail suspension test Immobility (s)
Mice were restrained daily for 3 weeks, and then subjected to only one of the following behavioral paradigms: open field, forced swimming test
or tail suspension test. Mice were exposed to the open field for 10 min, and the number of rearings, lines crossed and the immobility time were
recorded. In the case of the forced swimming test, the latency to begin floating and the immobility time during the last 4 min were registered. In
the tail suspension test, the immobility time was registered during the total 6 min. Values are expressed as group means ^ SEM. for each
parameter. Statistical significance was determined using unpaired t-tests (n 10 mice per group, *p , 0.05, **p , 0.01).
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are retained in filters when harvested, and their
radioactivity is inversely related to cytolytic activity of
effector cells. We found no differences in NK specific
cytotoxicity against YAC-1 cells between mice control
and stressed mice in the target:effector ratios
(Figure 2).
Chronic stress alters T-lymphocyte subsets distribution
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We also evaluated whether chronic stress induces any
modification in CD4T-helper/inducer and CD8T-
cytotoxic/suppressor lymphocytes. These populations
were determined in lymph node cell suspensions by
dual fluorescence flow cytometry using specific a-
CD4-FITC and a-CD8-PE antibodies. Chronic stress
resulted in a significant reduction of CD4 lympho-
cytes but did not affect CD8 lymphocytes (Table II;
p , 0.05). As expected, the CD4/CD8 ratio was also
reduced in stressed mice (Control 2.99 ^ 0.31;
Stress 1.80 ^ 0.17; p , 0.05).
Figure 1. Effects of chronic stress on lymphoproliferative response
to selective T- and B- mitogens. Cells from lymph nodes and spleens
of stressed mice and untreated controls were stimulated with Con A
and LPS respectively, labeled with [3H]thymidine for 72 h and
harvested. Representative results from three independent
experiments are shown. Values are expressed as
group means ^ SEM. of the stimulated/basal ratio (proliferation
index). Statistical significance was determined using unpaired t-test
(n 6 mice per group, **p , 0.01).
Control Stress
181.9 ^ 19.04 271.9 ^ 21.13**
37.9 ^ 4.69 58.3 ^ 8.36**
37.3 ^ 5.24 13.4 ^ 4.33*
58.2 ^ 3.12 23.8 ^ 4.93*
89.4 ^ 9.28 144.7 ^ 10.87*
162.6 ^ 15.07 217.8 ^ 17.92*
Chronic stress reduces cytokine expression
We next tested if chronic stress affects the production
of cytokines involved in T-cell mediated anti-tumoral
immunity. We measured TNF-a and IFN-g mRNA
expression in lymph nodes of normal and stressed
mice using real time RT-PCR according to the SYBR
Green method. Real time PCRs revealed a reduction
of TNF-a and IFN-g mRNA expression levels relative
to b-actin in lymphocytes of stressed mice compared
with controls (Figure 3A and B; p , 0.01).
Chronic stress promotes LBC lymphoma growth
We also asked whether immune system depression
induced by chronic stress exposure could promote
tumor growth. With this purpose, stressed and normal
syngeneic mice were subcutaneously injected with
1 106 LBC T-lymphoma cells to generate a solid
tumor. Measures of tumor volume indicated that
growth was increased in chronically stressed mice as
compared to control animals at several time points
tested (Figure 4A; p , 0.01). To evaluate whether
tumors growing in stressed mice proliferated faster
than those growing in control animals, LBC cells were
stained with CFSE prior to injection. CFSE is a
colorant that binds spontaneously and irreversibly to
intracellular proteins. As cells proliferate, CFSE
distributes equally between daughter cells, thus the
amount of dye present in the cytosol is reduced by half
with each division (Lyons 1999). After two weeks,
cells were recovered and analyzed by flow cytometry.
LBC cells growing in stressed mice had a smaller
mean fluorescence intensity (Gmean) than those grown
in control mice (Figure 4B; Control 32.33 ^ 1.40
arbitrary units of CFSE fluorescence at 492 nm (AU);
Stress 12.59 ^ 1.58 AU; p , 0.01), indicating that
tumor cells had an accelerated proliferation rate in
stressed mice. Additionally, KaplanMeier plots of
animal survival were compared with the LogRank
analysis. Stressed mice displayed a significant reduction
of survival, indicating a worse prognosis of the neoplastic
 Stress alters T-immunity and tumor growth 139

chronic stress impaired T-cell mediated immunity and

Figure 2. Effects of chronic stress on NK activity. Cells
suspensions from spleens of stressed mice and untreated controls
were co-incubated with YAC-1 cells labeled with [3H]-thymidine at
different target:effector ratios, cultured for 3.5 h and harvested.
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enhanced syngeneic lymphoma progression. With
respect to the effects of stress on cognate immunity, we
found that chronic stress significantly impaired T-cell
proliferation, without affecting B-cell proliferation.
Reduction of T-cell proliferation has been reported in
other models of chronic stress. For example, chronic
mild stress in mice and chronic social confrontation in
rats results in a reduced T-cell response to Con A
(Edgar et al. 2003; Silberman et al. 2002; Stefanski
and Engler 1999). Instead, mice subjected to chronic
mild stress display an increased B-cell proliferation
(Edgar et al. 2003; Silberman et al. 2002). Here, we
found no changes on B-cell reactivity to LPS in mice
subjected to restraint stress. The main effector
components of anti-tumoral immunity are T and NK

Representative results from three independent experiments are
shown. Values are expressed as group means ^ SEM. of NK specific
cytolysis, calculated as 100 (SR-ER)/SR, where SR is the
spontaneous release and ER is the experimental release of
[3H]thymidine. Statistical significance was determined using
unpaired t-test (n 4 mice per group, no significant difference).
pathology (Figure 4C, Control 26.80 ^ 0.70 days;
Stress 22.10 ^ 1.40 days; p , 0.01).
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cells (Abbas et al. 2000). Our results are in accordance
with these functions, given that we found a reduction
of T-but not B-cell proliferation. However, the role of
antibodies in anti-tumor immunity can not be ruled
out, because they participate in the elimination of
tumors (Paschen et al. 2004). Indeed, we have found
that T-cell depression induced by chronic unpredict-
able stress results in an impairment of the T-cell
dependent humoral response (Silberman et al. 2004).
Therefore, in our restraint stress model, a reduced

Chronic stress reduces specific cytotoxicity against

LBC cells
We finally tested whether cytotoxic activity against
LBC cells was altered in stressed mice bearing tumors.
Control and stressed mice were injected with LBC
cells, and two weeks later spleen cells were co-
incubated with LBC cells. Stressed mice showed a
reduced cytotoxicity against LBC cells in comparison
to control mice (Figure 5), supporting a worse tumor
prognosis induced by immune system depression
associated to chronic stress.
Discussion
To investigate the effects of chronic stress on
immunity and cancer prognosis, we used the well
known model of repeated restraint stress in mice
(Alfonso et al. 2006). Stressed mice showed the
common behavioral changes observed in this and
other models of chronic stress (Palumbo et al. 2007;
Morgulis et al. 2004). Briefly, here we showed that
T-lymphocyte response could result in a decrease of
T-cell dependent antibody production directed to
tumor antigens.
With regard to T-lymphocyte subset distribution,
here we demonstrated that chronic stress resulted in a
reduction of CD4, but not CD8, T-lymphocytes in
the lymph nodes, suggesting that stress mainly affects
T-helper immunity. This is in agreement with a report
by Saul et al. (2005), who found a reduction of tumor
infiltrating CD4 but not CD8- T cells in restraint-
stressed mice bearing squamous cell carcinoma
tumors. Stefanski and Engler (1999) found a reduction
of both CD4 and CD8T-lymphocytes in blood from
socially stressed rats. However, we found no changes in
CD4 and CD8T-lymphocyte subsets in lymph
nodes from mice exposed to chronic mild stress
(Silberman et al. 2002). Differences observed between
chronic mild and chronic restraint models could be due
to the type of stressor applied in each case. The chronic
mild stress model is a heterotrophic model that
includes different stressors of moderate intensity

Table II. T lymphocyte subsets distribution in stressed and control mice.

CD4 CD42
CD4 CD8 CD8 CD82

Control 43.8 ^ 2.27% 15.2 ^ 2.89% 2.65 ^ 1.02% 38.3 ^ 5.05%

Stress 28.4 ^ 3.53%* 16.2 ^ 2.92% 0.85 ^ 0.34% 54.5 ^ 6.24%

Mice were restrained daily for 3 weeks. CD4 T-helper/inducer and CD8 T-cytotoxic/suppressor lymphocyte populations were determined in
lymph node cell suspensions by dual fluorescence flow cytometry using specific antibodies (a-CD4-FITC and a-CD8-PE). Representative
results from three independent experiments are shown. Values are expressed as group means ^ SEM. of percentage of lymphocytes expressing
CD4 and/or CD8. Statistical significance was determined using unpaired t-test (n 6 mice per group, *p , 0.05).
 140 L. R. Frick et al.

Figure 3. Effects of chronic stress on TNF-a and IFN-g mRNA

expression in lymphocytes. Total RNA and mRNA were isolated
from lymph nodes of stressed mice, and untreated mice, and used
for cDNA synthesis. Cytokine expression was evaluated by Real
Time RT-PCR using SYBR Green dye. Representative results from
three independent experiments are shown. Values are expressed as
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group means ^ SEM. normalized with b-actin as housekeeping

gene. Statistical significance was determined using unpaired t-test
(n 5 mice per group, **p , 0.01).

applied randomly. Instead, chronic restraint stress and

social stress are homotrophic models that involve one
type of strong stressor. However, it is known that stress
affects the redistribution of immune cells among the
different lymphoid organs (Dhabhar 2003). Therefore,
the discrepancies between these studies could also be
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due to the fact that CD4/CD8 subsets were measured

in different immune compartments. It is important to
note that even though we did not find any difference in
CD8T-cell number, the participation of T-cytotoxic
activity can not be ruled out. In this respect, CD4T-
helper cells modulate cytotoxic activity of CD8T cells
(Dredge et al. 2002). Thus, the reduction of CD4T-
lymphocytes observed in stressed mice could at least
impair CD8T-cell activity. This is in accordance with
a report indicating that chronic stress reduces T-
cytotoxic activity (Nunez et al. 2006). Nevertheless, it
is important to highlight that it was recently demon-
strated that CD4T-helper cells are more efficient in
the elimination of tumors compared to CD8T-
cytotoxic cells (Perez-Diez et al. 2007).
However, we found no changes in cytotoxic activity
against YAC-1 cells between normal and chronically
stressed mice. Although some reports have shown a
reduction of NK activity induced by stress (Nunez
et al. 2006; Stefanski and Engler 1999), other reports
indicate that NK activity can remain unaltered after
stress exposure depending on the stressor applied and
its duration (Oishi et al. 2003). In particular, it was
demonstrated that chronic restraint stress did not
modify NK activity in rats (Steplewski and Vogel
1986). Although the participation of NK cells in these
processes can not be ruled out, NK activity has
classically been related to the inhibition of metastasis
(Christianson et al. 1996) rather than to the control of
tumor cell proliferation, which is undoubtedly
mediated by T cells (Turner et al. 1990), in
accordance with the present results.
Figure 4. Effect of chronic stress on LBC lymphoma cell
proliferation and prognosis. Mice were subjected to chronic
restraint stress, or control conditions, for three weeks, and then
were inoculated subcutaneously with 1 106 LBC cells to generate
a solid tumor. Stress procedures continued until the mice died.
Representative results from three independent experiments are
shown. (A) Tumor progression after appearance. Tumor length and
width were measured and tumor volume was calculated as
V p/6 L W2. Values are expressed as group means ^ SEM.
for each day post-tumor injection (p.t.i.). Statistical significance was
determined using unpaired t-test (n 10 mice per group,
**p , 0.01). (B) CFSE fluorescence intensity of LBC cells
growing in stressed and control mice. The graph shows the
number of events against the mean fluorescence intensity for each
treatment, for a representative experiment of one stressed and one
control mouse. Statistical significance was determined using
unpaired t-test (n 3 mice per group, p , 0.01). (C) Kaplan-
Meier plot of survival of stressed and control mice (n 10 mice per
group, p , 0.01, Log-Rank analysis).
Changes in the patterns of cytokine production have
been reported in stress models. Here, we observed an
important decrease in TNF-a and IFN-g, two key
cytokines involved in T-cell mediated anti-tumoral
immunity, in lymphocytes from chronically restrained
mice compared to controls. It was demonstrated that

Figure 5. Effects of chronic stress on specific cytotoxic activity
against LBC cells. Cells from spleens of stressed mice and untreated
controls were co-cultured with LBC cells labeled with
[3H]thymidine, and its release was evaluated as an indicator of
cytolysis. Representative results from three independent experiments
Stress Downloaded from informahealthcare.com by University of California Irvine on 10/30/14
are shown. Values are expressed as group means ^ SEM. of the
percentage of cytotoxic activity at different effector:target ratios.
Statistical significance was determined using unpaired t-test (n 4
mice per group, *p , 0.05, **p , 0.01).
chronic restraint stress exposure reduces IFN-g
production (Saul et al. 2005). In the case of TNF-a,
it was also shown that combined acoustic and restraint
stress decreases this proinflammatory cytokine (Kiank
et al. 2006), in agreement with the present results.
For personal use only.
Furthermore, stress hormones have been reported to
inhibit systemically TNF-a and IFN-g (Calcagni and
Elenkov 2006). The present results indicating a
reduction of TNF-a and IFN-g mRNA expression
are consistent with the promotion of tumor growth
observed in restrained mice, and could be associated
with a subsequent impairment of T-cell proliferation,
NK activity and specific cytotoxicity against tumor
cells.
We also asked whether this impairment of immunity
induced by chronic stress could be associated with an
alteration of tumor evolution. We observed an
enhanced syngeneic T-lymphoma growth in chroni-
cally restrained mice. Stressed mice had an increase of
tumor volume and of tumor cell proliferation, and also
a reduced survival. Moreover, specific cytolytic
activity against this lymphoma cell line was reduced
in stressed mice bearing a tumor. Recent findings
indicate that chronically restrained stressed mice have
a higher susceptibility to skin cancer (Saul et al. 2005)
and that behavioral stress results in the promotion of
ovarian carcinoma growth and angiogenesis (Thaker
et al. 2006). In addition, Freire-Garabal et al. (2004)
found that chronic auditory stress results in a
modification of the incidence of breast cancer in
mice and development of lung metastases in rats. It
was also shown that prolonged psychosocial stress
decreases resistance to B16F10 melanoma (Sa-Rocha
et al. 2006) and Ehrlich tumor (Morgulis et al. 2004).
Our results also contribute to the hypothesis of
coexistence between stress and cancer (Reiche et al.
2004; Calcagni and Elenkov 2006; Glaser and
Stress alters T-immunity and tumor growth 141
Kiecolt-Glaser 2005), and point to a key role of
T-cell mediated immunity in these processes. Indeed,
the importance of T-cell response impairment in
pathological challenges during exposure to stressful
situations has already been demonstrated (Bonneau
et al. 1997; 1998; Sheridan et al. 1991; 1998).
Moreover, pioneer work in this field suggested that
stress compromise immunological function, predis-
posing the individual to an increased risk of cancer
(Riley 1975; 1981). Th1 CD4 cells are well known to
modulate the activities of cytotoxic lymphocytes,
natural killer cells and macrophages, and these are
mediated by the production of IFN-g, TNF-a and IL-
2 (Dredge et al. 2002). Thus, chronic stress could be
affecting anti-tumoral immunity by diminishing the
production of these cytokines as observed here, hence
impairing the cellular immune response against tumor
cells. In particular, T-cell mediated immunity is
essential in the control of T-lymphomas, and TNF-a
and IFN-g are involved in these processes (Rook et al.
1999; Krawczyk et al. 1995), further supporting the
results of the present work. It is important to mention
that although other mechanisms can be involved,
during the exposure to a stressful situation undoubt-
edly there is a co-existence of T-cell immunity
impairment and tumor growth enhancement. Finally,
taking into account that stress is a widespread aspect
of modern life, the results emerging from this study
highlight the importance of the therapeutic control of
stress to improve cancer prognosis.
Acknowledgements
This work was supported by grants from CONICET,
UBA, ANPCyT, and HHMI. LRF, MLBA, and MPZ
are research fellows from CONICET, MR is a
research fellow from ANPCyT and MB, CM, AMG,
and GAC are researchers from CONICET. We wish to
thank Dr. Alberto C. Frasch for his invaluable
help with real-time RT-PCRs.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
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