Foxp3-Mediated Immunity of Human Pancreatic Cancer Cell Line PANC-1

American Journal of Immunology 5 (4): 101-107, 2009
ISSN 1553-619X
© 2009 Science Publications

Foxp3-Mediated Immunity of Human Pancreatic Cancer Cell Line PANC-1

1,3Renxi Wang, 1,3Gencheng Han, 1Jianan Wang, 1Guojiang Chen, 1Ruonan Xu,
1Liyan Wang, 2Xia Li, 1Beifen Shen and 1Yan Li
1Department of Molecular Immunology, Institute of Basic Medical Sciences,
Taiping Road, No. 27, Beijing 100850, People’s Republic of China
2Institute of Immunology, Medical School of Henan University,
Kaifeng, People’s Republic of China

Abstract: Problem statement: More and more evidences have shown that human cancer cells can
express Foxp3, the regulatory T cell-associated transcription factor. The role of Foxp3 in human cancer
cells is still unclear. We detected Foxp3 expression in human pancreatic cancer cell line PANC-1.
SiRNA assays showed that Foxp3 expression suppressed the expression of TGF-β and IL-10 which
could induce immune tolerance. Approach: Thus, we proposed that Foxp3 expression in PANC-1
cells controlled the response but not tolerance characteristics of the cells. To prove the proposal, we
first developed the PANC-1 cell-induced the immune response model. PANC-1 cells induced response
in autoimmune Non-Obese Diabetes (NOD) mice aged of >12 weeks. In vitro cell co-cultured assay
demonstrated that PANC-1 induced response in splenocytes from NOD mice aged of >12 weeks.
Results: In the same time, we found that inflammatory cytokine such as INF-γ, IL-2 and IL-17
increased and anti-inflammatory cytokine such as IL-4, IL-10 and TGF-β decreased in the co-cultured
supernatant. These results demonstrate that PANC-1 cells induced response in splenocytes from NOD
mice aged of >12 weeks. SiRNA assays showed that Foxp3 in PANC-1 cells controlled the response in
the co-cultured cells. Conclusion: The results suggested that Foxp3 control the immunological
response characteristics of PANC-1 cells in autoimmune condition.

Key words: PANC-1, tolerance, response, nod mice, foxp3

INTRODUCTION
expressed Foxp3[7-9]. The function of Foxp3 in human

tumor cells is studied by many groups. In breast cancer,

Foxp3 has been regarded as a key factor in
Foxp3 was regarded as an important suppressor for
regulatory T cell (Treg)[1]. Mutations in Foxp3
human breast cancer by suppressing HER-2/ErbB2
completely omit Treg in mouse and greatly reduce
oncogene expression[10]. Another paper found that
suppressive activity of Treg in human[2]. Moreover,
Foxp3-expressing pancreatic carcinoma cell line
ectopic expression of Foxp3 makes conventional
PANC-89 (also T3M4) suppressed T-cell proliferation,
murine T cells to gain the function of Treg[3], and
and knochdown of Foxp3 using RNA interference could
human T cells to acquire many characteristics of
effectively reduce the suppressive activity of these cells
Treg[4,5]. The molecular mechanisms through which
by up-regulation of IL-6 and IL-8[7].
Foxp3 determined the function and development of

The role of Foxp3 in human tumor cells may be
Treg are now studied by many groups. Foxp3 can form
different in different tumor cells. This may be in fact
a complex with the nuclear factor of activated T (NF-
that Foxp3 as suppressive transcription factor
AT) cells and regulates the transcription of several
suppresses the different targeted molecular in different
target genes including interleukin 2[6]. The results are
tumor cells. Previous study has found that PANC-89
consent with the characteristics of Foxp3 as suppressive
expresses IL-6 but not TGF-β, whereas PANC-1
transcription factor.
expresses TGF-β but not IL-6[11]. Thus, we propose that

Until recently, Foxp3 expression is found only in
if it was expressed in PANC-1 cells, Foxp3 may be an
suppressive T cells including CD4+Foxp3+Treg, and
important factor inducing tumor immunity by
CD8+Foxp3+Treg. Now, there are more and more
suppressing TGF-β expression. We here study the
evidences suggesting that human tumor cells also
proposal.
Corresponding Author: Yan Li, Department of Molecular Immunology, Institute of Basic Medical Sciences, Taiping Road,
No.27, Beijing, 100850, People’s Republic of China
101

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Am. J. Immunol., 5 (4): 101-107, 2009

MATERIALS AND METHODS
cells were harvested 24h later for IL-2 and 48h later for

IFN-γ, IL-17, IL-4, IL-10 and TGF-β1 assay using a
Mice: NOD were obtained from the Jackson
sandwich ELISA. The ELISA kits used in this study
Laboratory and bred in our animal facilities under
were purchased from Biosource (IL-2), R and D (TGF-
specific pathogen-free conditions. In female NOD mice,
β1), eBioscience (IL-17A), and BD Pharmingen (IFN-γ,
spontaneous diabetes began to appear by 12-weeks of
IL-4, IL-10), respectively.
age (increasing to 80-90% incidence at 30 weeks of

age). Mice were screened for glucose levels every week
Flow cytometry: Cells (1×105 cells/sample) were washed
and considered diabetic when glucose levels were
with fluorescence-activated cell sorting staining buffer
≥
−
13.8 mMol L 1. This study was approved by the
(phosphate-buffered saline, 2% fetal bovine serum or 1%
Animal Care and Use Committee of the Beijing
bovine serum albumin, 0.1% sodium azide). All samples
Institute of Basic Medical Science.
were incubated with the 2.4G2 anti-Fc receptors (BD

Pharmingen), prior to incubation with other Abs diluted in
Antigen preparation: We predicted T-cell epitope of
fluorescence-activated cell sorting buffer supplemented
Glutamic Acid Decarboxylase 65
with 2% anti-Fc receptor Ab. For the intracellular staining
(GAD 65) and proved
that GAD
of Foxp3 protein, fixed cells were permeabilized with
500-585 (representing the corresponding amino
acids of GAD65) was a main T-cell epitope[24,25].
Perm Buffer (eBioscience) for 1 h at 4°C. Cells were
Antigen preparation has been described previously[24].
incubated for 30 min at 4°C with phycoerythrin-
Briefly, the recombinant peptide GAD
conjugated anti-human Foxp3 Ab or isotype Ab
500-585
was
produced from Escherichia coli BL-21 containing the
(eBioscience, San Diego, CA, USA) at the concentration
bacterial
expression
vector
pET28a+
vector.
recommended by the manufacturer, washed, and analyzed
Recombinant
GAD
by flow cytometry. Cells were washed and fixed with 1%
500-585
was
purified
using
Co2+/TALONIMAC resin (Clontech) according to the
paraformaldehyde prior to analysis in a FACSCalibur
manufacturer’s instructions. Eluted GAD
flow cytometer using CellQuest version 3.3 software (BD
500-585 fractions
were dialyzed against PBS (pH7.2).
Biosciences, San Jose, CA).


Cell preparation: Human pancreatic carcinoma cell
Introduction of Foxp3-siRNA into PANC-1 cells: To
lines PANC-1 (American Type Culture Collection,
introduce the human Foxp3 siRNAs (SC-43569, Santa
Manassas, VA) were grown in RPMI (Biochrom,
Cruz Biotechnology, INC) into cultured PANC-1 cells,
Berlin, Germany) 10% FCS (Atlanta Biologicals,
we used Lipofectamine Reagent and Plus Reagent
Atlanta, GA). Lymphocytes from the spleen were
(Invitrogen, Carlsbad, CA, USA) according to the
collected under sterile conditions, obtained with mouse
manufacturer’s procedure. Briefly, siRNAs were
Ficoll separation liquid, washed twice with an
incubated with the Plus Reagent in serum-free DMEM
incomplete RPMI medium and then re-suspended at
for 15min at room temperature, and then 350µl of the
−
5×106 cells mL 1 in 10% FCS in an RPMI1640
mixture was incubated with an equal volume of the
medium.
Lipofectamine reagent (16.8 µg) for another 15min in

order for them to form the complex of the siRNA with
In vitro proliferation: For analysis of cellular
liposome. The PANC-1 cells on a 35×10 mm Petri dish
response, lymphocytes were collected and cultured
(Becton Dickinson Labware, Franklin Lakes, NJ, USA)
(5×105 cells/well) in triplicate in 0.1 ml of RPMI-1640
were then incubated in the mixture of siRNAs with the
medium supplement with 1% horse serum and
Lipofectamine reagents for 6h at 37°C, then added to
−
restimulated with 30 µg mL 1 GAD
700 µL of DMEM containing 10% FBS and incubated
500-585 without or in
the presence of PANC-1 or Foxp3-/-PANC-1 cells
for another 42 h. To confirm the introduction of Foxp3-
(5×104 cells/well). On day 3, cultures were pulsed with
siRNA into cultured PANC-1 cells, cells were stained
1µCi/well of [3H] thymidine for the last 16h, and the
with PE-conjugated anti-human Foxp3 and analyzed by
cells harvested and counted by standard liquid
flow cytometry.
scintillation. The results were expressed as the

stimulation index (SI, c.p.m. with antigen divided by
Statistical analysis: Results of cytokine ELISA were
c.p.m. with medium alone) or CPM.
analyzed by the Student’s t-test. Differences in diabetes

incidence were analyzed by χ quadrate test. The
Determination
of
cytokines
production:
The
cumulative diabetes onset was compared by Kaplan-
splenocytes were collected and cultured as for
Meier analysis. All differences reported in the results
lymphocyte proliferation assay. Supernatants of the
were significant (p<0.05).

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Am. J. Immunol., 5 (4): 101-107, 2009

RESULTS
Foxp3 suppressed IL-10 and TGF-β in PANC-1

cells: Whether does Foxp3 as a suppressive
Foxp3 expression in PANC-1 cells: Despite many
transcription factor suppress TGF-β expression? Firstly,
advances in understanding the mechanisms leading to
we set up SiRNA assays to knock down Foxp3
tumor immunity, malignant tumors are tolerated,
expression. By flow cytometry, we found that the
progress, and ultimately kill their host. Tumor cells
percentage
of
Foxp3-expressing
PANC-1
cells
employed different strategies to thwart immune
decreased from 2-11% (Fig. 2A).
responses: tumor-induced impairment of antigen

Next, we determined the effect of Foxp3 down-
presentation, the activation of negative co-stimulatory
regulation on IL-10 and TGF-β synthesized by PANC-
signals, and the elaboration of immunosuppressive
1. PANC-1 cells expressed much higher level of TGF-β
factors[12]. IL-10 and TGF-β synthesized by tumor
and IL-10 when Foxp3 was knocked down by SiRNA
which exerts suppressive effects on the immune
assays (Fig. 2B). These results suggest that Foxp3
system[12-14]. We found PANC-1 cells also secreted high
expression suppressed TGF-β and IL-10 expression in
level of TGF-β and low level of IL-10 (Fig. 1A). In
PANC-1 cells.
vitro studies have shown that TGF-β can impose a

−
regulatory phenotype on CD4+CD25 T cells through
the induction of Foxp3 expression[15,16]. This makes us
propose that PANC-1 expressed Foxp3. As expected,
we first detect the expression of Foxp3 in PANC-1 cells
by cell flow. We found that 11% of PANC-1 cells
expressed Foxp3 (Fig. 1B).


(A)


(A)


(B)

Fig. 2: Foxp3 suppressed TGF-β and IL-10 expression

in PANC-1 cells
(B)

Note:
Human Foxp3 siRNAs were used to transduce
Fig. 1: Foxp3 expression in PANC-1 cells
PANC-1 cells (called as Foxp3-/-PANC-1
Note: PANC-1 cells were cultured. After 48 h, cells
cells). At 48 h after transduction, cells and
and supernatant were collected. (A) The
supernatant were collected. (A) Cells were
production of IL-10 and TGF-β was determined
stained for Foxp3 and analyzed by flow
by ELISA. (B) Cells were stained for Foxp3
cytometry. The percentage of Foxp3+ cells was
and analyzed by flow cytometry. The
showed. (B) The production of IL-10 and
percentage of Foxp3+ cells was showed. The
TGF-β was determined by ELISA. The results
results are representative of four separate
are
representative
of
three
separate
experiments
experiments (**: p﹤0.01)

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Am. J. Immunol., 5 (4): 101-107, 2009

PANC-1 cells induced tolerance and response model:
Above results makes us propose that PANC-1 cells
Thus, we propose that in PANC-1 cells, Foxp3 may be
induce immune tolerance of splenocytes from 7-week-
an important factor inducing tumor immunity by
old female NOD mice and response of splenocytes from
suppressing TGF-β and IL-10 expression. To prove the
12-week-old female NOD mice to autoantigen
proposal, we first develop PANC-1 cells induced
Glutamic Acid Decarboxylase 65 (GAD65)[18]. We used
tolerance and immunity model. The autoimmune Non-
splenocytes from 7 or 12-week-old female NOD mice
Obese Diabetic (NOD) mouse, characterized by T-cell
and PANC-1 cells to co-culture in vitro. As expected,
mediated destruction of the insulin-producing β-cells in
PANC-1 induced immune tolerance of splenocytes from
the pancreatic islets, were used[17]. About 1×107 PANC-
7 week-old female NOD mice and response of
1 cells were injected into 7-weeks-old female NOD
splenocytes from 12 week-old female NOD mice to
mice. As shown in Fig. 3A, the suppression of diabetes
GAD500-585 (Fig. 4A). In the parallel with cell
in PANC-1-treated group persisted for 18 weeks,
proliferation, PANC-1 cells induced anti-inflammatory
compared to recipients of the control (p<0.05). After 24
cytokines IL-10, TGF-β and suppressed inflammatory
weeks of age, PANC-1-treated NOD mice showed

higher diabetes incidence than that in control groups
(p<0.05). About 1×107 PANC-1 cells were injected into
12 weeks-old female NOD mice. As shown in Fig. 3B,
diabetes incidence was significantly higher in PANC-1-
treated NOD mice than that in control groups (p<0.01).



(A)



(A)


(B)


Fig. 4: PANC-1-induced tolerance and response model
(B)
Note: Splenocytes were collected from 7 or 12 weeks-

old female NOD mice and stimulated for 48 h
Fig. 3: PANC-1 cells were injected into autoimmune
−1
diabetic NOD mice
with 30 µg mL GAD500-585 without or in the
Note: (A) 7 week-old or (B) 12-week-old female
presence of PANC-1 (PANC-1). (A) Cell
NOD mice were injected with 1×107 PANC-1
proliferation was measured by incorporation of
cells per mouse (◊ PANC-1, 10 mice per group)
[3H] thymidine during the final 16 h of culture.
or Saline (■ Control, 10 mice per group). Mice
Data are presented as stimulation index (the ratio
were screened for glucose levels every 1-2
of c.p.m. with Ag to c.p.m. with medium alone)
weeks after gene therapy and considered
with the background of 1000– 3000 c.p.m. (B)
−
The production of IL-2, IFN-γ, IL-4, IL-17A, IL-
diabetic when glucose levels 13.8 mMol L 1
10 and TGF-β were determined by sandwich
were
detected
at
two
consecutive
ELISA in the co-culture supernatants. The results
measurements. Results of diabetes incidence
are representative of three separate experiments,
(number of diabetic out of total mice) represent
(**: p<0.01 and *: p<0.05)
a typical experiment

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Am. J. Immunol., 5 (4): 101-107, 2009

Foxp3 were critical for PANC-1 cells immunity: To
further show the role of Foxp3 in PANC-1 cells, we use
Foxp3-/-PANC-1 cells (Foxp3 expression was knocked
down in PANC-1 cells by SiRNA assays) to co-culture
with splenocytes from 7 or 12-week-old female NOD
mice in the presence of GAD500-585. We found that
Foxp3-/-PANC-1 cells induced tolerance in GAD500-585-
stimulated splenocytes from 7 or 12-week-old female
NOD mice (Fig. 5A). In addition, Foxp3-/-PANC-1 cells

(A)
upregulated anti-inflammatory cytokines IL-10, TGF-β


and downregulated inflammatory cytokine IL-2, IFN-
γ in GAD500-585-stimulated splenocytes from 7 or 12
week-old female NOD mice (Fig. 5B). These results
suggest that Foxp3 is an important factor for PANC-1
cells-induced immune response.

DISCUSSION


Foxp3 expression has been thought to be restricted
to the T-cell lineage. Until recently, several reports were
published about Foxp3 expression in human cancer
cells[7-9]. In these studies, they showed Foxp3

(B)
expression in many tumor cell lines of different tissues

origins by RT-PCR and Western blot assay. We also
Fig. 5: Foxp3
determined
PANC-1
cells-inducing
detected Foxp3 in human pancreatic cancer cell line
immunity
PANC-1 by cell flow (Fig. 1B). Except for in tumor
Note: PANC-1 cells were transduced with human
cells,
Foxp3
is
expressed
in
normal
breast
Foxp3 SiRNA, after 48 h, Foxp3 SiRNA-
epithelium[10].
transduced PANC-1 cells (Foxp3-/-PANC-1)

TGF-β and IL-10 were factors most frequently
were used to co-culture for 48 h with
released by pancreatic ductal carcinoma cell line[11]. We
splenocytes from 7 or 12-weeks-old female
also found that PANC-1 expressed TGF-β and IL-10
−
NOD mice in the presence of 30 µg mL 1
(Fig. 1A). TGF-β can converse peripheral CD4+CD25-
GAD
naive T cells to CD4+CD25- regulatory T cells by
500-585. (A) Cell proliferation was measured
by incorporation of [3H]thymidine during the
inducing Foxp3 expression[16]. These results suggest
final 16 h of culture. Data are presented as
that auto-secreted TGF-β may induce Foxp3 expression
c.p.m. (B) The production of IL-2, IFN-γ, IL-4,
in PANC-1 cells. A report found that Foxp3 expression
IL-17A, IL-10 and TGF-β were determined by
was induced by TGF-β2, but not TGF-β1 stimulation in
sandwich ELISA in the co-culture supernatants.
pancreatic ductal adenocarcinoma cells[7].
The results are representative of three separate

The role of Foxp3 in TGF-β expression is still
experiments, (**: p<0.01 and *: p<0.05)
unclear. We have known that Foxp3 is a suppressive

transcription factor[19]. Foxp3 suppress the transcription
cytokine IL-2, IFN-γ, and IL-17A in GAD
of several target genes including IL-2, IFN-γ, IL-4, and
500-585-
stimulated splenocytes from 7-week-old NOD, whereas
IL-10[1,6] in Treg, HER-2/ErbB2 oncogene expression in
PANC-1 cells induced inflammatory cytokine IL-2,
breast cancer[16], IL-6 and IL-8 in pancreatic carcinoma
IFN-γ, and IL-17A and suppressed anti-inflammatory
cell line PANC-89[7]. Can Foxp3 as suppressive
cytokines IL-10, TGF-β in GAD
transcription factor suppress TGF-β expression? Foxp3-
500-585-stimulated
splenocytes from 12 week-old NOD response (Fig. 4B).
expressing CD4+CD25+Treg cells-mediated tolerance is
These results suggest that co-cultured PANC-1 and
dependent on TGF-β-dependent[20] suggesting that
GAD
Foxp3 may promote TGF-β expression. Foxp3
500-585-activated
splenocytes from 7 week-old
female NOD mice could be as PAN-1-induced tolerance
expression was knocked down in PANC-1 cells by
model and co-cultured PANC-1 and GAD
SiRNA assays to study the role of Foxp3 in TGF-β
500-585-
activated splenocytes from 12 week-old female NOD
secreted by PANC-1. TGF-β and IL-10 were up-
mice could be as PAN-1-induced immune model.
regulated in PANC-1 cells when Foxp3 was knocked

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Am. J. Immunol., 5 (4): 101-107, 2009

down (Fig. 2B). These results suggest that Foxp3
function may vary. There are PANC-1-induced immune
expression suppressed TGF-β and IL-10 expression in
response and tolerance model make us gain more
PANC-1 cells.
information in the function of Foxp3. We found that

To further explore the role of Foxp3 in the
Foxp3-/-PANC-1 cells significantly induced tolerance in
immunity of PANC-1 cells, we develop the PANC-1
GAD500-585-stimulated splenocytes from 12 week-old
induced tolerance and immunity model. The NOD
and 7-week-old female NOD mice (Fig. 5A). In
strain of mouse is an increasingly useful and important
addition,
Foxp3-/-PANC-1
cells
induced
anti-
model of autoimmune type 1 diabetes[17]. The
inflammatory cytokines IL-10, TGF-β and suppressed
autoimmune disease characterized by progressive
inflammatory cytokine such as IL-2, IFN-γ and IL-17A
destruction of pancreatic beta cells we found that
in GAD500-585-stimulated splenocytes from 12-week-old
PANC-1 cells could control diabetes until 13 weeks
and 7 week-old female NOD mice (Fig. 5B). These
after therapy when injected into 7-weeks-old female
results suggest that Foxp3 control the capacity of
NOD mice (Fig 3A). In vitro experiment further proved
PANC-1 cells in inducing immune response. In
that PANC-1 cells induced tolerance (Fig. 4A) and
addition, the extent to reducing response in PANC-1-
immunoregulatory cytokines IL-10 and TGF-β, and
induced response model was obviously bigger than that
suppressed inflammatory cytokine IL-2, IFN-γ and IL-
in PANC-1-induced tolerance model. This may explain
17 in splenocytes from 7 week-old female NOD mice
for the necessary of building PANC-1-induced response
(Fig. 4A and B). These results show that PANC-1 cells
model.
induced tolerance in splenocyte from 7 weeks-old

female NOD mice. Tumor cells employed different
CONCLUSSION
strategies to thwart immune responses: tumor-induced

impairment of antigen presentation, the activation of

In conclusion, human pancreatic cancer cell line
negative co-stimulatory signals, and the elaboration of
PANC-1
exhibits
immunological
response
immunosuppressive factors[12]. IL-10 and TGF-β are
characteristics by expressing Foxp3. The effect may act
immunoregulatory cytokines that are involved in
partly by suppressing TGF-β expression. Thus, we
induction and maintenance of peripheral tolerance by
proved our proposal that Foxp3 may be an important
affecting autoreactive T-cell-mediated response[21-23].
factor inducing tumor immunity by suppressing TGF-β
The studies suggest that PANC-1 cells induce immune
expression in PANC-1 cells. It is under study that
tolerance partly by auto-secreted TGF-β and IL-10.
immunogenic response of PANC-1 cells is enhanced by

When PANC-1 cells were injected into 12 weeks-
Foxp3 over-expression.
old female NOD mice, diabetes incidence was

significantly higher in PANC-1-treated NOD mice
ACKNOWLEDGEMENT
(Fig. 3B). In vitro experiment further proved that


We gratefully acknowledge Dr. Jiannan Feng for
PANC-1 cells induced response (Fig. 4A) and
useful advice. This work was supported by National
inflammatory cytokine IL-2, IFN-γ and IL-17, and
Nature and Science Funds (30571732) and National
suppressed immunoregulatory cytokines IL-10 and
“973” Fund Grant 2007CB512406 and 2009CB522408.
TGF-β in splenocytes from 12 week-old female NOD
The authors have no conflicts of interest.
mice (Fig. 4B). The fact that most mice demonstrate

severe insulitis by 10 weeks of age explained that
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