Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation

Abstract

The human breast tumor microenvironment can display features of T helper type 2 (Th2) inflammation, and Th2 inflammation can promote tumor development. However, the molecular and cellular mechanisms contributing to Th2 inflammation in breast tumors remain unclear. Here, we show that human breast cancer cells produce thymic stromal lymphopoietin (TSLP). Breast tumor supernatants, in a TSLP-dependent manner, induce expression of OX40L on dendritic cells (DCs). OX40L(+) DCs are found in primary breast tumor infiltrates. OX40L(+) DCs drive development of inflammatory Th2 cells producing interleukin-13 and tumor necrosis factor in vitro. Antibodies neutralizing TSLP or OX40L inhibit breast tumor growth and interleukin-13 production in a xenograft model. Thus, breast cancer cell-derived TSLP contributes to the inflammatory Th2 microenvironment conducive to breast tumor development by inducing OX40L expression on DCs.

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J. Exp. Med. Vol. 208 No. 3 479-490
www.jem.org/cgi/doi/10.1084/jem.20102131
479
There is accumulating evidence that inamma-
tion plays a key role in the initiation and pro-
gression of cancer (Grivennikov et al., 2010).
There are two types of inammation that have
opposing eects on tumors: (a) chronic inam-
mation, which promotes cancer cell survival
and metastasis (Coussens and Werb, 2002;
Condeelis and Pollard, 2006; Mantovani et al.,
2008), and (b) acute inammation, which can
trigger cancer cell destruction as illustrated by
regressions of bladder cancer after treatment
with microbial preparations (Rako-Nahoum
and Medzhitov, 2009). Although chronic in-
ammation is often linked with the presence of
type 2–polarized macrophages (M2), acute in-
ammation associated with cancer destruction
is linked with type 1–polarized macrophages
(M1). M1 macrophages are induced by the type
1 cytokine IFN-, whereas, M2 macrophages
are induced by the type 2 cytokines IL-4 and
IL-13 (Mantovani and Sica, 2010).
Type 2 cytokines can contribute to tumori-
genesis in several ways. For example, IL-13 pro-
duced by NKT cells induces myeloid cells to
CORRESPONDENCE
A. Karolina Palucka:
karolinp@baylorhealth.edu
Abbreviations used: HPC,
hematopoietic progenitor cell;
mDC, myeloid DC; NOD/
SCID/2m/, nonobese
diabetic/LtSz-scid/scid 2
microglobulin–decient; TSLP,
thymic stromal lymphopoietin.
A. Pedroza-Gonzalez’s present address is Dept. of
Gastroenterology and Hepatology, Erasmus MC,
Rotterdam, Netherlands.
T.-C. Wu’s present address is College of Life Sciences,
Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
C. Aspord’s present address is Institut National de la Santé
et de la Recherche Médicale U823, Immunobiology and
Immunotherapy of Cancers, 38701, La Tronche, France.
J. Banchereau’s present address is Homan-La Roche, Inc.,
Nutley, NJ.
Thymic stromal lymphopoietin fosters
human breast tumor growth by promoting
type 2 inammation
Alexander Pedroza-Gonzalez,1 Kangling Xu,1,2 Te-Chia Wu,1,2
Caroline Aspord,1 Sasha Tindle,1 Florentina Marches,1 Michael Gallegos,1
Elizabeth C. Burton,4 Daniel Savino,4 Toshiyuki Hori,5 Yuetsu Tanaka,6
Sandra Zurawski,1 Gerard Zurawski,1 Laura Bover,7 Yong-Jun Liu,7
Jacques Banchereau,1,8,9 and A. Karolina Palucka1,3,8,9
1Baylor Institute for Immunology Research, Baylor Research Institute, Dallas, TX 75204
2Department of Biomedical Studies, Baylor University, Waco, TX 76706
3Sammons Cancer Center, 4Baylor University Medical Center, Dallas, TX 75246
5Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507, Japan
6Department of Immunology, University of the Ryukyus, Okinawa 903-0215, Japan
7MD Anderson Cancer Center, Houston, TX 77030
8Department of Gene and Cell Medicine and 9Department of Medicine, Immunology Institute, Mount Sinai School of Medicine,
New York, NY 10029
The human breast tumor microenvironment can display features of T helper type 2 (Th2)
inammation, and Th2 inammation can promote tumor development. However, the
molecular and cellular mechanisms contributing to Th2 inammation in breast tumors
remain unclear. Here, we show that human breast cancer cells produce thymic stromal
lymphopoietin (TSLP). Breast tumor supernatants, in a TSLP-dependent manner, induce
expression of OX40L on dendritic cells (DCs). OX40L+ DCs are found in primary breast
tumor inltrates. OX40L+ DCs drive development of inammatory Th2 cells producing
interleukin-13 and tumor necrosis factor in vitro. Antibodies neutralizing TSLP or OX40L
inhibit breast tumor growth and interleukin-13 production in a xenograft model. Thus,
breast cancer cell–derived TSLP contributes to the inammatory Th2 microenvironment
conducive to breast tumor development by inducing OX40L expression on DCs.
© 2011 Pedroza-Gonzalez et al. This article is distributed under the terms of
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rst six months after the publication date (see http://www.rupress.org/terms).
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The Journal of Experimental Medicine

480 TSLP–OX40L–IL-13 axis in human breast cancer | Pedroza-Gonzalez et al.
>410,000 will die from the disease (Coughlin and Ekwueme,
2009). Here, we show that inammatory Th2 cells that promote
tumor development are driven by breast cancer–derived TSLP,
which induces and maintains OX40L-expressing DCs in the
tumor microenvironment. Thus, TSLP, and down-stream mole-
cules, might represent novel potential therapeutic targets.
RESULTS
Inammatory Th2 cells in primary breast cancer tumors
Our earlier study using a pilot cohort of 19 samples of pri-
mary breast cancer tumors revealed the secretion, upon acti-
vation with PMA and ionomycin, of both type 1 (IFN-) and
type 2 (IL-4 and IL-13) cytokines (Aspord et al., 2007). The
current study extends the analysis to a total of 99 consecutive
samples (Table S1). Supernatants of activated tumor fragments
display high levels of IFN-, IL-2, IL-4, IL-13, and TNF
(Fig. 1 A and Table S1). Supernatants from tumor sites
contained signicantly higher levels of IL-2, type 2 (IL-4 and
IL-13), and inammatory (TNF) cytokines than those from
macroscopically uninvolved surrounding tissue (Fig. 1 B and
Table S1). Whereas a signicant correlation can be observed
between IL-2 levels in tumor and adjacent tissue (P = 0.02),
IL-4 and IL-13 levels are not correlated (P = 0.5), further
suggesting polarization of cytokine environment in breast
tumors (Fig. 1 B). IFN- did not correlate with other cyto-
kine levels (Table S1). However, levels of TNF were correlated
with those of IL-13 (P < 0.0001; r = 0.62; n = 98) and IL-4
(P = 0.0175; r = 0.31; n = 59; Fig. 1 C). Thus, this survey of
cytokine expression suggested Th2 polarization in the breast
tumor microenvironment.
To identify the cells producing these cytokines, single-cell
suspensions were prepared from tumors; activated for 5 h with
PMA and ionomycin; stained with antibodies against T cells
and cytokines; and analyzed by ow cytometry. Gated viable
CD4+CD3+ T cells expressed IL-13 (3.67%), most of them
coexpressing IFN- and TNF (Fig. 1 D and Fig. S1 A).
A small fraction of IL-13+CD4+ T cells coexpressed IL-4, but
none expressed IL-10 (Fig. 1 D). Such T cells have been re-
ferred to as inammatory Th2 cells that are involved in aller-
gic inammatory diseases (Liu et al., 2007). Flow cytometry
analysis of consecutive tumor inltrates (n = 22) shows the
overall increased percentages of IL-4– and IL-13–secreting
CD4+CD3+ T cells (P = 0.0313 and P = 0.0156, respectively)
when compared with adjacent tissue samples (Fig. 1 D and
Fig. S1 B). Thus, the dierence between tumor tissue and ad-
jacent tissue appears to be caused by both the increased num-
bers of inltrating T cells and enhanced polarization. The
analysis of frozen tissue sections further demonstrated that in-
ltrating T cells in primary breast cancer tumors express IL-13
(Fig. 1 E). Thus, breast cancer tumors are inltrated with in-
ammatory Th2 cells.
DCs inltrating breast cancer tumors express OX40 ligand
Because OX40 ligation drives the dierentiation of CD4+
T cells into inammatory Th2 (Ito et al., 2005), we analyzed
the presence of OX40L in primary breast cancer tumors.
make TGF-, which ultimately inhibits CTL functions
(Berzofsky and Terabe, 2008). Spontaneous autochthonous
breast carcinomas arising in Her-2/neu transgenic mice ap-
pear more quickly when the mice are depleted of T cells,
which is evidence of T cell–mediated immunosurveillance
slowing tumor growth (Park et al., 2008). This immuno-
surveillance could be further enhanced by blockade of IL-13,
which slowed the appearance of these autologous tumors
compared with control antibody-treated mice (Park et al.,
2008). A spontaneous mouse breast cancer model recently
highlighted the role of Th2 cells which facilitate the develop-
ment of lung metastasis through macrophage activation
(DeNardo et al., 2009). We identied CD4+ T cells secreting
IFN- and IL-13 in breast cancer tumors (Aspord et al.,
2007). We also found that breast cancer cells express IL-13 on
cell surface. Autocrine IL-13 has been shown to be important
in the pathophysiology of Hodgkin’s disease (Kapp et al.,
1999; Skinnider et al., 2001, 2002). IL-13 and IL-13R are fre-
quently expressed by Hodgkin’s and Reed-Sternberg cells
(Skinnider et al., 2001), and IL-13 stimulates their growth
(Kapp et al., 1999; Trieu et al., 2004). Similar to Hodgkin’s
cells (Skinnider et al., 2002), breast cancer cells express
pSTAT6 (Aspord et al., 2007), suggesting that IL-13 actually
delivers signals to cancer cells. However, the mechanisms un-
derlying the development of Th2 inammation in breast can-
cer are unknown.
Like many other features of the immune response, Th1/
Th2 polarization is regulated by DCs. In the steady state, non-
activated (immature) DCs present self-antigens to T cells,
which leads to tolerance (Hawiger et al., 2001; Steinman et al.,
2003). Once activated (mature), antigen-loaded DCs are
geared toward the launching of antigen-specic immunity
(Finkelman et al., 1996; Brimnes et al., 2003) leading to the
proliferation of T cells and their dierentiation into helper
and eector cells. DCs are composed of distinct subsets, in-
cluding myeloid DCs (mDCs) and plasmacytoid DCs (Caux
et al., 1997; Maldonado-López et al., 1999; Pulendran et al., 1999;
Luft et al., 2002; Dudziak et al., 2007; Klechevsky et al.,
2008). DCs are also endowed with functional plasticity, i.e.,
they respond dierentially to distinct activation signals
(Steinman and Banchereau, 2007). For example, IL-10–
polarized mDCs generate anergic CD8+ T cells that are un-
able to lyse tumors (Steinbrink et al., 1999), as well as CD4+
T cells with regulatory/suppressor function (Levings et al.,
2005). In contrast, thymic stromal lymphopoietin (TSLP)–
polarized mDCs are conditioned to express OX40 ligand
(OX40L) and to expand T cells producing type 2 cytokines
(Soumelis et al., 2002; Gilliet et al., 2003). Both the distinct DC
subsets and their distinct response to microenvironment contrib-
ute to the generation of unique adaptive immune responses.
Unraveling the mechanisms by which breast cancer po-
larizes the immune responses might oer novel therapeutic
options. This is important because despite declining mortality
rates, breast cancer ranks second among cancer-related deaths
in women. Worldwide, it is estimated that more than 1 million
women are diagnosed with breast cancer every year, and

JEM VOL. 208, March 14, 2011
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481
mean ± SE for surrounding tissue = 1.5 ± 0.8% [n = 7]
and for breast cancer tumors 11 ± 1.67% [n = 12], respec-
tively; Fig. 2 B). Thus, breast cancer tumors are inltrated
with OX40L+ mDCs.
Breast cancer tumors produce soluble factors that induce
functional OX40L expression on DCs
To identify the breast cancer tumor factors that induce
OX40L on mDCs, LINnegHLA-DR+ CD123 CD11c+
mDCs were sorted from blood of healthy volunteers and
Immunouorescence staining of frozen tissue sections of pri-
mary breast cancer tumors showed the expression, in 57 out
of 60 analyzed tumors, of OX40L by a majority of HLA-
DRhigh cells (Fig. 2 A). These OX40L+ cells are located in
peritumoral areas (Fig. 2 A). Flow cytometry analysis of
single-cell suspensions further conrmed the expression of
OX40L by a fraction of HLA-DRhigh CD14neg CD11chigh
mDCs (Fig. 2 B). Paired analysis demonstrated that the tumor
beds express higher percentages of OX40L+ mDCs than
the surrounding tissue (P = 0.0156; n = 7 paired samples;
Figure 1. Inammatory Th2 in breast cancer immune environment. (A) Cytokine proles as determined by Luminex in supernatants of human
breast tumor fragments stimulated for 16 h with PMA and ionomycin. Numbers on the x-axis indicate the number of tissue samples from different pa-
tients tested. (B) Cytokine proles as determined by Luminex in supernatants of tumor fragments (T) and surrounding tissue (ST) from the same patient
after PMA and ionomycin stimulation. Cytokine concentration values of IL-2, IL-4, and IL-13 from T and ST samples were plotted and analyzed using lin-
ear regression to determine the level of correlation between cytokine concentration in T and ST samples. (C) Cytokine proles as determined by Luminex
in supernatants of tumor fragments after PMA and ionomycin stimulation. Cytokine concentration values of TNF and IL-13 and of TNF and IL-4 were
plotted and analyzed using nonparametric Spearman correlation to determine the level of correlation of two cytokines concentration in tumor samples.
(D, top) Single-cell suspensions from tumor samples were stimulated for 5 h with PMA and ionomycin. Cytokine production was measured by ow
cytometry. Dot plots are gated on CD3+CD4+ T cells. (top right dot plot) Blue indicates gate on CD3+CD4+IL-13+ T cells that coexpress IFN- and TNF. Rep-
resentative of four different patients from whom we have been able to obtain sufcient numbers of cells for 10-color analysis (patient nos. 148, 155, 164,
and 169). Bottom, percentages of CD4+ T cells expressing IL-4 and IL-13 in tumor inltrates and surrounding tissue (ST) were analyzed by ow cytometry.
Dotted lines indicate paired samples from the same patient (n = 7, Wilcoxon matched-pairs ranked test). Single points indicate the percentage of cytokine
expressing cells in tumor samples analyzed by ow cytometry for which we did not obtain sufcient number of cells from surrounding tissue to allow the
analysis. (E) Frozen tissue sections from the same patient as in D were analyzed by immunouorescence. Triple staining with anti-CD3-FITC (green), anti–
IL-13–Texas red (red), and DAPI nuclear staining (blue). Bar, 90 µm.

482 TSLP–OX40L–IL-13 axis in human breast cancer | Pedroza-Gonzalez et al.
To test whether primary breast cancer tumors could also
regulate OX40L expression, fragments of primary tumors
were sonicated, centrifuged, ltered, and used in cultures with
blood mDCs. As illustrated in Fig. 2 D, mDCs acquired both
CD83 (a DC maturation marker) and OX40L. To determine
the impact of OX40L on the generation of inammatory Th2
responses in breast cancer, blood mDCs were rst exposed for
48 h to either TSLP or breast tumor–soluble fractions. Exposed
mDCs were then used to stimulate naive allogeneic CD4+
T cells with either the anti-OX40L antibody or a relevant
exposed to breast cancer supernatants. These were generated
from established breast cancer cell lines expanded in vitro
(Hs578T, MDA-MB-231, MDA-MB-468, MCF7, HCC-
1806, and T47D; Table S2) and breast cancer tumors estab-
lished in vivo by implanting breast cancer cell lines in
immunodecient mice (Aspord et al., 2007). As illustrated in
Fig. 2 C, mDCs exposed for 48 h to Hs578T and HCC-1806
supernatants expressed OX40L. Four of the ve breast cancer
cell lines, with the notable exception of T47D, induced
OX40L expression on mDCs (Fig. S1 C).
Figure 2. OX40L in breast cancer immune environment. (A) Immunouorescence of primary breast tumor with indicated antibodies. Bar, 180 µm.
Representative of 57/60 tumors analyzed. (B) Flow cytometry analysis of single-cell suspensions of primary breast tumors and surrounding tissue. Dot
plots are gated on CD14neg nonlymphocytes. OX40L expression is analyzed on HLA-DRhighCD11chigh DCs. Graph summarizes percentages of OX40L-expressing
DCs in tumor inltrates and surrounding tissue (ST) analyzed by ow cytometry. Dotted lines indicate paired samples from the same patient (Wilcoxon
matched-pairs ranked test). Single points indicate the percentage of OX40L+ DCs in tumor samples for which we did not obtain sufcient number of cells
from surrounding tissue to allow the analysis. (C and D) mDCs were exposed to media alone, to supernatant of breast cancer cell lines (1806 or Hs587T),
or to sonicate of primary breast cancer tissue from patients (tumor 43). OX40L and CD83 were measured by ow cytometry. FMO, uorescence minus one
indicates controls where one staining uorescence is omitted to set negative gate. (E and F) mDCs were exposed for 48 h to supernatants of breast cancer
cells Hs578T, and then co-cultured with allogeneic naive CD4+ T cells in the presence of 40 µg/ml of anti-OX40L (Ik-5 clone) or isotype control antibody.
After 1 wk, cells were collected and restimulated for 5 h with PMA/ionomycin for intracellular cytokine staining. Data in E are representative of four ex-
periments. (F) Summary of the effect of blocking OX40L during T cell stimulation by tumor-activated DCs. Graph shows the proportion of IL-13–secreting
cells induced by DCs activated with supernatants from breast cancer cell line Hs578T (left) or primary breast tumors (right, T15, T29, and T53).

JEM VOL. 208, March 14, 2011
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483
and secreted TSLP (Fig. 3 A). Supernatants of some pr imary
breast cancer tumors stimulated with PMA and ionomycin
displayed up to 300 pg/ml TSLP (Fig. 3 B). The expression of
TSLP by cancer cells was further analyzed using an anti-TSLP
antibody and immunouorescence of frozen breast cancer
tumors generated in the xenograft model (Aspord et al., 2007).
There, subcutaneous MDA-MB-231 tumors transplanted in
mice expressed TSLP (Fig. 3 C). The specicity of the staining
is demonstrated by pretreatment of the antibody with recom-
binant TSLP (Fig. S3).
Importantly, TSLP is expressed in 35 out of 38 analyzed
primary breast cancer tumors obtained from patients regard-
less of grade, histology, or stage of analyzed tumors. Fig. 3 D
illustrates the pattern of TSLP staining and coexpression with
isotype control. Blocking OX40L prevented the expansion of
IL13+CD4+ or TNF+CD4+ T cells by TSLP-primed mDCs
(>50% inhibition; Fig. S2 A), mDCs exposed to Hs578T
breast cancer cells (n = 4; median inhibition of IL13+CD4+
cells = 74%; range = 67–80%; Fig. 2, E and F), and mDCs ex-
posed to sonicates of randomly selected primary breast cancer
tumors (Fig. 2 F and Fig. S2 B). Thus, breast cancer cells pro-
duce factors that activate mDCs and induce them to express
OX40L and to elicit inammatory Th2 cells.
Breast cancer tumors express and secrete TSLP
OX40L can be induced on mDCs by TSLP, an IL-7–like cyto-
kine produced by epithelial cells (Liu et al., 2007; Ziegler
and Artis, 2010). All tested breast cancer cell lines expressed
Figure 3. TSLP in breast cancer environment. (A) Luminex analysis of TSLP in supernatants of breast cancer cell lines after 24 h of culture in the
presence of PMA and ionomycin. (B) Luminex analysis of TSLP levels in supernatants of primary breast tumors (from 44 patients) activated with PMA and
ionomycin. (C) NOD/SCID/2m/ mice were irradiated the day before tumor implantation and 10 × 106 MDA-MB-231 cells were implanted by subcuta-
neous injection. Tumors were harvested at 4 wk after implant. Frozen tissue sections were analyzed by immunouorescence for expression of TSLP (red).
Actively dividing cells were identied by expression of Ki67 (green). Bar, 45 µm. (D and E) Frozen tissue sections from primary breast tumors from patients
(38 patient samples) were analyzed by immunouorescence for expression of TSLP. Tissues were also stained for the expression of IL-13 and cytokeratin
19, as indicated, to conrm TSLP expression by cancer cells. Staining pattern is representative of 35 out of 38 analyzed tumor samples from different
patients. Bars: (D) 180 µm; (E) 15 µm. (F) NOD/SCID/2m/ mice were sublethally irradiated and transplanted with human CD34+ HPCs by intravenous
injection. 4 wk after HPC transplant, 5 × 106 MDA-MB-231 breast cancer cells were implanted subcutaneously. Tumors at the site of implantation, as well
as lungs and kidneys, were harvested at 3 mo after implant. Frozen tissue sections were analyzed by immunouorescence for expression of TSLP (green)
and cytokeratin (red). Staining pattern is representative of tumors from three different mice. Bar, 90 µm.

484 TSLP–OX40L–IL-13 axis in human breast cancer | Pedroza-Gonzalez et al.
is specic to epithelial cells as no staining can be found in
tumor-inltrating broblasts (Fig. S4 B). Furthermore, non-
malignant breast epithelia can also express TSLP (Fig. S4 C).
Thus, similar to normal skin or lung epithelium, breast
epithelial cells and breast cancer cells have the capacity to ex-
press, produce, and secrete TSLP.
cytokeratin 19–positive cells. It demonstrates that TSLP is ex-
pressed in the cytoplasm and the nucleus of breast cancer cells
that display IL-13 on their surface (Fig. 3 E). Importantly,
TSLP is also expressed in lung and kidney metastasis of MDA-
MB-231 tumors in humanized mice (Fig. 3 F) and in breast can-
cer tumor metastasis from patients (Fig. S4 A). TSLP expression
Figure 4. Blocking TSLP in vitro. (A) mDCs were incubated with supernatant of breast cancer cell line Hs578T in the presence or absence of 20 µg/ml
of anti-TSLP (AB 19024; rabbit IgG). OX40L expression was measured by ow cytometry after 48 h of incubation. (B) mDCs treated as in A were co-
cultured with naive allogeneic CD4+ T cells for 7 d. IL-13 production was measured by intracellular cytokine staining and ow cytometry after cells were
restimulated for 5 h with PMA and ionomycin. Data are representative of three experiments. (C) mDCs were incubated with soluble factors from sonicated
human breast tumors (T53, T60, and T97) in the presence or absence of 20 µg/ml of anti-TSLP (AB 19024; rabbit IgG). OX40L expression was measured by
ow cytometry after 48 h of incubation. (D) mDCs treated as in C were co-cultured with naive allogeneic CD4+ T cells for 7 d. IL-13 production was mea-
sured by intracellular cytokine staining and ow cytometry after cells were restimulated for 5 h with PMA and ionomycin. Representative of three pa-
tients tested. Blue dots represent IL-13+ T cells gated in the same sample. (E) Graph shows data from three independent experiments as described in A–D.

JEM VOL. 208, March 14, 2011
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485
whether this axis might actually contribute to breast cancer
tumor development. To address this question, humanized mice
were reconstituted with both Hs578T cells and T cells with
or without anti-OX40L–, anti-TSLPR–, and anti-TSLP–
neutralizing antibodies. As shown in Fig. 6 A, the administra-
tion of neutralizing anti-OX40L antibodies leads to signicant
inhibition of tumor development.
The administration of a neutralizing anti-TSLP antibody
also results in the inhibition of tumor development (Fig. 6 B).
TSLP blockade also leads to decreased secretion of IL-4 and
IL-13 by tumor inltrating T cells upon PMA and ionomycin
activation (Fig. 6 C). Finally, the administration of antibody
blocking TSLPR nearly completely blocks tumor develop-
ment driven by CD4+ T cells (Fig. 6 D). These results indicate
that the TSLP contributes to breast cancer pathogenesis.
DISCUSSION
Here, we show that breast cancer is inltrated with inamma-
tory Th2 cells and that such T cells are driven by OX40L on
DCs. Blocking OX40L in vitro prevents generation of these
CD4+ T cells without impact on IL-10–producing CD4+
T cells. Blocking OX40L in vivo partially prevents T cell–
dependent acceleration of breast cancer tumor development.
OX40L is not constitutively expressed, but can be induced on
DCs, macrophages, and B cells; e.g., upon CD40 engagement
or cytokine signals such as TSLP or IL-18, as well as upon
TLR stimulation (Ito et al., 2005; Croft et al., 2009). Thus, the
Anti-TSLP antibodies block the generation of inammatory
Th2 responses in vitro
To determine the impact of blocking TSLP on the generation
of inammatory Th2 responses in breast cancer, blood mDCs
were rst exposed for 48 h to either TSLP or breast tumor–
soluble fractions. Addition of anti-TSLP–neutralizing anti-
bodies to breast cancer tumor supernatants inhibited their
ability to induce OX40L on mDCs (Fig. 4 A). Such mDCs
displayed a diminished capacity to expand IL13+CD4+ or
TNF+CD4+ T cells (n = 3; median inhibition = 73%, range =
72–77%; Fig. 4, B and E). Likewise, adding anti-TSLP–
neutralizing antibody to sonicate of randomly selected primary
breast cancer tumors led to down-regulation of OX40L ex-
pression by mDCs (Fig. 4 C) and decreased expansion of
IL13+TNF+CD4+ T cells (Fig. 4, D and E). Finally, when
anti-TSLP receptor  chain (TSLPR) antibody was added to
mDCs during their exposure to the supernatant of three dif-
ferent breast cancer cell lines (Hs578T, MDA-MB-231, and
MCF7; Fig. 5 A), resulting mDCs showed a much diminished
expansion of IL13+ CD4+ T cells (Fig. 5 B). Thus, TSLP is the
factor secreted by breast cancer cells that contributes to gen-
eration of inammatory Th2 responses.
Antibodies neutralizing TSLP–OX40L axis block tumor
development in vivo
Our results thus far suggest a role for the TSLP–OX40L axis
in generation of IL13+TNF+CD4+ T cells, but do not establish
Figure 5. Blocking TSLP-R in vitro. (A) mDCs were treated with anti-TSLP-R (clone AB81_85.1F11, mouse IgG1), media or control antibody during
activation with TSLP or with supernatant of one of the three different breast cancer cell lines (Hs578T, MDA-MB-231, and MCF7). mDCs were then co-
cultured with allogeneic naive CD4+ T cells. After 1 wk, cells were collected, restimulated for 5 h with PMA and ionomycin, and analyzed by ow cytom-
etry. (B) Analysis of different experiments showing the effect of blocking TSLP-R on the induction of IL-13 secreting cells as described in A.

486 TSLP–OX40L–IL-13 axis in human breast cancer | Pedroza-Gonzalez et al.
cells markedly in-
crease TSLP expres-
sion (Liu et al.,
2007). The TSLP-
activated DCs mi-
grate to the draining lymph nodes, prime CD4+ T cells via
OX40L to dierentiate into inammatory Th2 eector
and memory cells, and thus initiate the adaptive phase of
allergic immune responses. Interestingly, in breast cancer,
OX40L+ mDCs are present in the tumor. It remains to be
determined whether this reects their inability to migrate
from the tumor to draining lymph nodes. Indeed, such
an evasion mechanism has been documented recently in
human and mouse tumors showing an inhibition of DC
migration from tumors to tumor-draining lymph nodes
(Villablanca et al., 2010). This eect depends on tumor-
derived ligands of the liver X receptors (Villablanca et al.,
2010). It also remains to be determined whether these DCs
are able to prime Th2 immunity in situ in tertiary lymphoid
structures or whether their main role is to maintain the ac-
tivation and survival of Th2 cells at the tumor site. Their
ability to maintain Th2 cell phenotype and eector func-
tion is supported by our earlier studies showing that T cells
isolated from experimental breast tumors and transferred to
naive tumor-bearing humanized mice can promote tumor
development even at low numbers and upon single injec-
tion (Aspord et al., 2007).
presence of OX40L+ mDCs in breast tumors indicate sus-
tained activation of DCs in tumor environment. Indeed,
OX40L expression by DCs is driven by TSLP secreted from
breast cancer cells. Accordingly, TSLP expression can be found
in primary and metastatic tumors. Blocking TSLP reduces in-
ammation and partially inhibits tumor development.
Based on our results presented herein and in our earlier
studies, we propose a vicious circle of smoldering type 2 in-
ammation that perpetuates breast cancer and which is main-
tained by TSLP. There, breast cancer attracts DCs, possibly
through macrophage inammatory protein 3  (MIP3-;
Bell et al., 1999). Tumor-inltrating DCs are then exposed to
TSLP secreted by breast cancer cells, which triggers their
maturation and OX40L expression. This might explain the
aseptic mDC maturation that we found in breast cancer (Bell
et al., 1999; Aspord et al., 2007). OX40L+ mDCs induce
CD4+ T cells to secrete IL-13, as well as TNF. These inam-
matory CD4+ T cells contribute to tumor development in an
IL-13–dependent pathway (Aspord et al., 2007). Thus far,
TSLP represents the only factor that activates mDCs without
inducing them to produce Th1-polarizing cytokines (Liu
et al., 2007). Under normal physiological conditions, TSLP
appears to play a critical role in CD4+ T cell homeostasis in
the peripheral mucosa-associated lymphoid tissues and in the
positive selection and/or expansion of regulatory T (T reg)
cells in the thymus (Watanabe et al., 2005a,b). In inamma-
tory conditions such as atopic dermatitis and asthma, epithelial
Figure 6. Blocking OX40L-TSLP in vivo.
(A) NOD/SCID/2m/ mice were sublethally
irradiated and transplanted with human
CD34+ HPCs by intravenous injection. 4 wk
after HPC transplant, 10 × 106 Hs578T breast
cancer cells were implanted subcutaneously.
3, 6, and 9 d after, mice were reconstituted
with autologous total T cells together with
200 µg per injection of blocking anti-OX40L
or isotype control antibody (mouse Ig; red
arrows). PBS group was injected with tumor
cells but not with T cells (gray line). Tumor
size was measured at indicated times. Mean
values from three experiments representing
nine mice per each condition. Anti-OX40L and
isotype-treated cohorts were compared sta-
tistically. (B) NOD/SCID/2m/ mice were
irradiated and implanted with 10 × 106
Hs578T breast cancer cells together with 200 µg
per injection of neutralizing anti-TSLP
(rabbit), rabbit isotype control antibody, or
PBS. 3, 6, and 9 d after, mice were reconsti-
tuted with immature DCs and autologous
total T cells together with 200 µg per injection
of neutralizing anti-TSLP (rabbit), rabbit iso-
type control antibody or PBS. Representative
of three independent experiments with a total
of nine mice in TSLP blockade group. (C) Cyto-
kine secretion in single-cell suspensions from
tumors after 16-h restimulation with PMA
and ionomycin. (D) Same as in B, but mice
were injected with anti-TSLPR or isotype con-
trol at days 3, 6, and 9. Representative of two
independent experiments. n indicates number
of mice per cohort in this representative
experiment.

JEM VOL. 208, March 14, 2011
Article
487
Interestingly, studies in mice suggest the role of Th2 cyto-
kines in mammary gland development. Indeed, the dierenti-
ation of the luminal epithelial lineage appears to require
autocrine signaling by Th2 cytokines, as shown by reduced
dierentiation and alveolar morphogenesis in both Stat6 and
IL-4–/IL-13–decient mice during pregnancy (Khaled et al.,
2007). Yet, in the murine model of endogenous breast cancer
type 2, polarized CD4+ T cells are essential to the metastatic
process via secretion of IL-4, which induces macrophages to
secrete epidermal growth factor (DeNardo et al., 2009). Simi-
lar to endogenous mouse model, in our model of transplanted
metastatic human breast cancer IL-13 is derived from micro-
environment. IL-13 can exert pro-cancer activity in several
ways, including the triggering of TGF- secretion (Terabe
et al., 2000, 2003; Park et al., 2005; Shimamura et al., 2010).
Furthermore, IL-4 exposure of cancer cells leads to the up-
regulation of antiapoptotic pathways via mobilization of
STAT6 (Zhang et al., 2008). We have shown that STAT6 is
phosphorylated in primary breast cancer tumors (Aspord et al.,
2007). All these antiapoptotic pathways are likely to synergize
to promote the survival of cancer cells and facilitate metastasis.
Importantly, such a protective eect on cancer cell suscepti-
bility to apoptosis might increase their resistance to chemother-
apy (Todaro et al., 2008) and immune-mediated cytotoxicity
driven by Granzyme B (Sarin et al., 1997; Heibein et al.,
2000). Thus, the TSLP–OX40L–IL13 axis might oer a novel
therapeutic target.
MATERIALS AND METHODS
Isolation and culture of myeloid dendritic cells. DCs were pur ied
from buy coat of blood from healthy donors. In brief, DCs were enriched
from mononuclear cells by negative selection using a mixture of antibodies
against linage markers for CD3, CD14, CD16, CD19, CD56, and glycopho-
rin A (Dynabeads Human DC Enrichment kit; Invitrogen). Cells from nega-
tive fraction were immunolabeled with anti–human FITC-labeled lienage
cocktail (CD3, CD14, CD16, CD19, CD20, and CD56; BD); PE-labeled
CD123 (mIgG1; clone 9F5; BD), QR-labeled HLA-DR (mIgG2a; clone
HK14; Sigma-Aldrich) and APC-labeled CD11c (mIgG2b; clone S-HCL-3;
BD). DCs (lin, CD123, HLA-DR+, CD11c+) were sorted in a FACSAria
cytometer (BD). DCs were seeded at 100 × 103 cells/well in 200 µl of me-
dium (RPMI supplemented with 2 mM glutamine, 50 U/ml penicillin,
50 µg/ml streptomycin, 0.1 mM MEM nonessential amino acids, 10 mM
Hepes buer, 0.1 mM sodium pyruvate, and 10% of human AB serum). DCs
were cultured with medium alone or in the presence of 20 ng/ml of TSLP,
or dierent tumor derived products. After 48 h, DCs were harvested and
washed. The stimulated cells were stained for phenotype analysis or co-
cultured with allogeneic naive CD4+ T cells.
Immunouorescence. 6-µm-frozen sections from tissues were xed with
cold acetone for 5 min. The sections were labeled with 5 µg/ml of anti-
OX40L antibody (mouse IgG1, 8F4), followed by anti–mouse IgG conju-
gated to Texas red (Jackson ImmunoResearch Laboratories). For IL-13, tissue
was labeled with 10 µg/ml of anti–IL-13 (polyclonal goat IgG; AF-123-NA;
R&D System) followed with Texas red anti–goat IgG (Jackson Immuno-
Research Laboratories). TSLP was detected with 10 µg/ml of mouse anti-TSLP
antibody prepared in-house (mIgG1; clone 14C3.2E11). Cytokeratin 19 was
labeled with monoclonal antibody clone A53-BA2 (IgG2a; Abcam), followed
by Alexa Fluor 568 goat anti–mouse IgG2a (Invitrogen). The following di-
rect-labeled antibodies used were as follows: FITC anti–HLA-DR (mouse
IgG2a; L243; BD), FITC anti-CD11c (clone; KB 90; Dako), FITC anti-Ki67
Our results add another feature to the role of OX40L in
tumors. Indeed, several studies in mouse models of transplant-
able tumors suggested that engaging OX40 via an agonist anti-
body, OX40L.Fc, or transfected tumor cells and DCs appears
to promote antitumor eects (Weinberg et al., 2000; Morris
et al., 2001; Ali et al., 2004; Piconese et al., 2008). However,
Tnfsf4 (gene coding OX40L) is regulated by the mRNA
MIRN125B (Smirnov and Cheung, 2008), whose expression
is down-regulated in breast cancer (Iorio et al., 2005). Tnfsf4
is up-regulated in ataxia telangiectasia carriers and patients
(Smirnov and Cheung, 2008), who have been shown to have
an increased risk of breast cancer (Swift et al., 1987). Further-
more, a recent pilot study described the expression of OX40
and OX40L in >100 tissue samples from invasive ductal car-
cinomas, and suggested a possible association of OX40 ex-
pression and lymph node metastatic status (Xie et al., 2010).
OX40L signaling has several important features that might
help explain the results observed in our and other studies.
Thus, OX40L triggers Th2 polarization independent of IL-4,
promotes TNF production, and inhibits IL-10 production by
the developing Th2 cells, but only in the absence of IL-12. In
the presence of IL-12, OX40L signaling instead promotes the
development of Th1 cells that, like inammatory Th2 cells,
produce TNF but not IL-10 (Liu et al., 2007).
Interestingly, inammatory Th2 cells coexist within the
tumor immune environment alongside IL-10–secreting CD4+
T cells. Recent studies demonstrate a colocalization of cells
with the phenotype of T reg cells) with mature DCs in lym-
phoid inltrates in breast cancer (Gobert et al., 2009). A high
number of FoxP3+ T reg cells was associated with disease re-
lapse (Gobert et al., 2009). Thus, the niche nding and metas-
tasis formation might be facilitated by inammatory Th2
response, whereas in the established tumor a T reg cell re-
sponse might prevail. Interestingly, CCL22 (a macrophage-
derived chemokine) appears involved in the attraction of
T reg cells (Gobert et al., 2009), but also, in cooperation with
CCL17 (TARC), of inammatory Th2 cells (Liu et al., 2007).
How this ne balance between T reg cells and Th2 responses
is regulated will require further study.
Two key questions arise from our work: (1) what are the
mechanisms allowing TSLP release from cancer cells; and
(2) what is the impact of IL-13 (and IL-4) on cancer cells,
on the stroma, and on the immune inltrate? Our data show
that nonmalignant breast epithelia can express TSLP. This fur-
ther demonstrates that cancer cells can exploit pathways that
are present in the normal tissue to establish a microenviron-
ment facilitating tumor development. The mechanisms
regulating TSLP expression and secretion from cancer cells,
including a potential link with oncogenic events, remain to
be established. It will also be important to determine the
impact of TSLP and inammatory Th2 environment on the
stromal broblasts. Indeed, recent studies point to the critical
role of cross talk between cancer cells and broblasts in deter-
mining the type of microenvironment established by cancer
cells originating from dierent types of breast cancers (Camp
et al., 2011).

488 TSLP–OX40L–IL-13 axis in human breast cancer | Pedroza-Gonzalez et al.
followed by surface and intracellular staining. For blocking OX40L, tumor-
activated mDCs were co-cultured with naive CD4 T cells in the presence of
50 µg of anti-OX40L (Ik-5 clone) or control IgG2a isotype antibody. For
blocking TSLP, tumor-derived factors were preincubated with 20 µg/ml of
anti-TSLP antibody (rabbit; AB 19024) or normal rabbit IgG (R&D Sys-
tems) at room temperature for 30 min after DC activation. DCs were acti-
vated with the neutralized tumor-derived factors and nally co-cultured
with naive CD4 T cells for 7 d. For TSLPR blocking, DCs were preincubated
with anti-TSLP receptor antibody (clone AB81_85.1F11; mouse IgG1) for
3 min at room temperature.
Tumor-bear ing mice. Mice were humanized either by CD34+ hemato-
poietic progenitor cell (HPC) transplant or by co-administration of DCs and
T cells as described previously (Aspord et al., 2007; Institutional Animal Care
and Use Committee no. A01-005). CD34+HPCs were obtained from apher-
esis of adult healthy volunteers mobilized with G-CSF and puried as de-
scribed previously (Aspord et al., 2007). The CD34 fraction of apheresis was
Ficoll pur ied, and the PBMCs obtained were stored frozen and used as a
source of autologous T cells. 3 million CD34+ HPCs were transplanted intra-
venously into sublethally irradiated (12 cGy/g body weight of 137Cs  irra-
diation) nonobese diabetic/LtSz-scid/scid 2 microglobulin–decient
(NOD/SCID/2m/) mice (Jackson ImmunoResearch Laboratories).
After 4 wk of engraftment, 10 million Hs578T breast cancer cells were har-
vested from cultures and injected subcutaneously into the anks of the mice.
Mice were reconstituted with 10 million CD4+ T cells and 10 million CD8+
T cells autologous to the grafted CD34+ HPCs. CD4+ and CD8+ T cells
were positively selected from thawed PBMCs using magnetic selection ac-
cording to the manufacturer’s instructions (Miltenyi Biotec). The purity was
routinely >90%. T cells were transferred at days 3, 6, and 9 after tumor im-
plantation. For experiments with NOD/SCID/2m/ mice, they were
sublethally irradiated the day before tumor implantation. Mice were then re-
constituted with 1 million monocyte-derived DCs (MDDCs) and autolo-
gous T cells as described in the previous paragraph. MDDCs were generated
from the adherent fraction of PBMCs by culturing with 100 ng/ml GM-
CSF (Immunex) and 10 ng/ml IL-4 (R&D Systems). Tumor size was moni-
tored every 2–3 d. Tumor volume (ellipsoid) was calculated as follows: ([short
diameter]2 × long diameter)/2.
Blocking in vivo experiments. For dierent experimental purposes,
tumor-bearing mice transferred with autologous T cells were injected intra-
tumorally with 200 µg of anti-OX40L (clone IK-5; mIgG2a) blocking anti-
body, 100 µg of anti-TSLP antibody (clone AB19024; rabbit IgG), 100 µg of
anti-IL-13 neutralizing antibody prepared in-house (clone 13G1.B2; mIgG1),
200 µg of anti-TSLPR neutralizing antibody (clone AB81_85.1F11; mouse
IgG1), or isotype control, respectively at days 3, 6, and 9 after tumor implan-
tation. For blocking TSLP, anti-TSLP antibody injection was also given at day
0 while the tumor was implanted.
Online supplemental material. Fig. S1 illustrates gating strategy for ow
cytometry analysis of tumor-inltrating lymphocytes and the accumulation
of lymphocytes in tumor beds as compared with surrounding tissue. Fig. S2
shows that mDCs exposed to TSLP or to soluble tumor factors in the pres-
ence of anti-OX40L are unable to drive the dierentiation of inammatory
Th2 cells. Fig. S3 shows the specicity of anti-TSLP antibody staining in fro-
zen tissue sections. Fig. S4 shows the expression of TSLP in breast cancer cells
and in nonmalignant breast tissue. Table S1 provides the pathological diagno-
sis information and Luminex data for cytokine levels of all 99 breast cancer
patients used for cytokine analysis. Table S2 describes the characteristics of
breast cancer cell lines studied. Online supplemental material is available at
http://www.jem.org/cg i/content/full/jem.20102131/DC1.
We are grateful to Albert Barnes, Sebastien Coquery, Elizabeth Kraus, Mark
Michnevitz, Gina Stella Garcia-Romo, Jenny Smith, and Lynnette Walters for help;
Dr. Joseph Fay for help with healthy volunteers; Cindy Samuelsen for continuous
support; and Drs. Sally M. Knox and Michael Grant at the Department of Surgery
(clone ki-67; Dako), and Alexa Fluor 488 anti-CD3 (mIgG1; UCHT1; BD).
Finally, sections were counterstained for 2 min with 3 µM of the nuclear stain
DAPI (in PBS; Invitrogen). To conr m specicity of TSLP staining, pr imary
anti-TSLP antibody was preincubated with 100 µg of recombinant human
TSLP (R&D Systems) for 30 min at room temperature before staining of
tissue sections that previously showed to be TSLP positive.
Flow cytometry analysis. Cell suspensions from human breast carcinoma
tissue and tumor or draining lymph nodes from humanized mice were used
for phenotypic characterization of leukocytes. Cell suspensions were ob-
tained by digestion with 2.5 mg/ml of collagenase D (Roche), and 200 U/ml
of DNase I (Sigma-Aldrich) for 30–60 min at 37°C. The anti–human anti-
bodies used were as follows: labeled FITC-labeled lineage cocktail (CD3,
CD14, CD16, CD19, CD20, and CD56; BD); PE-labeled OX40L (mIgG1;
clone Ik-1; BD); QR-labeled HLA-DR (mIgG2a; clone HK14; Sigma-
Aldrich); APC-labeled CD11c (mIgG2b; clone S-HCL-3; BD); PerCP-labeled
CD3 (mIgG1; clone SK7; BD); PECy7-labeled CD4 (mIgG1; clone SK3;
BD); APCCy7-labeled CD8 (mIgG1; clone SK1; BD); CFS-labeled IL-4
(mIgG1; clone 3007; R&D Systems); Pacic blue–labeled IL-10 (rat IgG1;
clone JES3-9D7; eBioscience); PE-labeled IL-13 (rat IgG1; clone JES10-5A2
BD); APC-labeled TNF (mIgG1; clone 6401.1111; BD); Alexa Fluor 700–
labeled IFN- (mIgG1; clone B27; BD). Cells were incubated with the
antibodies for 30 min at 4°C in the dark, and then washed three times and
xed with 1% paraformaldehyde to be analyzed in a FACSCalibur or LSR-II
cytometer (BD). For intracellular cytokines, cells were stained using BD
Cytox/Cytoperm xation/permeabilization kit according to the manufac-
turer’s directions.
Tumor factors preparation. Tumor factors were obtained from superna-
tant of established breast cancer cell lines cultured in vitro (Table S2; Soule
et al., 1973; Hackett et al., 1977; Lacroix and Leclercq, 2004; Neve et al.,
2006) or by sonication from tumor cell lines, human breast tumor tissue, or
tumors from humanized mice. In brief, cell lines were culture in medium
(RPMI supplemented with 2 mM glutamine, 50 U/ml penicillin, 50 µg/ml
streptomycin, 0.1 mM MEM nonessential amino acids, 10 mM Hepes buer,
0.1 mM sodium pyruvate, and 10% fetal calf serum), and when the cells
reached 90% of conuence fresh medium was added and the cells were left
in culture for an additional 48 h. For sonication, cells or tissues were placed
in PBS and disrupted for 30 s at 4°C, with the power output adjusted at 4.5
level of the 60 sonic dismembrator (Thermo Fisher Scientic). Cellular de-
bris was removed by centr ifugation, and the supernatant was collected and
stored at 80°C.
Cytokine analysis. Tumor samples from patients diagnosed with breast car-
cinoma (in situ, invasive duct, and/or mucinous carcinoma of the breast, as
well as lobular carcinoma) were obtained from the Baylor University Medical
Center Tissue Bank (Institutional Review Board no. 005–145). Tumors and
draining lymph nodes from mice implanted with breast cancer cell lines
H578T and MDA-MB-231 were also analyzed. Whole-tissue fragments (4 ×
4 × 4 mm, 0.02 g, approximately) were placed in culture medium with
50 ng/ml of PMA (Sigma-Aldrich) and 1 µg/ml of ionomycin (Sigma-Aldrich)
for 16 h. Cytokine production was analyzed in the culture super natant by
Luminex. For intracellular staining, cells were resuspended at a concentration
of 106 cells/ml in medium and activated for 5 h with PMA and ionomycin;
brefeldin A (GolgiPlug; BD) and monensin (GolgiStop; BD) were added for
the last 2.5 h.
DC–T cell co-cultures. Total CD4+ T cells were enriched from PBMCs of
healthy donors using magnetic depletion of other leukocytes (EasySep
Human CD4+ T Cell Enrichment kit; STEMCELL Technologies, Inc.).
Naive CD4+ T cells were sorted based on the expression of CD4+ CD27+ and
CD45RA+. Activated mDCs with medium, TSLP, or tumor-derived factors
were co-cultured with naive CD4+ T cells at a ratio of 1:5 for 7 d. After that,
the cells were washed and restimulated for 5 h with 50 ng/ml PMA and
1 µl/ml ionomycin. Brefeldin A and monensin were added for the last 2.5 h,

JEM VOL. 208, March 14, 2011
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and Ms. Dan Su at the Department of Pathology at Baylor University Medical
Center. We thank Dr. Michael Ramsay for continuous support.
This work was supported by the Baylor Health Care Systems Foundation, and the
National Institutes of Health (grants R0-1 CA89440 and R21 AI056001 to A. Karolina
Palucka; grants U19 AI057234, R0-1 CA78846, and CA85540 to J. Banchereau).
J. Banchereau holds the Caruth Chair for Transplantation Immunology Research.
A. Karolina Palucka holds the Ramsay Chair for Cancer Immunology Research.
The authors have no conicting nancial interests.
Submitted: 7 October 2010
Accepted: 27 January 2011
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