Professional Documents
Culture Documents
Zhijian Cai, Fei Yang, Lei Yu, Zhou Yu, Lingling Jiang,
Qingqing Wang, Yunshan Yang, Lie Wang, Xuetao Cao and
Jianli Wang
J Immunol 2012; 188:5954-5961; Prepublished online 9 May
2012;
doi: 10.4049/jimmunol.1103466
http://www.jimmunol.org/content/188/12/5954
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Received for publication December 2, 2011. Accepted for publication April 5, 2012.
This work was supported by the National Key Basic Research Program of China
(2007CB512400), the National Natural Science Foundation of China (30671909 and
30972725), and the Natural Science Foundation of Zhejiang Province (Z2090042).
Address correspondence and reprint requests to Dr. Jianli Wang, Institute of Immunology, School of Medicine, Zhejiang University, Hangzhou 310058, China. E-mail
address: jlwang@zju.edu.cn
Abbreviations used in this article: c-FLIP, cellular FLICE inhibitory protein;
COX2, cyclooxygenase-2; DC, dendritic cell; EXO, exosome derived from activated CD8+ OT-I T cell; EXOcont, exosome derived from naive CD8+ OT-I T cell;
EXOgld, exosome derived from Con A-activated CD8+ T cell of gld mice; EXOwt,
exosome derived from Con A-activated CD8+ T cell of C57BL/6 mice; FasL, Fas
ligand; MMP, matrix metalloproteinase; OT-I, C57BL/6-Tg (Tcra Tcrb) 1100Mjb/J;
PDTC, ammonium pyrrolidinedithiocarbamate; siRNA, small interfering RNA;
WT, wild-type.
Copyright 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103466
Activation of T cells is a pivotal step in the process of host antitumor immunity. During this course, T cells release large numbers
of membrane vesicular bodies termed exosomes (15), which were
initially described by Johnstone et al. (16). Exosomes are nanoscale membrane vesicles (50100 nm) released by various live
cells, which have a lipid bilayer structure (17). They are formed by
membrane budding into the lumen of an endocytic compartment,
leading to the formation of multivesicular bodies. Fusion of multivesicular bodies to the plasma membrane leads to the extracellular release of exosomes (18). The exosomes secreted by activated
human T cells express bioactive FasL and can induce the apoptosis
of Jurkat T cells (15). Exosomes purified from CD8+ T cells,
which are generated by cultivation of OVA-pulsed dendritic cells
(DCs) with naive CD8+ T cells derived from OT-I mice, also express FasL and can inhibit DCs to stimulate CD8+ CTL responses
(19). However, the potential effects of FasL in activated T cell
exosomes on Fas-resistant tumor cells are not well understood.
Matrix metalloproteinase (MMP)9 plays a critical role in the
breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue
remodeling, as well as in disease processes, such as tumor metastasis (20). MMP9 was reported to be an important factor in
facilitating lung cancer invasion and metastases in the B16 melanoma model (21). It was also demonstrated that the activation of
tumor Fas signaling can induce the expression of MMP9 (22).
Therefore, we hypothesize that FasL in activated T cell exosomes
probably upregulates MMP9 expression and promotes the invasive
ability of Fas-resistant tumor cells, which may provide a novel
view for understanding tumor escape from the immune system.
Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However,
their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from
activated CD8+ T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of
tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes
increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-kB pathways, which
subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that
the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb
significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor
mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma
and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer
cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune
escape. The Journal of Immunology, 2012, 188: 59545961.
5955
Electron microscopy
For electron microscopy observations, exosome pellets were fixed in 4%
paraformaldehyde at 4C for 1 h. Then, the pellets were loaded onto
electron microscopy grids coated with formvar carbon, contrasted, and
embedded in a mixture of uranyl acetate and methylcellulose. Sections
were observed with a Philips Tecnai-10 transmission electron microscope operating at 80 kV (Phillips Electronic Instruments, Mahwah,
NJ).
Western blot
A total of 50 mg exosomes or crude proteins extracted from cell lysates
was separated by 12% SDS-PAGE and transferred onto polyvinylidene
difluoride membrane (Millipore, Billerica, MA). Membrane was blocked
with 5% BSA in TBST and then incubated with corresponding primary
Abs overnight at 4C. After incubating with HRP-coupled secondary Ab
Real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen), according to the
manufacturers instructions. The specific primers for b-actin for real-time
PCR were 59-CGTTGACATCCGTAAAGACC-39 (sense) and 59-AACAGTCCGCCTAGAAGCAC-39 (antisense). The specific primers for MMP9
for real-time PCR were 59-CGGCACGCCTTGGTGTAGCA-39 (sense)
and 59-GGCGCACCAGCGGTAACCAT-39 (antisense). The following
PCR conditions were used: 1 cycle at 95C for 30 s and then 40 cycles
of 5 s at 95C and 34 s at 60C. Real-time PCR was performed on an
Applied Biosystems 7500 real-time PCR system (Foster City, CA).
A total of 2 3 106 cells/ml naive lymphocytes derived from OT-I mice was
cultured in RPMI 1640 complete medium (containing 50 mM 2-ME, 1 mM
sodium pyruvate, 10 mM HEPES, and 10 U/ml IL-2) and stimulated or not
with OVA257264 peptide (2 mg/ml) for 48 h. Active CD8+ T cells were
purified using CD8+ Dynabeads (Invitrogen, Carlsbad, CA). Purified OVAspecific active CD8+ T cells were then cultured in exosome-free RPMI
1640 complete medium for 24 h. Exosomes in T cell culture supernatants
were isolated, as previously described (23). Briefly, collected culture supernatants were differentially centrifuged at 300 3 g for 10 min, 1,200 3 g
for 20 min, and 10,000 3 g for 30 min at 4C. The supernatant from the
final centrifugation was ultracentrifuged at 100,000 3 g for 1 h at 4C.
After removing the supernatant, the exosome pellets were washed in large
volume of ice-cold PBS and centrifuged at 100,000 3 g for another 1 h at
4C. To isolate the FasL+ exosomes released by CD8+ T cells of C57BL/6
and FasL-deficient gld mice, CD8+ T cells were stimulated with 50 mg/ml
Con A for 15 min and then washed and cultured in exosome-free RPMI
1640 complete medium for 1 h. Supernatant was collected, and exosomes
were purified, as previously described (23). Exosomes in the pellet were
resuspended in PBS. Exosomes from CD8+ T cells, stimulated or not with
OVA257264 peptide, were termed EXO or EXOcont; exosomes from Con
A-activated CD8+ T cells of C57BL/6 or gld mice were termed EXOwt or
EXOgld, respectively.
for 1 h, the membranes were scanned using Tanon 4500 (Shanghai, China),
according to the manufacturers instructions.
5956
Statistical analysis
Experimental data were analyzed using one-way ANOVA and the t test
using SPSS 11.5. Differences were considered significant when the p
values were , 0.05.
Results
FasL+ EXO showed no effect on B16 tumor cell apoptosis and
proliferation
FIGURE 2. EXO promoted tumor cell invasion via Fas signaling in vitro. (A) B16 or 3LL tumor cells were incubated with 10 mg of EXO, EXOcont, or
PBS for 2 h, and the cells were plated in the bottom chamber precoated with 50 ml of Matrigel. Forty-eight hours later, the number of cells on the bottom of
the Transwell filter was imaged and quantified. (B) Same experimental procedure as in (A), except that the cells were preincubated with 20 mg/ml of FasLneutralized mAbs or ISO for 2 h (n = 5). Data are representative of three independent experiments. *p , 0.05 versus Cont and EXOcont (A) or EXO+antiFasL (B).
5957
5958
FIGURE 3. EXO-mediated activation of Fas signaling induced upregulation of MMP9 expression in tumor cells. (A) B16 tumor cells were treated with
the indicated doses of EXO for 24 h or 10 mg/ml EXO for the indicated times. mRNA expression levels of MMP9 were assayed by quantitative PCR. (B)
Western blot analysis of MMP9 expression in B16 tumor cells stimulated with 10 mg/ml of EXO for the indicated times. (C) Western blot analysis of MMP9
expression in B16 tumor cells stimulated with 10 mg/ml of EXO, EXO preincubated with 20 mg/ml of FasL-neutralized mAbs, or isotype mAbs (ISO) for
2 h. (D) Invasive assay of B16 tumor cells after stimulation with 10 mg/ml of EXO or EXO plus MMP9 inhibitor (MMP9i) (n = 5). Data are representative
of three independent experiments. *p , 0.05, **p , 0.01.
EXO increased the tumor cell lung invasion via Fas signaling
in vivo
To further clarify the effect of EXO on tumor invasion, we performed tumor invasive analysis with the B16 tumor cell-induced
murine lung cancer model. We found that EXO greatly increased the tumor cell lung invasion, as indicated by gross morphology of the lungs (Fig. 6A, left panels) and the number of
invasive nodules (Fig. 6A, right panel). Histological assessment of
lungs showed few tumor colonies in EXOcont and PBS groups but
more numerous tumor colonies in EXO groups (Fig. 6B). After
preincubation with FasL mAbs, the ability of EXO to promote
FIGURE 4. EXO promoted MMP9 expression and tumor cell invasion through ERK and NF-kB pathways. (A) B16 tumor cells were stimulated with
10 mg/ml of EXO or EXOcont for the indicated times and harvested; the extracted crude proteins were subjected to Western blot with Abs against NF-kB,
phosphoNF-kB, ERK, or phospho-ERK. (B) B16 tumor cells were pretreated with NF-kBspecific inhibitor PDTC or ERK1/2-specific inhibitor U0126 for
30 min and then stimulated with 10 mg/ml EXO for 24 h. The expression of MMP9 in B16 tumor cells was detected by Western blot. (C) Using the same
experimental conditions as in (B), the number of cells that migrated through Matrigel and adhered to the bottom of Transwell filter was quantified (n = 5).
Data are representative of three independent experiments. **p , 0.01.
down (Fig. 5C), which excludes the possibility that the changes
in signal pathways may be caused by increasing cell apoptosis.
Subsequent Western blot analysis showed that the phosphorylation
of ERK and NF-kB decreased significantly when the expression
of c-FLIPL was inhibited by siRNA (Fig. 5D). Moreover, cell
invasion and the expression of MMP9 were also inhibited when
c-FLIPL expression was knocked down (Fig. 5E, 5F). It is known
that NF-kB signals can induce the expression of c-FLIP (33),
which is related to the expression of MMP9 (8). We propose that
a similar pattern might also appear in Fas signaling-activated tumor cells induced by EXO. As shown in Fig. 5G, inhibition of the
NF-kBsignaling pathway decreased the expression of c-FLIPL.
Interestingly, the same result was observed when B16 tumor cells
were treated by specific inhibitors of ERK (Fig. 5G). These results
suggest that c-FLIPL plays an important role in the increase in
MMP9 and tumor invasion induced by EXO.
5959
Discussion
Stimulation of FasR normally induces an apoptotic death signal.
However, the interaction between Fas and FasL in tumor cells does
not always induce a death signal, and conversely, promotes tumorigenesis (11). Activated CD8+ T cells secrete FasL+ exosomes,
which can induce DC apoptosis and inhibit their ability to stimulate CD8+ CTL responses (19). Until now, the effect of FasL+
exosomes from activated CD8+ CTLs on Fas-resistant tumors was
unknown. In this study, we found that FasL+ EXO did not affect
the apoptosis and proliferation of tumor cells but accelerated their
invasion in vivo via upregulation of MMP9 expression. Moreover,
we identified a counterpart of EXO in tumor-bearing mice that
FIGURE 5. c-FLIP was critical to the increase in MMP9 expression and tumor invasion induced by EXO. (A) Western blot analysis of c-FLIPL and
c-FLIPS expression in B16 tumor cells stimulated with 10 mg/ml of EXO or EXOcont for the indicated times. (B) B16 cells were transfected with c-FLIPL
(Si) or control (Cont Si) siRNA duplexes for 24 h, and c-FLIPL expression in the cells was detected by Western blot. (C) The apoptosis of Si- or Cont Sitransfected B16 tumor cells treated by EXO were analyzed by FACS (n = 3). (D) After inhibition of c-FLIPL expression by siRNA, the phosphorylation of
NF-kB and ERK in EXO- or EXOcont-stimulated B16 tumor cells was detected by Western blot. (E) After inhibition of c-FLIPL expression by siRNA,
MMP9 expression was detected by Western blot in B16 cells stimulated with 10 mg/ml of EXO or EXOcont for 24 h. (F) After inhibition of c-FLIPL
expression by siRNA, the number of B16 tumor cells, stimulated with 10 mg/ml of EXO or EXOcont for 2 h, which migrated through Matrigel to the bottom
of the Transwell filter was quantified (n = 5). (G) Western blot analysis of c-FLIPL expression in EXO-stimulated B16 tumor cells pretreated with NF-kB
specific inhibitor PDTC or ERK1/2-specific inhibitor U0126. Data are representative of three independent experiments. **p , 0.01 versus Cont or Si+EXO.
5960
a guard into an accessory of tumor progression under such circumstances). In the FACS results in Fig. 1A, EXO is adsorbed
onto latex with an intact membrane structure, and if FasL can be
detected, it is probably membrane-bound. Moreover, in the results
from Western blotting, we detected a band at 40 kDa, which is
similar to the molecular mass of membrane-bound FasL, whereas
we detected no signal at 26 kDa, which is similar to the molecular
mass of soluble FasL (data not shown). CTLs themselves also
express FasL and may trigger Fas signal in tumor cells, which
probably promotes MMP9 expression and the invasive capability
of tumor cells. However, there are very limited numbers of CTLs
in tumor sites (35). Furthermore, unlike exosomes, whose diameters are ,100 nm, allowing them to freely pass through basement
membrane (36), immigration of CTLs from spleen and lymph
nodes into the tumor site is more complicated. It can be presumed
that more FasL+ EXO will accumulate in the tumor location than
CTLs, which will probably exert a stronger effect of invasive
promotion on tumors than their parent CTLs.
Many molecules were reported to be associated with tumor invasion. Among them, MMP2, MMP9, cyclooxygenase-2 (COX2),
b-catenin, and urokinase plasminogen activator, are considered
the primary tumor-invasive effectors (13, 20, 3739). In our study,
we found that, after stimulation by EXO, expression of the mRNA
levels of MMP2, MMP9, and COX2 was significantly elevated in
3LL and B16 tumor cells. However, we could not detect any
change in the protein expression of MMP2 in either cell type (data
not shown). Although the expression of COX2 mRNA increased in
both cells, almost no PGE2 secretion could be detected with or
without EXO stimulation in B16 tumor cells (data not shown).
Interestingly, urokinase plasminogen activator, which is reported
to be related to tumor invasion mediated by Fas signaling (13),
showed no increase in either cell type after EXO stimulation (data
not shown). Among those tumor-invasive effectors, we detected
only elevated mRNA and protein levels of MMP9 in both cancer
cell lines. It is likely that MMP9 is a universal effector molecule
that promotes tumor invasion induced by FasL in EXO.
Although increasing tumors have been found to possess Fas
resistance, the signaling mechanisms of tumor Fas resistance remain unknown. c-FLIP is an endogenous inhibitor of death
receptor-induced apoptosis, and it plays a critical role in tumor-
FIGURE 7. Identification of a counterpart of EXO in vivo from tumor-activated CD8+ T cells. CD8+ exosomes from spleens and draining lymph nodes of
control (EXOcln) and tumor mice (EXOtln), as well as tumor tissue of tumor mice (EXOts), were isolated by CD8+ magnetic beads. (A) Western blot
analysis of FasL, CD8, HSP70, and TSG101 expression in EXOcln, EXOtln, and EXOts. (B) Western blot analysis of MMP9 expression in tumor cells after
stimulation with 10 mg/ml of EXOcln, EXOtln, and EXOts for 24 h. (C) The number of invasive tumor cells stimulated with EXOcln, EXOtln, and EXOts
through Matrigel to the bottom of the Transwell filter was quantified (n = 5). *p , 0.05, **p , 0.01 versus Cont and EXOcln. (D) A total of 5 3 105 B16
tumor cells was injected into C57BL/6 (WT) or gld (GLD) mice via the tail vein. The number of lung nodules was measured (n = 5). Data are representative
of three independent experiments. **p , 0.01.
Disclosures
The authors have no financial conflicts of interest.
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