Tumor exosomes inhibit differentiation of bone marrow dendritic cells.
ABSTRACT The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b(+) myeloid precursors in the bone marrow (BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective. Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6 to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation.
- [show abstract] [hide abstract]
ABSTRACT: The possible involvement of the endothelium in the vasodilator action of eugenol was investigated in the mesenteric vascular bed (MVB) of the rat. Bolus injections of eugenol (0.2, 2 and 20 micromol) and acetylcholine (ACh; 10, 30 and 100 pmol) induced dose-dependent vasodilator responses in noradrenaline-precontracted beds that were partially inhibited by pretreatment of the MVB with deoxycholate (1 mg mL(-1)) to remove the endothelium (approximately 14% and approximately 30% of the control response remaining at the lowest doses of ACh and eugenol, respectively). The vasodilator effect of glyceryl trinitrate (1 micromol) was unaltered by deoxycholate. In the presence of either N(omega)-nitro-L-arginine methyl ester (300 microM) or tetraethylammonium (1 mM)the response to ACh was partially reduced, whereas eugenol-induced vasodilation was unaffected. Similarly the vasodilator effect of eugenol was not inhibited by indometacin (3 microM). Under calcium-free conditions the vasoconstrictor response elicited by bolus injections of noradrenaline (10 nmol) was dose-dependently and completely inhibited by eugenol (0.1-1 mM). Additionally, the pressor effects of bolus injections of calcium chloride in potassium-depolarized MVBs were greatly reduced in the presence of eugenol (0.1 mM), with a maximal reduction of approximately 71% of the control response. Our data showed that eugenol induced dose-dependent, reversible vasodilator responses in the rat MVB, that were partially dependent on the endothelium, although apparently independent of nitric oxide, endothelium-derived hyperpolarizing factor or prostacyclin. Furthermore, an endothelium-independent intracellular site of action seemed likely to participate in its smooth muscle relaxant properties.Journal of Pharmacy and Pharmacology 04/2003; 55(3):359-65. · 2.03 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In the present study, we investigated the differentiation of human NK cells from bone marrow, cord blood and mobilized peripheral blood purified CD34+ stem cells using a potent culture system. Elutriated CD34+ stem cells were grown for several weeks in medium supplemented with stem cell factor (SCF) and IL-15 in the presence or absence of a murine stromal cell line (MS-5). Our data indicate that IL-15 induced the proliferation and maturation of highly positive CD56+ NK cells in both types of culture, although murine stromal cells slightly increased the proliferation of NK cells. NK cells differentiated in the presence of MS-5 were mostly CD56+ CD7 and a small subset expressed CD16. These in vitro differentiated CD56+ NK cells displayed cytolytic activity against the HLA class I- target K562. The CD56+ CD16+ subset also lysed NK-resistant Daudi cells. Neither of these NK subsets were shown to express Fas ligand. Total CD56+ cells expressed high amounts of transforming growth factor-beta and granulocyte-macrophage colony-stimulating factor, but no IFN-gamma. Investigation of NK receptor expression showed that most CD56+ cells expressed membrane CD94 and NKG2-A mRNA. PCR analysis revealed that p58 was also expressed in these cells. The role of CD94 in NK cell-mediated cytotoxicity was assessed on human HLA-B7-transfected murine L cells. While a low cytotoxic activity towards HLA-B7 cells was observed, the HLA-DR4 control cells were killed with high efficiency. These studies demonstrate that cytolytic and cytokine-producing NK cells may be derived from adult and fetal precursors by IL-15 and that these cells express a CD94 receptor which may influence their lytic potential.European Journal of Immunology 07/1998; 28(6):1991-2002. · 4.97 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Chronic myeloid leukemia (CML) is characterized by expression of the BCR-ABL fusion gene that encodes a 210-kDa protein, which is a constitutively active tyrosine kinase. At least 70% of the oncoprotein is localized to the cytoskeleton, and several of the most prominent tyrosine kinase substrates for p210(BCR-ABL) are cytoskeletal proteins. Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells responsible for the initiation of immune responses. In CML patients, up to 98% of myeloid DCs generated from peripheral blood mononuclear cells are BCR-ABL positive. In this study we have compared the morphology and behavior of myeloid DCs derived from CML patients with control DCs from healthy individuals. We show that the actin cytoskeleton and shape of CML-DCs of myeloid origin adherent to fibronectin differ significantly from those of normal DCs. CML-DCs are also defective in processing and presentation of exogenous antigens such as tetanus toxoid. The antigen-processing defect may be a consequence of the reduced capacity of CML-DCs to capture antigen via macropinocytosis or via mannose receptors when compared with DCs generated from healthy individuals. Furthermore, chemokine-induced migration of CML-DCs in vitro was significantly reduced. These observations cannot be explained by a difference in the maturation status of CML and normal DCs, because phenotypic analysis by flow cytometry showed a similar surface expression of maturation makers. Taken together, these results suggest that the defects in antigen processing and migration we have observed in CML-DCs may be related to underlying cytoskeletal changes induced by the p210(BCR-ABL) fusion protein.Blood 06/2003; 101(9):3560-7. · 9.06 Impact Factor
of June 11, 2013.
This information is current as
Bone Marrow Dendritic Cells
Tumor Exosomes Inhibit Differentiation of
Robert Kimberly, William E. Grizzle, Carla Falkson and
Yuelong Liu, Liming Zhang, Chuanyu Li, Yingzi Cong,
Shaohua Yu, Cunren Liu, Kaihong Su, Jianhua Wang,
2007; 178:6867-6875; ;
, 18 of which you can access for free at:
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2007 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 11, 2013
Tumor Exosomes Inhibit Differentiation of Bone Marrow
Shaohua Yu,* Cunren Liu,* Kaihong Su,* Jianhua Wang,* Yuelong Liu,* Liming Zhang,*
Chuanyu Li,* Yingzi Cong,‡Robert Kimberly,* William E. Grizzle,†Carla Falkson,§
and Huang-Ge Zhang2*¶
The production of exosomes by tumor cells has been implicated in tumor-associated immune suppression. In this study, we show
that, in mice, exosomes produced by TS/A murine mammary tumor cells target CD11b?myeloid precursors in the bone marrow
(BM) in vivo, and that this is associated with an accumulation of myeloid precursors in the spleen. Moreover, we demonstrate that
TS/A exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) in vitro. Addition of tumor
exosomes at day 0 led to a significant block of differentiation into DC, whereas addition at later time points was less effective.
Similarly, exosomes produced by human breast tumor cells inhibited the differentiation of human monocytes in vitro. The levels
of IL-6 and phosphorylated Stat3 were elevated 12 h after the tumor exosome stimulation of murine myeloid precursors, and
tumor exosomes were less effective in inhibiting differentiation of BM cells isolated from IL-6 knockout mice. Addition of a rIL-6
to the IL-6 knockout BM cell culture restored the tumor exosome-mediated inhibition of DC differentiation. These data suggest
that tumor exosome-mediated induction of IL-6 plays a role in blocking BM DC differentiation. The Journal of Immunology,
2007, 178: 6867–6875.
that subsequently circulate via the blood and are delivered to pe-
ripheral tissues, where they sample Ags of diverse origin, thus
acting as APCs to stimulate the host antitumor immune response.
It has been demonstrated that DC are not activated in human
cancers and that their function is compromised by the tumor (1–6).
Several lines of evidence indicate that the ability of tumors to
affect DC maturation and differentiation may result in a general-
ized failure of the host to mount an effective antitumor response.
These findings and additional data (5, 7–9) are relevant clinically,
as an association with significantly poorer prognoses in patients
with several types of cancer has been described. Although these
observations suggest that DC play a critical role in determining the
final outcome of the immune response, the mechanisms responsi-
ble for this phenomenon remain uncharacterized, and the effects of
tumors on DC function are poorly understood.
endritic cells (DC)3originate from hemopoietic stem
cells within the bone marrow (BM). Under physiological
conditions, progenitors differentiate into immature DC
The decreased frequency and immature phenotype of DC in the
tumor tissues and in the peripheral blood of patients with tumors of
the head and neck, lung, and breast, as well as those with multiple
myeloma (8–10), suggest a systemic alteration in DC function.
This, in turn, suggests that the release of soluble factors by the
tumors contributes to the tumor-associated alterations in the activ-
ity of DC. Several reports have now confirmed that the release of
IL-10, IL-6, macrophage colony-stimulating factor, vascular en-
dothelial growth factor, and gangliosides and/or prostanoids by
tumors can prevent DC differentiation and function in vitro and in
vivo (7, 11–15).
In this study, we found that exosomes produced by tumor cells
target CD11b?myeloid precursor cells in the BM. Interaction of
the exosomes with the myeloid cells in vitro induced the produc-
tion of IL-6 preferentially and suppressed their differentiation into
DC. Tumor exosomes only partially blocked DC differentiation
when BM cells isolated from IL-6 knockout (KO) mice were used
in these experiments, indicating that the exosome-induced produc-
tion of IL-6 plays a role in blocking DC differentiation.
Materials and Methods
Seven-week-old BALB/c and C57BL/6j IL-6 KO mice (The Jackson Lab-
oratory) were housed under standard laboratory conditions in the facility of
Laboratory Animal Care at the University of Alabama at Birmingham.
Animal care was in accordance with institutional guidelines, and all ex-
perimental protocols involving animals were performed using an institu-
tional protocol that was approved by the Animal Care and Use Committee.
The TS/A cell line, a moderately differentiated and immunogenic murine
mammary adenocarcinoma of spontaneous BALB/c origin that is MHC
class I?(H-2Dd, H-2Kd) was maintained in vitro at 37°C in a humidified
5% CO2atmosphere in air in complete medium (DMEM with 5% FBS) as
described previously (16). The B16 melanoma cell line was purchased
from the American Type Culture Collection and cultured in DMEM. The
human breast cancer cell line MDA-MB-231 was purchased from the
American Type Culture Collection and maintained in DMEM supple-
mented with 10% FBS, 2 mM glutamine, 10 mM HEPES (pH 7.4), and
*Department of Medicine, Division of Clinical Immunology and Rheumatology,†De-
partment of Pathology,‡Department of Medicine, Division of Gastroenterology and
Hepatology, and§Department of Medicine, Division of Hematology and Oncology,
University of Alabama at Birmingham, Birmingham, Alabama 35294; and¶Birming-
ham Veterans Administration Medical Center, Birmingham, AL 35233
Received for publication October 20, 2006. Accepted for publication March 21, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from the National Institutes of Health
(P30 AR48311, R01 CA116092, and R01 CA107181) and by Birmingham Veterans
Administration Medical Center Merit Review Grants (to H.-G.Z.).
2Address correspondence and reprint requests to Dr. Huang-Ge Zhang, University of
Alabama at Birmingham, 1825 University Boulevard SIRB 306 Birmingham, AL
35294. E-mail address: Huang-Ge.Zhang@ccc.uab.edu
3Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; KO, knock-
out; E-control, exosome control; WT, wild type; PI, propidium iodide; MHCII, MHC
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
by guest on June 11, 2013
antibiotics (100 U of penicillin/ml and 100 ?g of streptomycin/ml). FBS
for use in cell cultures was exosome depleted by differential centrifugation
using a method as described previously (17).
Purification of tumor exosomes
Exosomes derived from TS/A, B16, or MDA-MB-231 cells were isolated
from 1000 ml of culture medium containing ?108tumor cells using a
sucrose-gradient purification procedure as described previously (17). The
tumor-derived exosomes were collected in fraction 3 of the sucrose gra-
dient. The nonbanded fractions (fractions 6 and 7), which contain non-
membrane protein complexes, also were collected and concentrated using
a protein concentrator with a 100-kDa cutoff (Millipore) for use as the
exosome control (E-control). The protein content of the exosomes and
E-control was determined using a bicinchoninic acid protein assay kit (Bio-
Rad). The samples were then aliquoted and stored at ?80°C until use. The
E-control was used in assays at the same protein concentration as the
Labeling of exosomes and analysis of their target cells in vivo
TS/A tumor exosomes were labeled with the PKH67 green fluorescent dye
using a commercially available kit (Sigma-Aldrich) according to the man-
ufacturer’s instructions. The efficiency of labeling of the TS/A exosomes
(?90%) with PKH67 was determined by FACS analysis as described pre-
viously (17). BALB/c mice 7 wk of age were injected i.v. with PKH67-
labeled exosomes obtained from TS/A cells (10 mice/group) or unlabeled
exosomes obtained from the same cells. The mice were sacrificed 24 h after
injection, and the spleen, lung, lymph nodes, and liver were resected and
retained and the BM was collected. Single-cell suspensions of each tissue
were prepared in RPMI 1640 medium and subjected to FACS analysis. The
percentages of cells containing exosomes were determined by counting
green fluorescent-positive cells.
In vitro differentiation of DC from BM precursors
BM-derived DC were generated from primary cultures of femoral marrow
from 6- to 8-wk-old female wild-type (WT) and IL-6?/?mice as described
previously (18). In brief, BM cells were flushed from the femurs of 6- to
8-wk-old mice using an RPMI 1640-filled syringe to obtain a single-cell
suspension. After erythrocytes were depleted, the cells were washed twice
with RPMI 1640 (Invitrogen Life Technologies) containing 1% heat-inac-
tivated FBS, and then resuspended in RPMI 1640 supplemented with 10%
FBS, 1 mM pyruvate (Sigma-Aldrich), 1? nonessential amino acids (Sigma-
Aldrich), 2 mM glutamine (Sigma-Aldrich), 50 nM 2-ME (Invitrogen Life
Technologies), and 20 ng/ml recombinant mouse GM-CSF (PeproTech),
plated at a density of 2 ? 106cells/ml in 6-well plates and cultured at 37°C
in a 5% CO2atmosphere. Various concentrations of TS/A tumor exosomes
were added to the BM cell culture medium on days 0, 3, or 5. Fresh
medium (0.7 ml) was added to each well every 2 days. Some cultures of
BM precursor cells isolated from IL-6?/?C57BL/6j mice had rIL-6
(eBioscience) added to some of the wells every 2 days. After in ex vivo
culture, the percentages of differentiated DCs were determined by FACS
analysis of the expression of CD11c, CD86, CD80, and MHC class II
(MHCII). To determine whether the differentiated DCs were active bio-
logically, their ability to stimulate T cell proliferation was determined as
described below. Cell viability was tested routinely before experimental
analysis using trypan blue exclusion. In addition, we validated the lack of
toxicity of the inhibitors used in the experiments by FACS analysis of
propidium iodide (PI)-stained cells to identify necrosis or PI and Annexin
VFITCdouble-positive cells as an indicator of apoptosis (19).
ELISA and mixed lymphoid reaction
CD11C?cells were isolated from BM cells that had been cultured for 7
days, as described above, using anti-CD11c-coated magnetic beads (Milte-
nyi Biotec). The CD11C?cells were then resuspended in fresh RPMI 1640
medium supplemented with 10% FCS, cultured for 24 h in the presence of
LPS (1 ?g/ml). The ability of the DC to stimulate T cell proliferation was
determined using a MLR as described previously (16). Responder T lym-
phocytes were obtained by a nylon wool column enrichment of single-cell
suspensions of splenocytes from C57BL/6j mice and resuspended at 2 ?
106/ml in RPMI 1640 supplemented with 10% FBS. CD11C?DC that had
been cultured for 7 days, as described above, were resuspended in RPMI
1640 at various concentrations (3 ? 103–3 ? 105cells/ml). Equal volumes
of responding and stimulating cells (100 ?l/well) were added to flat-bot-
tom, 96-microtiter plates (BD Biosciences) and incubated at 37°C in hu-
midified 5% CO2air for 5 days. The absence of the proliferation of purified
DC populations alone, or with syngeneic T cells, made irradiation unnec-
essary. Cultures were pulsed with 1 ?Ci [3H]thymidine per well for the last
18 h of culture (Amersham Biosciences). The cells were collected on cel-
lulose filters with an automated harvester (Tomtech MacIII; PerkinElmer)
and the incorporated thymidine was measured using a scintillation counter
(MicroBeta 1450 Trimux; PerkinElmer Wallac).
Single-cell suspensions of BM-derived cells that had been cultured, as
described above, for 6 days were analyzed by FACScan flow cytometry
(BD Biosciences) using four-color analysis with a combination of allophy-
cocyanin-conjugated-CD11C (clone N418), PE-anti-CD86 (clone GL1),
CD11b (Mac-1), and FITC-conjugated-anti-MHCII (clone 14-4-4S). Sin-
gle-cell suspensions of cultured BM cells (1 ? 106) were washed once with
FACS buffer (5% FCS and 0.1% sodium azide in PBS) and incubated first
with unconjugated anti-CD16/CD32 (Fc Block; BD Pharmingen) at 22°C
for 20 min. Cells were then incubated with Ab coupled directly to fluoro-
chromes, at dilutions determined by pretitration, for 20 min at 22°C. After
staining, the cells were washed twice with FACS buffer and stored in PBS
containing 2% paraformaldehyde until FACS analysis. Cells (30,000/sam-
ple) were analyzed by flow cytometry using a FACScan (BD Biosciences
protocol; BD Pharmingen). Histogram analysis was performed using Win-
MDI software (email@example.com). Three to five samples were analyzed
for each treatment group. The results are presented as fluorescence histo-
grams, with the relative number of cells on a linear scale plotted vs the
relative fluorescence intensity on a log scale.
TS/A exosomes or E-control-treated BM cells were lysed in radioimmu-
noprecipitation assay lysis buffer containing 1% Triton X-100, 0.1% SDS,
150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 5 mM
sodium molybdate, and 20 mM phenylphosphate with protease and phos-
phatase inhibitors (1 mM PMSF, 10 ?g/ml aprotinin, 20 ?g/ml leupeptin,
20 ?g/ml pepstatin A, 50 mM NaF, and 1 mM sodium orthovanadate).
Total cell lysate (50 ?g protein) was loaded per lane on a 10% SDS-
polyacrylamide gel and subjected to electrophoresis. Proteins were blotted
onto nitrocellulose membranes using a Bio-Rad mini protein II transfer
apparatus. After overnight saturation at 4°C in PBS/0.05% Tween 20 con-
taining 5% BSA (Sigma-Aldrich), the membranes were probed with Abs
against Stat3, phosphorylated Stat3 (BD Biosciences), ?-tubulin, and ?-ac-
tin (Santa Cruz Biotechnology) for 1 h at 22°C. The membranes were
washed five times with PBS/0.05% Tween 20 and then probed with goat
anti-mouse or anti-rabbit secondary Abs conjugated to Alexa Fluor 680
(Molecular Probes) or IRdye 800 (Rockland). Blotted proteins were de-
tected using the Odyssey infrared imaging system (LI-COR).
Generation of human monocyte-derived DC
PBMC were obtained from healthy adult volunteers who signed informed
consent forms approved by the Ethics Committee of the University of
Alabama at Birmingham. In brief, PBMCs were isolated using Ficoll, as
described previously (20), and anti-CD14 magnetic beads were used ac-
cording to the manufacturer’s instructions (Miltenyi Biotec) to isolate
monocytes from the PBMC. The purity of these monocytes, as determined
by flow cytometry, was always ?97%. Monocytes were placed in the wells
of 12-well plates at a concentration of 1.25 ? 106/ml RPMI 1640 medium
(Invitrogen Life Technologies), containing GM-CSF (100 ng/ml) and IL-4
(1000 U/ml; PeproTech), in the presence or absence of exosomes purified
from the supernatants of human breast tumor MDA-MB-231 cells. Every
2 days, 0.15 ml of medium was removed, and 0.25 ml of a medium con-
taining IL-4 and GM-CSF was added. On day 6, the cells were washed with
0.5% PBS/0.02% BSA/sodium azide staining buffer (Mallinckrodt Baker),
and stained using different combinations of fluorochrome-labeled mAbs
against the following human proteins: CD14?(FITC) (clone M5E2),
CD1a?(PE) (clone HB15e), and isotype-control mAbs (BD Biosciences).
The cells were then washed once with staining buffer and fixed with 1%
paraformaldehyde (Electron Microscopy Sciences). At least 20,000 cells
were acquired on a FACSCalibur (BD Biosciences) and analyzed with
CellQuest software (version 3.1). Dead cells and debris were excluded by
forward and side scatter gating.
Oligonucleotide microarray analysis
RNA was prepared from CD11b?BM cells that had been treated with
either TS/A exosomes (n ? 3) or E-control (n ? 3) or PBS at a protein
concentration of 1 ?g/ml (n ? 3) for 0 and 2 h. Each RNA sample was
analyzed using an Affymetrix oligonucleotide microarray (Affymetrix).
The gene microarray assay was conducted at the Differential Gene Expres-
sion Core Facility of the University of Alabama at Birmingham. Data were
6868TUMOR EXOSOME SUPPRESSION OF DC DIFFERENTIATION
by guest on June 11, 2013
analyzed using three workstations running Affymetrix Microarray Analysis
Suite software (version 4.01). The fold induction of each gene at given
time points was calculated and expressed as exosome-treated sample ?
PBS-treated sample/E-control-treated sample ? sample treated with
Results are expressed as the mean ? SEM. The means of different treat-
ment groups were compared by two-way ANOVA. Individual differences
were further analyzed using the Bonferroni post tests.
BM myeloid precursor cells take up tumor exosomes
We have shown previously that sucrose density-purified tumor
exosomes, in contrast to fractions that contain membrane proteins
but not exosomes (E-control), promote tumor growth and metas-
tasis by suppressing the host immune response (17). To determine
whether BM cells are targeted by tumor exosomes in vivo, we
injected mice i.v. with exosomes (50 and 100 ?g/mouse) derived
from TS/A tumor cells that had been labeled with PKH67 or
nonlabeled TS/A exosomes (unlabeled exosomes served as an
autofluorescent background control). The cells that took up the
injected PKH67-labeled exosomes were identified by FACS anal-
ysis 24 h later. We found that a significant portion of PKH67-
positive exosomes were located in the BM (Fig. 1A). Within the
gated R1 region (Fig. 1A, inset), FACS analysis of the PKH67-
positive cells revealed that the majority of these cells (94 ? 4% in
BM; Fig. 1B) were CD11b?Gr-1?cells, indicating that they were
myeloid cells. The staining was CD11b specific, as a control
mouse IgG1 Ab did not stain the cells (Fig. 1B).
Tumor exosomes inhibit the differentiation of BM myeloid
precursor cells into DC in vitro
To determine the effects of the TS/A exosomes on the differenti-
ation of BM cells, exosomes were added to cultures of mononu-
clear cells, isolated from the BM of female BALB/c mice, and
TS/A tumor exosomes. Ten BALB/c mice were injected
i.v. with PKH67-labeled TS/A exosomes (50 ?g and
100 ?g of each mouse) or unlabeled TS/A exosomes
(100 ?g/mouse). BM cells were recovered after 24 h
and subjected to FACS analysis. A, Fluorescent-positive
cells. B, Within the gated R1 region, cell surface im-
munostaining with PE-conjugated anti-mouse CD11b
and Cy5-Gr-1 and normal mouse serum IgG1 used as a
control. A representative FACS graph of BM cells is
shown (A and B, inset). The data represent the mean ?
SEM from 10 mice from each group.
CD11b?BM precursors are targeted by
lysis buffer to eliminate erythrocytes were cultured in RPMI 1640 supplemented with 10% FBS and 20 ng/ml recombinant mouse GM-CSF. TS/A exosomes
or E-control (100 ?g/ml) were added to the cultured cells at different times after the addition of the GM-CSF to the cultures (A and B). After 7 days of
culture, cultured cells were analyzed by FACS for the expression of CD11c and IAd. One representative of four independent experiments is shown (A).
The proportion of apoptotic (PI?Annexin-V?) plus dead cells (PI?Annexin-V?) (percentage) was determined by flow cytometric analysis following
annexin V and PI staining 1 and 3 days after culturing. Representative figures are shown (C, inset). All FACS analysis results are presented as the average
values ? SEM obtained for three samples in four independent experiments (B and C).
TS/A exosomes block the differentiation of GM-CSF-stimulated BM precursor cells. Erythrocyte-depleted BM cells incubated with RBC
6869 The Journal of Immunology
by guest on June 11, 2013
grown in the presence of the differentiation-inducing agent GM-
CSF. On day 7, the expression of the DC surface marker, CD11c,
and an MHCII Ag was determined by FACS analysis (Fig. 2A).
The number of cells expressing the DC marker CD11c was sig-
nificantly lower in the cultures of mononuclear cells in which
TS/A exosomes were added at the time the cultures were set up
(day 0) than in the cultures of mononuclear cells treated with the
E-control (Fig. 2B). The addition of TS/A exosomes at later time
points (days 1 and 3) also resulted in a reduction in the size of the
CD11c?cell population, but the effect declined with the delay in
addition of the TS/A exosomes (Fig. 2, A and B). These results
suggest that the TS/A exosome-induced reduction in the size of the
CD11c?cell population was not due to a nonspecific cytotoxic
effect on the BM cells. It also is unlikely that the reduction in the
numbers of CD11c?is due to a discrepancy in the sensitivity of
BM precursor cells to apoptosis because there was not a significant
difference in the percentages of PI?AnnexinV?cells on treatment
with exosomes or E-control (Fig. 2C) at the 1-day or 3-day culture
after BM cells were treated with TS/A exosomes. The number
of CD11c?is reduced, but the total cell numbers of exosome-
treated cells compared with untreated cells are not significantly
different (5.1 ? 0.7 ? 106and 5.7 ? 0.1 ? 106, respectively;
n ? 10; p ? 0.05), and flow cytometric analysis of the resulting
cells showed that the majority of these cells subsequently gave
rise to substantial numbers of F4/80?macrophages. Total cell
numbers of F4/80?cells went up to 3.4 ? 0.4 ? 106in TS/A
exosome-treated cells, compared with 0.8 ? 0.2 ? 106in un-
treated cells (n ? 10; p ? 0.01).
The myeloid DC that differentiate from TS/A exosome-treated
myeloid precursors are incapable of maturation
To determine whether the CD11c?cells that escape the TS/A exo-
some-induced block of myeloid cell precursor differentiation are
capable of maturation, we harvested the TS/A exosome-treated
BM cells on day 6 and stimulated the cells with LPS overnight.
FACS analysis for the DC surface marker CD11c and the costimu-
latory molecules CD86 and CD80 indicated that significantly
fewer BM cells were CD11c?, CD11c?CD80?, or CD11c?
CD86?(Fig. 3A) than of BM cells treated with E-control. The degree
of reduction of CD11c?CD80?(Fig. 3B) and CD11c?CD86?
(Fig. 3C) was correlated with the concentrations of TS/A exosomes
added to the culture, suggesting that the reduction of DC maturation
is tumor exosome specific.
To further characterize the functional capacity of the CD11c?
DC that had been treated with TS/A exosomes, we tested the abil-
ity of the CD11c?DC to stimulate T cell proliferation in a primary
MLR-specific T cell activation response. BM-differentiated
prevent DC maturation. Erythrocyte-
depleted BM cells (1 ? 106) were
cultured in RPMI 1640 supplemented
with 10% FBS and 20 ng/ml recom-
binant mouse GM-CSF. TS/A exo-
somes or E-control at 100 ng/ml (A)
or various concentrations (B and C)
were added to the cultured cells at day
0 of culture. Seven days after culture,
the cells stimulated with LPS over-
night were analyzed for the expres-
sion of CD11c?(A), CD11c?CD80?
(B), or CD11c?CD86?(C) using
FACS. Alternatively, CD11c?cells
were isolated from the 7-day cultures
using beads coated with anti-CD11c
Ab. The purified CD11c?cells were
then used in a standard MLR reaction
(D, CD11c:T cells ? 1:10) or in a
MLR, where increasing numbers of
CD11c cells purified from 7-day stim-
ulated BM DC pretreated with TS/A
exosomes (10 ?g/ml) (E) were used.
Data are presented as the means of trip-
licate stimulated/unstimulated cpm of
five mice from each group. Results are
obtained using five samples in three in-
TS/A tumor exosomes
6870 TUMOR EXOSOME SUPPRESSION OF DC DIFFERENTIATION
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