of June 13, 2013.
This information is current as
and Collagen-Induced Arthritis
Dendritic Cells Can Suppress Inflammation
Exosomes Derived from IL-10-Treated
Watkins, Andrea Gambotto and Paul D. Robbins
C.Menon, Annahita Keravala, Joan Nash, Zhibao Mi, Simon
Seon-Hee Kim, Eric R. Lechman, Nicole Bianco, Rajasree
2005; 174:6440-6448; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2005 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 13, 2013
Exosomes Derived from IL-10-Treated Dendritic Cells Can
Suppress Inflammation and Collagen-Induced Arthritis1
Seon-Hee Kim,2* Eric R. Lechman,2* Nicole Bianco,* Rajasree Menon,* Annahita Keravala,*
Joan Nash,* Zhibao Mi,* Simon C. Watkins,§Andrea Gambotto,*†and Paul D. Robbins3*‡
We have demonstrated previously that local, adenoviral-mediated gene transfer of viral IL-10 to a single joint of rabbits and mice
with experimental arthritis can suppress disease in both the treated and untreated contralateral joints. This contralateral effect
is mediated in part by APCs able to traffic from the treated joint to lymph nodes as well as to untreated joints. Moreover, injection
of dendritic cells (DC) genetically modified to express IL-4 or Fas ligand was able to reverse established murine arthritis. To
examine the ability of exosomes derived from immunosuppressive DCs to reduce inflammation and autoimmunity, murine models
of delayed-type hypersensitivity and collagen-induced arthritis were used. In this study, we demonstrate that periarticular ad-
ministration of exosomes purified from either bone marrow-derived DCs transduced ex vivo with an adenovirus expressing viral
IL-10 or bone marrow-derived DCs treated with recombinant murine IL-10 were able to suppress delayed-type hypersensitivity
responses within injected and untreated contralateral joints. In addition, the systemic injection of IL-10-treated DC-derived
exosomes was able suppress the onset of murine collagen-induced arthritis as well as reduce severity of established arthritis. Taken
together, these data suggest that immature DCs are able to secrete exosomes that are involved in the suppression of inflammatory
and autoimmune responses. Thus DC-derived exosomes may represent a novel, cell-free therapy for the treatment of autoimmune
diseases. The Journal of Immunology, 2005, 174: 6440–6448.
established, the affected joints exhibit inflammatory cell infiltration
and synovial hyperplasia that contribute to the progressive degra-
dation of cartilage and bone, resulting in the complete loss of nor-
mal joint function. Recently, specific biological agents able to in-
hibit the proinflammatory cytokines IL-1 and TNF-? have been
shown to be therapeutic for treating RA. However, if treatment
with the IL-1 and TNF inhibitors is terminated, then disease pro-
gression and severity of symptoms return (3). Thus, there is still
need for an effective therapy able to reverse the course of arthritis
progression following a single or infrequent treatment.
We and others (4–6) have demonstrated that local, adenoviral-
mediated gene transfer of viral IL-10 (vIL-10) to a single joint of
rabbits and mice with experimental arthritis can suppress disease
he autoimmune disease, rheumatoid arthritis (RA),4is a
debilitating disease characterized by chronic inflamma-
tion of the distal diarthroidial joints (1–3). Once RA is
in both the treated and untreated contralateral joints. This con-
tralateral effect has been observed following both in vivo and ex
vivo delivery of a variety of different therapeutic genes in several
different animal models of arthritis (6–14). However, the immu-
nological mechanism by which local gene transfer to one joint can
distribute antiarthritic effects to untreated joints is currently poorly
understood. Recent analysis of the effect suggests that APCs are
able to traffic from the treated joint to not only the draining lymph
nodes but also untreated joints, suggesting a role for APC in the
generation and systemic dissemination of local anti-inflammatory
effects (6, 8, 9, 15). We and others (9, 12, 15) have also shown that
systemic administration of dendritic cells (DC), genetically mod-
ified to express IL-4 or Fas ligand (FasL), was able to reverse
established murine collagen-induced arthritis (CIA). These results
suggest that immature or certain genetically modified DCs are able
to suppress or reverse the course of autoimmune arthritis progres-
sion in a murine model.
The release of small lipid vesicles, termed exosomes, by a va-
riety of cells types, including DCs, has been well documented.
Exosomes are small membrane-bound vesicles (40–100 nm) that
are secreted in the extracellular medium by many cells of hemo-
poietic origin (16–18) as well certain nonhemopoietic cell types
(19). Exosomes are formed by inverse membrane budding into the
lumen of an endocytic compartment, which results in the formation
of multivesicular intracellular structures or multivesicular bodies.
Fusion of the multivesicular bodies with the plasma membrane
leads to the release of internal vesicles (exosomes) into the me-
dium. The protein content of exosomes varies depending on the
cell type from which they were derived but contains both mem-
brane-associated as well as cytosolic proteins (17, 19–21).
DC-derived exosomes have been characterized extensively at
the ultrastructural and protein levels (22–27). However, the in vivo
function of DC-derived exosomes remains unclear. Recent exper-
iments suggest that DC-derived exosomes are able to stimulate
Ag-specific T cell responses through their ability to interact or fuse
Departments of *Molecular Genetics and Biochemistry,†Surgery,‡Orthopaedic Sur-
gery, and§Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA
Received for publication August 26, 2004. Accepted for publication February
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 AI56374 and DK44935 from the Na-
tional Institutes of Health (to P.D.R.).
2S.-H.K. and E.R.L. contributed equally to the manuscript.
3Address correspondence and reprint requests to Dr. Paul D. Robbins, Department of
Molecular Genetics and Biochemistry, W1246 Biomedical Science Tower, University
of Pittsburgh School of Medicine, Pittsburgh, PA 15261. E-mail address:
4Abbreviations used in this paper: RA, rheumatoid arthritis; vIL-10, viral IL-10; DC,
dendritic cell; FasL, Fas ligand; CIA, collagen-induced arthritis; BM-DC, bone mar-
row-derived DC; DTH, delayed-type hypersensitivity; MHC I, MHC class I; MHC
II, MHC class II; eGFP, enhanced GFP; TEM, transmission electron microscopy;
KLH, keyhole limpet hemocyanin; exo/vDC-10, exosomes from the Ad-vIL-10
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
by guest on June 13, 2013
with endogenous or follicular DCs. Exosomes may fuse with or be
internalized into endogenous DCs and macrophages to transfer
class II molecules, as well as possibly other proteins, to the DCs
and thus indirectly regulate T cell function. Alternatively, DCs
also have been shown to secrete exosomes that carry peptide-
loaded MHC molecules that can stimulate T cell proliferation in
vitro, suggesting that exosomes alone may be able to present Ag to
T cells (28, 29). When used as a tumor vaccine, DC-derived exo-
somes loaded with acid-eluted tumor peptides eradicated estab-
lished tumors in mice (30). On the basis of these and other data,
two different clinical trials have been initiated using exosomes
derived from tumor peptide-pulsed DCs (31, 32).
The ability of DCs to induce or suppress immune responses is
dependent on several factors including Ag exposure, state of ma-
turity, and culture conditions (33, 34). Interestingly, it appears that
exosomes may have a similar dual capability as well. Several re-
ports have suggested that exosomes can suppress as well as stim-
ulate immune responses (35–38). For example, small particles pro-
duced by rat intestinal epithelial cells cultured in the presence of
IFN-? and digested OVA were able to induce the Ag-specific tol-
erance of that same Ag after injection into mice (38). It has also
been shown that both T cells and melanoma tumor cells are able to
generate exosomes expressing FasL, which can induce the apoptosis
of T cells that would otherwise counteract tumor growth (36, 37).
In this study, we have assessed the in vitro and in vivo immu-
nosuppressive capacity of bone marrow-derived DC (BM-DC)-
derived exosomes. Periarticular administration of exosomes de-
rived from BM-DCs that were either transduced ex vivo with an
adenovirus expressing vIL-10 or treated with recombinant murine
IL-10 was able to suppress murine delayed-type hypersensitivity
(DTH) responses within injected and untreated contralateral foot-
pads. In addition, the systemic injection of IL-10-treated exosomes
was able to suppress the onset of murine CIA. Collectively, these
data suggest that immature DCs are able to secrete exosomes that
are involved in the suppression of inflammatory responses. Given
that exosomes are unable to change their phenotypes following
purification and are stable, DC-derived exosomes represent a novel
therapeutic approach for the treatment of inflammatory and auto-
Materials and Methods
Female C57BL/6 (H-2Kb) mice and male DBA1/LacJ (H-2Kq) mice, all
7–8 wk of age, were purchased from The Jackson Laboratory. Animals
were maintained in a pathogen-free animal facility at the University of
Pittsburgh Biotechnology Center (Pittsburgh, PA).
Generation and culture of BM-DCs
BM-DCs were generated as described previously (9). Briefly, bone marrow
was harvested from mouse tibias and femurs and passed through a nylon
mesh to eliminate small pieces of bone and debris. Contaminating eryth-
rocytes were lysed with 0.83 M NH4Cl buffer, and lymphocytes were de-
pleted with a mixture of Abs (RA3-3A1/6.1, anti-B220; 2.43, anti-Lyt2;
and GK1.5, anti-L3T4; all from American Type Culture Collection) and
rabbit complement (Accurate Chemical and Scientific) on day 0. The cells
were then cultured for 24 h in complete medium (RPMI 1640 containing
10% FBS, 50 ?M 2-ME, 2 mM glutamine, 0.1 mM nonessential amino
acids, 100 ?g/ml streptomycin, and 100 IU/ml penicillin) to remove the
adherent macrophages. The nonadherent cells were then placed in fresh
complete medium containing recombinant murine GM-CSF (1000 U/ml)
and recombinant murine IL-4 (1000 U/ml) on day 1. Cells were cultured
for 4 days and harvested for adenoviral transduction or recombinant cyto-
kine treatment on day 5.
For adenoviral infection, 1 ? 106DC/well were plated on 24-well
plates, and 5 ? 107PFU of each recombinant adenovirus was added in a
total volume of 1 ml of serum-free medium. After incubation for 24 h at
37°C, the cells were collected and washed five times in PBS, and fresh
medium was added. On day 7, infected DCs and exosomes were recovered,
washed extensively, and injected into animals.
Vector construction and adenovirus generation
Adenoviruses expressing viral IL-10 (Ad.vIL-10) and enhanced GFP (Ad-
.eGFP) were constructed, propagated, and titered according to standard
protocols as described previously (8). Briefly, the recombinant adenovi-
ruses were generated by homologous recombination in 293 cells expressing
Cre recombinase (CRE8 cells), after cotransfection of DNA, an adenovirus
5-derived, E1- and E3-deleted adenoviral backbone (psi5) and pAdlox, the
adenoviral shuttle vector. The inserted cDNA sequences are expressed un-
der the human CMV promoter. The recombinant adenoviruses were puri-
fied by CsCl gradient ultracentrifugation, dialyzed in sterile virus storage
buffer, aliquoted, and stored at ?80°C until use. The CRE8 cells were
grown and maintained in DMEM (Invitrogen Life Technologies) supple-
mented with 10% FCS.
Exosomes were prepared from the cell culture supernatant of day 7
BM-DC cultures by differential centrifugation as described previously (22).
Briefly, recovered culture supernatant from each BM-DC culture was sub-
jected to three successive centrifugations at 300 ? g (5 min), 1,200 ? g (20
min), and 10,000 ? g (30 min) to eliminate cells and debris, followed by
centrifugation for 1 h at 100,000 ? g. To remove excess serum proteins,
the exosome pellet was washed with a large volume of PBS, centrifuged at
100,000 ? g for 1 h, and finally resuspended in 120 ?l of PBS for further
studies. The exosomes were quantified by a micro Bradford protein assay
(Bio-Rad). Each batch was standardized by protein content, and 1 ?g was
suspended in 20 ?l of PBS for in vivo mouse studies. For MHC class II
(MHC II) adsorption, 100 ?l of washed anti-mouse MHC II paramagnetic
beads (Miltenyi Biotech) were incubated with prediluted exosomes (1
?g/20 ?l) for 1 h, 4°C with gentle shaking. After magnetic separation, the
fraction not retained in the microcentrifuge tube was adjusted to the orig-
inal volume with PBS. The freeze/thaw of prediluted exosomes was per-
formed by three separate rounds of snap freezing in a dry ice/ethanol bath
and subsequent warming in 37°C bath. Contaminating concentrations of
IL-10 and vIL-10 in final exosome preparations were determined by IL-10
On day 0, mice were sensitized by s.c. injection of 100 of ?g Ag (OVA)
emulsified 1:1 in CFA (Difco). Two weeks later, presensitized mice were
given injections in one rear footpad with either 1 ? 106treated DCs (in 50
?l of PBS) or 1 ?g of purified exosomes derived from each experimental
DC group (in 50 ?l of PBS). The contralateral footpads were injected with
equal volumes of saline. One day later, the mice were challenged in both
rear footpads by injecting 20 ?g of Ag dissolved in 50 ?l of PBS, and the
footpads were measured with a spring-loaded caliper (Dyer) 24, 48, and
72 h later. The results were expressed as the difference in size due to
swelling (millimeters ? 10?2).
By stirring overnight at 4°C, bovine type II collagen (Chondrex) was dis-
solved in 0.05 M acetic acid at a concentration of 2 mg/ml and emulsified
in an equal volume of CFA. The mice were immunized intradermally at the
base of the tail with 100 ?g of collagen. On day 21 after priming, the mice
received an intradermal booster injection of type II collagen in IFA. For the
prevention of disease onset study, the mice received injections with exo-
somes from DCs at day 28 at the time of disease onset. Mice were mon-
itored every other day by an established macroscopic scoring system rang-
ing from 0 to 4 as follows: 0 ? normal; 1 ? detectable arthritis with
erythema; 2 ? significant swelling and redness; 3 ? severe swelling and
redness from joint to digit; and 4 ? maximal swelling and deformity with
ankylosis. The average of macroscopic score was expressed as a cumula-
tive value for all paws, with a maximum possible score of 16 per mouse.
For the treatment of established arthritis, mice were given injections with
20 ?g of LPS i.p. at day 28 to induce synchronous disease onset. Four days
after LPS injection (day 32), exosomes from Ad.vIL-10-transduced or rIL-
10-treated DCs were injected into the mice with evidence of disease. The
in vivo experiments were performed with 10 mice/group and repeated
twice to ensure reproducibility.
Exosomes were purified by differential centrifugation, 10 ?l loaded on a
Formvar/carbon coated grid, negatively stained with 10 ?l of neutral 1%
6441The Journal of Immunology
by guest on June 13, 2013
aqueous phosphotungstic acid, and viewed using JEOL-1210 computer-
controlled, high-contrast, 120-kV transmission electron microscope.
The cytosol of cells was separated from the total membranes by homoge-
nization in 10 mM triethanolamine, 1 mM EDTA, 10 mM acetic acid, 250
mM sucrose (pH 7.4), and supplemented with CLAP (chymotrypsin, leu-
peptin, aprotinin, and pepstatin, 100 ?M each), by 60 passages through a
25-gauge needle. The supernatant was cleared from nuclei and cell debris
by centrifugation at 1,200 ? g. Total membranes were recovered in the
pellet after centrifugation for 1 h at 100,000 ? g. Proteins (10 ?g) were
then separated on 5–20% gradient SDS-PAGE, transferred onto nitrocel-
lulose, and detected by Western blot using an ECL detection kit (Amer-
DCs were defined by phenotypic analysis for expression of CD11b,
CD11c, CD80, CD86, and MHC class I (MHC I) and MHC II in the
majority of the cultured cells (60–95%) by FACScan (BD Biosciences).
For exosomes, 30 ?g of pelleted exosomes were incubated with 10 ?l of
4-?m diameter aldehyde/sulfate latex beads (Interfacial Dynamics) for 15
min at room temperature in a 30 to 100-?l final volume, followed by 2 h
with gentle agitation in 1 ml of PBS. The reaction was stopped by 30-min
incubation in 100 mM glycine. Exosome-coated beads were washed three
times in FACS wash buffer (3% FCS, and 0.1% NaN3in PBS) and resus-
pended in 500 ?l of FACS wash. Beads were incubated for 1 h with each
primary Ab, followed when necessary by incubation in FITC-conjugated
secondary Ab, washed, and analyzed on a FACSCalibur (BD Biosciences).
Data acquisition and analysis were performed using Lysis II FACScan
software (BD Biosciences).
Mixed lymphocyte reaction
T cells were purified from the spleens of BALB/c mice for in vitro micro-
culture in round-bottom, 96-well plates. In each well, 5 ? 104splenic T
cells were seeded with either control C57BL/6-derived DCs or genetically
modified C57BL/6-derived DCs (vIL-10, rIL-10, or luciferase). On day 5
of culture, 1 ?Ci of [3H]thymidine was added to each well 16 h before
harvest. Radioactive labeling of proliferating T cells was measured on a
microplate beta counter (Wallac).
All data were analyzed using the Microsoft Excel software program. Group
comparisons were performed using both Student’s t test and ANOVA.
Characterization of murine BM-DC-derived exosomes
To examine the immunoregulatory role of DC-derived exosomes,
DCs were generated from C57BL/6 mouse bone marrow precur-
sors cultured at high density in GM-CSF/IL-4. Exosomes produced
by the DCs were then isolated from the culture medium by differ-
ential centrifugation and characterized by electron microscopy,
Western blot, and flow cytometry (Fig. 1). Ultrastructural analysis
of exosome pellets by whole mount transmission electron micros-
copy (TEM) showed a significant enrichment of the characteristic
saucer-shaped exosomes, 40–100 nm in diameter (Fig. 1A). West-
ern analysis demonstrated that the DC-derived exosomes were
positive for the exosome-associated proteins CD71 and Hsp70
(Fig. 1B) but negative for proteins not found in exosomes, such as
Hsp90, the invariant chain, and calnexin (data not shown). To fur-
ther demonstrate the intact vesicular nature of the exosome frac-
tion, exosomes were purified from BM-DCs of eGFP/C57 mice,
because the majority of cells within the animal constitutively express
the marker protein eGFP. Western analysis of the highly enriched
exosome fraction showed a significant level of full-length eGFP, sug-
gesting that the soluble eGFP is encapsulated within the protective
environment of the lumen of BM-DC-derived exosomes.
We further examined the surface proteins on the DC-derived
exosome fraction by flow cytometry. Exosomes were recovered
after the 100,000 ? g spin, bound to latex beads, and stained with
several mAbs against murine DC-associated leukocytic marker
proteins. The surface of exosomes stained positive for high levels
of MHC II, with more moderate levels of MHC I, CD11C, CD80
(B7.1), and CD86 (B7.2) detected (Fig. 1C). Taken together, these
data demonstrate the ability to enrich for intact exosomes that con-
tain many of the markers of DC-derived exosome-associated pro-
teins as described previously (21, 22, 24).
In vitro function of BM-DC-derived exosomes
To test the ability of BM-DC-derived exosomes to suppress T cell
proliferation, the effect of adding DC-derived exosomes to a MLR
exosomes from BM-DCs. A, Whole-
mount TEM of exosomes from murine
BM-DCs. BM-DCs were grown from
lymphocyte-depleted monocytic pre-
cursors and cultured in GM-CSF and
IL-4 for 7 days. The cell culture super-
natants were then sequentially centri-
fuged to obtain the pellet containing
exosomes. The 100,000 ? g pellet was
washed and analyzed by TEM. Bar,
200 nm. B, Western blot analysis of
exosome-associated proteins. Ten mi-
crograms of exosomes and BM-DC ly-
sates were separated by SDS-PAGE
and analyzed in parallel by Western
blotting for the presence of several
exosome-associated proteins. C, Flow
cytometric analysis of murine DC-de-
rived exosomes. BM-DC-derived exo-
somes were isolated by differential
centrifugation, coated onto beads,
and stained with FITC-coupled Abs
specific for MHC I and II, CD11c,
CD80 (B7.1), CD86 (B7.2), or the
6442DC-DERIVED EXOSOMES ARE IMMUNOSUPPRESSIVE IN VIVO
by guest on June 13, 2013
was examined. As a source of potentially immunosuppressive DC-
derived exosomes, BM-DCs transduced with an adenovirus ex-
pressing the EBV-encoded IL-10 gene, termed vIL-10, were used.
Intra-articular gene transfer of vIL-10 has been shown to suppress
inflammation in both rabbit Ag-induced arthritis and murine CIA
models (5, 6). As a control, BM-DCs transduced with adenoviral
vector expressing luciferase (Ad.Luc) were used. When added to
the MLR, the DCs transduced with Ad.vIL-10 were able to almost
completely suppress T cell proliferation, as measured by [3H]thy-
midine incorporation, whereas addition of nontransduced DCs dis-
played little or no effect (Fig. 2A). Exosomes secreted by BM-DCs
infected with Ad.vIL-10 exhibited a moderate 4-fold decrease in T
cell proliferation (Fig. 2B). These data suggest that exosomes iso-
lated from the immunosuppressive vIL-10-expressing DCs are
able to block T cell proliferation and that exosomes derived from
unmodified BM-DCs may themselves harbor partial anti-
Exosomes can suppress inflammation in a DTH model
To investigate the anti-inflammatory effect of BM-DC-derived
exosomes in vivo, a DTH model in C57BL/6 mice was used. Pre-
viously, we have used this model to show that injection of Ad-
.vIL-10 into one hind footpad results in suppression of inflamma-
tion in both the injected and contralateral footpad. Moreover, we
have shown by adoptive transfer experiments that the contralateral
effect observed following local Ad.vIL-10 delivery in the DTH
model was conferred by endogenous APC. Groups of sensitized
mice were given injections in the right rear footpad with either
untreated or genetically modified DCs or the exosomes derived
from the culture media of these cells. The contralateral footpads
received a saline injection of a similar volume. After 12 h, each
footpad was challenged with 20 ?g of keyhole limpet hemocyanin
(KLH), and footpad swelling was monitored at 24, 48, and 72 h
after disease induction. As shown in Fig. 3, the DTH response in
saline control animals was acute, with the average increase in paw
thickness ?2 mm. However, footpad swelling was reduced by
?50% in the injected footpads of mice receiving 1 ? 106BM-DCs
transduced with Ad.vIL-10. A reduction of inflammation (40%)
was also observed in the saline-treated contralateral footpads of
these same animals. Interestingly, injection of 1 ?g of secreted
MLR. A, The 5 ? 105cells/ml T cells from BALB/c (H-2kd) spleen were
stimulated with C57BL/6 (H-2kb) bone marrow DCs noninfected or in-
fected either Ad-vIL-10 or Ad-Luc at serial ratio. After 4 days of culture,
1 ?Ci of [3H]thymidine was added in each well 16 h before harvest. B,
Exosomes were isolated from C57BL/6 DCs that were noninfected or in-
fected with either Ad-vIL-10 or Ad-Luc. The serial concentrations of exo-
somes were added into culture of T cells isolated from BALB/c spleen and
naive DCs from C57BL/6 at 10:1 ratio. T cell proliferation was determined
by [3H]thymidine incorporation after 5 days of culture. Luc, Luciferase.
Analysis of function of BM-DC-derived exosomes in an
exosomes. A, Suppression of DTH response in Ad-vIL-10-transduced DCs
and exosomes derived from the DC/vIL-10. Groups of mice were sensi-
tized to KLH in CFA by s.c. injection on day 0. Two weeks later, sensitized
mice received injections into the right footpad with 1 ? 106untreated DCs,
DCs transduced with 50 multiplicity of infection of Ad-Luc, DCs trans-
duced Ad.vIL-10, or 1 ?g of exosomes derived from these cells. The con-
tralateral joints were injected with a similar volume of saline. The mice
were then challenged with 20 ?g of Ag in PBS 12 h later, and footpad
swelling was measured at 24-, 48-, and 72-h time points. B, Suppression of
DTH reaction with recombinant murine IL-10-treated BM-DCs and exo-
somes derived from DCs treated with recombinant murine IL-10. Day 5
BM-DCs were treated with 1 ?g/ml recombinant murine IL-10 protein for
24 h. After washing, treated DCs were cultivated for an additional 48 h
before harvest. Exosomes were isolated from either naive DCs or recom-
binant murine IL-10-treated DCs and injected into the right footpad of
KLH-immunized mice. After challenge of KLH into both footpads, swell-
ing of footpad was measured at 24-, 48-, and 72-h time points. ?, Signif-
icance at p ? 0.01. rmIL-10, Recombinant murine IL-10.
Suppression of the DTH response by DCs and DC-derived
6443The Journal of Immunology
by guest on June 13, 2013
exosomes derived from Ad.vIL-10-tranduced BM-DCs was even
more protective, suppressing paw swelling by 65% compared with
saline control mice. Furthermore, a significant reduction was also
observed in the footpads contralateral to the Ad.vIL-10/exosome-
treated joints (Fig. 3A). No significant reductions in footpad swell-
ing were observed following administration of Ad.Luc/DCs, Ad-
.Luc/exosomes, untreated DCs, or exosomes from untreated DCs.
These data suggest that exosomes derived from Ad.vIL-10-trans-
duced BM-DCs can suppress DTH in both treated and untreated
contralateral footpads when delivered locally to sensitized mice.
rIL-10-treated-DC-derived exosomes are immunosuppressive
Although the experiments performed above suggest that exosomes
derived from Ad.vIL-10-transduced DCs are immunosuppressive,
it is possible that a low level of Ad.vIL-10 or vIL-10 protein con-
taminated the exosome preparation. To show that adenovirus in-
fection or vIL-10 protein contamination did not contribute to the
observed effects, BM-DCs were treated with recombinant murine
IL-10 protein and the generated exosomes tested in vivo using the
DTH model in C57BL/6 mice (Fig. 3B). The exosomes derived
from recombinant murine IL-10-treated DCs produced a strong
immunosuppressive effect 48 h postchallenge, as demonstrated by
a 6-fold reduction in paw swelling in the treated paws and a 3-fold
reduction in the untreated contralateral paws. Taken together, these
results demonstrate that exosomes derived from murine IL-10-
treated BM-DCs can suppress DTH in both treated and untreated
contralateral footpads, effectively ruling out adenovirus contami-
nation as the mechanism for this effect. It is also important to note
that no rIL-10 protein was detected in the exosome preparations by
Membrane disruption causes loss of immunosuppressive ability
To confirm that the exosomes present in the enriched 100,000 ? g
pellet fraction were important for conferring the therapeutic effects
in the DTH model, the requirement for intact exosomes particles
for efficacy was examined. Initially, we demonstrated by electron
microscopy that the integrity of exosomes was disrupted by four
cycles of freeze/thaw (Fig. 4A). In addition, the soluble, exosome-
associated protein Hsc70 was not detected in the freeze/thaw-
treated exosome fractions, whereas it was associated with un-
treated exosomes (Fig. 4B). To test whether exosomes require an
intact membrane to suppress the DTH response, intact or freeze/
thawed exosomes from DCs transduced with Ad-vIL-10 were in-
jected into the footpad of one hind paw of KLH-immunized mice
(Fig. 4C). After 24 h, each footpad was boosted with KLH, and the
extent of footpad swelling was measured. Although reduction of
footpad swelling was observed in the group treated with intact
exosomes from the Ad-vIL-10 transduced DC (exo/vIL-10), the
group injected with freeze/thaw-treated exo/vIL-10 showed no re-
duction in paw swelling, similar to the control groups treated with
saline or exosomes from control DCs (also see Fig. 7B). These
results demonstrate that multiple cycles of freeze/thaw disrupted
membrane structure of exosomes, thereby eliminating the immu-
nosuppressive ability of exosomes in the DTH model. Thus, intact
vesicles appear to be required for the observed suppressive effect.
MHC II-containing exosomes required for suppression of the
To determine whether the exosomes able to suppress the DTH
response indeed were derived from DCs, the effect of depleting
class II-positive particles on suppression of the DTH response was
examined. Exosomes from Ad-vIL-10-transduced BM-DCs were
divided into four samples for pretreatment before injection into
sensitized mice (Fig. 5A). The first exosome sample was pread-
sorbed with paramagnetic beads specific for murine MHC II,
whereas the second sample was preadsorbed with paramagnetic
beads specific for NK1.1, a cell surface molecule not present on
DC-derived exosomes. The third sample was subjected to multiple
cycles of freeze/thaw, whereas the fourth sample was left un-
treated. The exosome samples were then injected into one hind
footpad of mice, and 12 h later, DTH-induced footpad swelling in
both hind footpads was measured over the next 72 h. Similar to
previous experiments, exosomes from control DCs had no effect
on footpad swelling, whereas Ad.vIL-10/exosomes were able to
dramatically block DTH in both the injected and untreated con-
tralateral footpads. The exosome preparation preadsorbed to the
NK1.1 beads exhibited immunosuppressive activity. However,
preadsorption of the Ad.vIL-10/exosome sample with class II
beads abrogated ?100% of the activity in vivo. Importantly, the
suppressive effects. A, Whole-mount TEM of intact or freeze/thawed exo-
somes from BM-DCs transduced with Ad-vIL-10. B, Western Blot against
anti-Hsc70 for intact or freeze/thawed exosomes from BM-DCs transduced
with Ad-vIL-10. Exosomes from either nontransduced DCs or DCs trans-
duced with Ad-Luc or Ad-vIL-10 were isolated and subjected to multiple
cycles of freeze/thaw. Total of 5 ?g of intact or freeze/thawed exosomes
were separated by SDS-PAGE and analyzed in parallel by Western blotting
for Hsc70. C, Analysis of membrane-disrupted exosomes in the DTH
model. Exosomes were isolated from either Ad-vIL-10- or Ad-Luc-trans-
duced mouse bone marrow DCs. After several cycles of freeze/thaw, 1 ?g
of intact or disrupted exosomes was injected into one hind paw of the
KLH-immunized mouse. After boost injection of KLH, swelling of both
hind paws was measured at 48-h time point. ?, Significance at p ? 0.01.
Luc, Luciferase; F/T, freeze/thaw.
Membrane disruption of exosomes abrogates the immuno-
6444DC-DERIVED EXOSOMES ARE IMMUNOSUPPRESSIVE IN VIVO
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injection of the actual paramagnetic beads with bound, class II-
positive exosomes resulted in immunosuppressive activity similar
to that seen with the nonadsorbed Ad-vIL-10 exosomes. Exosomes
from recombinant mouse IL-10 protein-treated DCs showed sim-
ilar results in the DTH response after MHC II depletion (Fig. 5B).
The results were consistent with each other. The formulation that
underwent several freeze/thaw cycles in both the exosomes from
Ad-vIL-10- or rIL-10-treated DCs also lost activity. These data
suggest that the in vivo anti-inflammatory effects are conferred by
MHC II-positive vesicles. Furthermore, it appears that the integrity
of the vesicle is also required. Given that the in vivo anti-
inflammatory effects also appear to be specific for structurally in-
tact MHC II-positive exosomes, this rules out adenovirus contam-
ination as the mechanism for the effect.
Exosomes can suppress CIA in mice
RA is a debilitating autoimmune disease characterized by chronic
inflammation of the distal diarthroidial joints and progressive de-
struction of cartilage tissue. Similar pathologies as well as inflam-
mation in joints can be induced in the DBA1/lacJ (H-2kq) strain
with injection of bovine type II collagen. To examine the ability of
DCs and DC-derived exosomes to treat CIA, DCs were infected
with Ad-vIL-10, and the resultant exosomes were injected i.v. into
DBA1 mice immunized with bovine type II collagen. Injection was
done at day 28, just before disease onset. A single injection of
either DC/vIL-10 or exosomes from DC/vIL-10 was able to delay
the onset and reduce the severity of arthritis, whereas disease pro-
gressed normally in the saline-injected control group (Fig. 6). This
result suggests that a single injection of exosomes derived from
DCs expressing IL-10 is comparable to an injection of genetically
modified parental DCs in preventing onset of CIA.
In addition to the analysis in DC-derived exosomes in the pre-
vention of disease study, exosomes from DC/IL-10 were tested in
the mice with established CIA. Exosomes from Ad.vIL-10-trans-
duced or rIL-10-treated DCs were injected i.v. into the mice with
established disease (Fig. 7). Although disease suppression in the
exo/IL-10-treated group was less than that shown in the prevention
study, exosomes from both the Ad.vIL-10-transduced and rIL-10-
treated DCs were able to reduce the severity of established disease
(Fig. 7A). Moreover, freeze/thaw treatment of the exosomes abro-
gated the therapeutic effect (Fig. 7B), whereas direct injection of
rIL-10 protein had no effect on disease progression (C).
Recently, exosomes have been shown to be involved in regulating
certain biological processes. In particular, exosomes can be used to
transfer proteins to cells through membrane fusion, potentially me-
diated by tetraspanins and MFG-E8 (39, 40). For example, mi-
crovesicles, possibly exosomes, can transfer the HIV coreceptor,
CCR5, from CHO cells to CD4?T cells that do not carry CCR5,
thus rendering the T cells susceptible to HIV infection. In addition,
exosomes are able to transfer MHC II molecules to follicular DCs,
which are normally void of class II proteins (41). Exosomes de-
rived from B cells have been shown to stimulate CD4?cells in an
Ag-specific manner, although the extent of stimulation is lower
than that achieved using B cells directly (42). It also has been
shown that exosomes produced by DCs pulsed with tumor Ag
peptides were able to stimulate an antitumor response in mice as
efficiently as the DCs themselves (43). However, the ability of the
exosomes carrying a specific class I peptide to stimulate T cell
proliferation in culture required unpulsed DCs, suggesting that
exosomes transfer the MHC I complex to the DCs (29). It also has
been shown that exosomes derived from tumor cells are able to
transfer tumor Ags to DCs, presumably through fusion (44). Re-
cently, two different clinical trials have been initiated using exo-
somes derived from tumor Ag peptide-pulsed DCs (31, 32).
Given the ability of DC-derived exosomes to stimulate immune
responses in culture and in vivo, we were interested in determining
whether exosomes derived from immunosuppressive DCs were
of exosomes in the DTH model. Before footpad injection, a single, large-
scale preparation of exosomes derived from BM-DCs that were transduced
with Ad.vIL-10 (A) or treated with recombinant murine IL-10 (B) were
diluted into parallel experimental groups. One group was preincubated with
either anti-MHC II or anti-NK1.1 paramagnetic beads, and another group
was disrupted by a series of freeze/thaw cycles. The effect of the exosome
fractions on footpad swelling is shown. ?, Significance at p ? 0.01. F/T,
Freeze/thaw;IP, immunoprecipitation; rmIL-10, recombinant murine IL-10.
MHC II depletion abrogates the immunosuppressive effect
tion of onset of murine CIA model. Exosomes were isolated from DBA1
mouse bone marrow that was infected with Ad-vIL-10. The purified exo-
somes were injected i.v. at day 28 (arrow indicates) into the DBA1 mice,
which were immunized with bovine type II collagen. Mice were monitored
periodically by an established macroscopic scoring system. The macro-
scopic score was expressed as a cumulative value for all paws, with a
maximum possible score of 16.
Analysis of exosomes derived from DC/vIL-10 in preven-
6445 The Journal of Immunology
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able to suppress inflammation. In this report, we have demon-
strated the ability of exosomes derived from DCs either expressing
vIL-10 or treated with recombinant murine IL-10 to suppress paw
inflammation in a murine DTH model. Interestingly, the injection
of DC-derived exosomes as well as DCs was able to suppress
inflammation in both the injected footpad and the untreated con-
tralateral footpad. These results are similar to our previous exper-
iments in which injection of an adenovirus expressing vIL-10 into
one hind footpad resulted in the treatment of both the injected and
the untreated contralateral footpad (5). This observation suggests
that both DCs and DC-derived exosomes are involved in confer-
ring the observed contralateral effect. Consistent with this obser-
vation is the fact that we have detected luciferase-positive mi-
crovesicles in the synovial fluid in both injected and contralateral
rabbit knee joints following intra-articular injection of Ad.Luc.
It is important to note that we have demonstrated that intact
exosomes are required for conferring the therapeutic effects in the
DTH model. Treating exosomes derived from DCs infected with
Ad.vIL-10 or from DCs treated with recombinant murine IL-10
with three cycles of freeze/thaw resulted in the complete loss of the
therapeutic effect in not only the DTH model but also in estab-
lished CIA. This result suggests that the observed therapeutic ef-
fect in the DTH and CIA models requires intact particles rather
than only membranes. Similarly, the observed suppression of in-
flammation or autoimmunity by the exosome preparation could be
abrogated by sonication (S. H. Kim, N. Bianco, and P. D. Robbins,
unpublished material), consistent with intact vesicles being impor-
tant for mediating the biological effects. The results with treatment
of DCs with rIL-10 also demonstrate that it is not necessary to
infect DCs with adenoviral vectors to generate immunosuppressive
exosomes. Thus, the observed therapeutic effects are not due to
contaminating adenovirus in the exosome preparation, a possibility
that was unlikely based on the fact that at least 105PFU of Ad-
.vIL-10 are required for a therapeutic effect in the DTH model. The
ability to generate immunosuppressive exosomes from DCs with-
out the need for gene transfer should facilitate their clinical
In addition to suppressing DTH, our exosome preparations were
able to suppress the onset and severity of CIA as well as partially
reverse established disease. We demonstrated that a single injec-
tion of exo/Ad.vIL-10 or exo/rIL-10 conferred these effects over
an extended period. Of all the different gene- and cell-based ther-
apies currently being tested in murine CIA, systemic injection of
exosomes appears to be the most effective. In fact, recent results
suggest that exosomes derived from DCs genetically modified to
express a membrane-bound IL-4 completely reverse established
disease with disease-free status maintained for an extended period
(S. H. Kim, N. Bianco, A. Morelli, and P. D. Robbins, unpublished
material). However, it is still unclear whether treatment with spe-
cific DC-derived exosomes results in tolerance induction to spe-
cific Ags. Interestingly, control exosomes derived from DC control
cells also were able to confer a weak therapeutic effect, suggesting
that optimizing the growth conditions of DCs could result in the
generation of immunosuppressive exosomes to treat arthritis. Un-
like DCs, which can undergo phenotypic changes following injec-
tion, exosomes appear to be static, presumably reflecting the phe-
notype of the DCs at the time of release. Thus, the use of exosomes
derived from DCs made to be immunosuppressive may be safer
and more effective than the use of modified DCs.
In preliminary experiments, the ability of both DCs and exo-
somes to suppress the DTH response appears to be MHC II de-
pendent, but MHC I independent as demonstrated using DCs, ge-
netically modified to express FasL, and exosomes from class I- and
II-deficient mice (S. H. Kim, N. Bianco, A. Morelli, and P. D.
Robbins, unpublished material). These data were confirmed by the
observation that syngeneic, but not allogeneic, DC-derived exo-
somes were able to suppress the DTH response. We also have
shown that exosomes from FasL-expressing DCs pulsed with spe-
cific Ag, but not unrelated Ag, were able to suppress the inflam-
matory responses, suggesting that exosomes confer Ag-specific
suppression (S. H. Kim, N. Bianco, A. Morelli, and P. D. Robbins,
DC/IL-10 in an established CIA model. Exosomes were isolated from
DBA1 mouse bone marrow DCs that were infected with either Ad.vIL-10
or pulsed with recombinant mouse IL-10 (A). Exosomes from rIL-10-
pulsed DCs were divided into two groups, and one of them was subjected
for three cycles of freeze and thaw to disrupt the membrane (B). Exosomes
from DC treated with recombinant murine IL-10 were tested in the estab-
lished CIA mouse compared with direct injection of recombinant murine
IL-10 (C). The purified exosomes were injected i.v. at day 32 (as indicated
by the arrow) into the DBA1 mice, which were immunized with bovine
type II collagen and given LPS at day 28. Mice were monitored periodi-
cally by an established macroscopic scoring system expressed as a cumu-
lative value for all paws, with a maximum possible score of 16. rmIL-10,
Recombinant murine IL-10; F/T, freeze/thaw.
Analysis of the therapeutic effect of exosomes derived from
6446DC-DERIVED EXOSOMES ARE IMMUNOSUPPRESSIVE IN VIVO
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The mechanism(s) through which exosomes function to sup-
press DTH as well as CIA is unclear. Presumably the DCs ex-
pressing vIL-10 are able to regulate T cell responses directly by the
down-regulation of costimulatory molecules such as B7.1 and
B7.2. However, exosomes are less effective in the regulation of T
cells responses in vitro compared with DCs, suggesting that a dif-
ferent, indirect mechanism may be at work in vivo. It is possible
that exosomes are able to bind and perhaps fuse with endogenous
cells, macrophages, or APC, to modulate their activity. However,
a recent report suggests that DC-derived exosomes are internalized
into macrophages and DCs (45). It is also possible that the exo-
somes function at several levels such as fusion and direct interac-
tion with T cells. Interestingly, exosomes from DCs expressing
vIL-10 showed decreased levels of heat shock protein Hsc70 (see
Fig. 4B). Hsp70 is known to convert T cell tolerance to autoim-
munity in vivo (46), and Hsc70 showed accumulative expression
in RA synovial tissue (47).
Recently, we have initiated adoptive transfer experiments to ex-
amine the mechanism of action of the DC-derived exosomes. In
these experiments, different cell populations from spleens and
lymph nodes of mice treated with DCs or DC-derived exosomes
were transferred to mice before injection of the Ag used to induce
a DTH response. In preliminary experiments, both T cells and DCs
from exosome-treated mice were able to suppress significantly the
DTH response following injection into the footpad. This suggests
that DC-derived exosomes are able to modulate, either directly or
indirectly, the activity of both endogenous DCs and T cells, ren-
dering them able to confer anti-inflammatory effects. Taken to-
gether, these results imply that DC-derived exosomes suppress in-
flammation and autoimmunity through a class II-dependent
pathway in an Ag-specific manner by modulating the activity of
both endogenous T cells and APCs. The strong immunosuppres-
sive effects conferred by exosomes suggest that they could have a
significant therapeutic effect in clinical studies.
We thank Dr. M. Hitchens for helpful comment on the manuscript, and
Ana Bursick for technical assistance with the electron microscope.
P. D. Robbins is a member of the Scientific Advisory Boards for Tissue-
gene, Inc., and Orthogen AG, which are developing biological therapies for
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