Download Production Which Suppress T Cells through Adenosine Cancer

Cancer Exosomes Express CD39 and CD73,
Which Suppress T Cells through Adenosine
Production
This information is current as
of June 17, 2017.
Aled Clayton, Saly Al-Taei, Jason Webber, Malcolm D.
Mason and Zsuzsanna Tabi
J Immunol published online 15 June 2011
http://www.jimmunol.org/content/early/2011/06/15/jimmun
ol.1003884
http://www.jimmunol.org/content/suppl/2011/06/15/jimmunol.100388
4.DC1
Subscription
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Permissions
Email Alerts
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Supplementary
Material
Published June 15, 2011, doi:10.4049/jimmunol.1003884
The Journal of Immunology
Cancer Exosomes Express CD39 and CD73, Which Suppress
T Cells through Adenosine Production
Aled Clayton, Saly Al-Taei, Jason Webber, Malcolm D. Mason, and Zsuzsanna Tabi
A
denosine present in the extracellular environment is a
potent immune regulatory factor protecting cells and
tissues from excessive immune-mediated damage. This
role has been highlighted by numerous pathology models including
colitis (1), diabetes (2), asthma (3), sepsis (4), and ischemic injury
(5, 6). Adenosine also represses immune responses in the tumor
microenvironment, assisting tumor immune evasion (7, 8). The
mechanisms involved in adenosine formation and subsequent signaling are therefore of considerable therapeutic interest (9).
Adenosine is formed by ATP metabolism and accumulates in the
tumor microenvironment by a combination of processes. For example, adenosine is exported from the cell by various nucleoside
transport proteins (10). Its extracellular degradation, mediated by
adenosine deaminase, may be impaired in cancer, partly through
downregulation of CD26, which binds adenosine deaminase to the
cell surface (11). However, the dominant pathway leading to high
extracellular adenosine is by the action of membrane associated
ectoenzymes that produce adenosine by phosphohydrolysis of
ATP or ADP released from dead/dying cells. This aspect of adenosine generation has received much attention in recent years
(12–15).
Department of Pharmacology, Radiology and Oncology, School of Medicine, Cardiff
University, Velindre Cancer Centre, Whitchurch, Cardiff CF14 2TL, United Kingdom
Received for publication November 24, 2010. Accepted for publication May 10,
2011.
This work was supported by grants from Cancer Research Wales and the British Lung
Foundation.
Address correspondence and reprint requests to Dr. Aled Clayton, School of Medicine, Cardiff University, Velindre Cancer Centre, Whitchurch, Cardiff CF14 2TL,
United Kingdom. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: APCP, a,b-methylene-ADP; MES, 2-(N-morpholino)
ethanesulfonic acid; POM-1, sodium metatungstate; Treg, regulatory T cells.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003884
The sequential activities of a pair of molecules catalyze the twostep process to produce adenosine extracellularly. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) converts extracellular ATP (or ADP) to 59-AMP. Thereafter, CD73 (59-nucleotidase) converts 59AMP to adenosine. Natural regulatory T cells
of mice express both CD39 and CD73, whose coordinated enzymatic actions produce adenosine as a mechanism for immune
suppression that is independent of IL-10 and TGF-b (12, 13).
Several cancer cell types also express CD73 (14, 15) and CD39
(15) suggesting cancers can produce adenosine from extracellular
substrate directly, without the requirement of nucleotidases expressed by infiltrating cells or by endothelial cells. In murine
cancer models, targeting cancer cell CD73 inhibits tumor growth
not only by direct inhibition of tumor cell migration and metastasis (16) but also by freeing antitumor T cells from the suppressive effects of extracellular adenosine (15, 17).
Adenosine receptors (of four known subtypes A1, A2A, A2B, and
A3) are expressed broadly by leukocytes such as neutrophils and
macrophages (expressing all four receptors), B lymphocytes (A2A),
and T cells (A2A, A2B, and A3). These primarily drive anti-inflammatory effects such as reduced TNF-a, IL-1b, IL-2, IFN-g,
and elevated IL-10 secretion (5, 18–21), although the precise
cellular response can be influenced by other factors (22). The predominant adenosine receptor expressed by T cells is the highaffinity A2A receptor, which becomes elevated after cell activation
(23). This induction may render activated T cells more sensitive to
adenosine-mediated suppression when they migrate into the tumor
microenvironment. Signals emanating from A2A trigger intracellular cAMP production (23) and increase Src-homology region 2
domain-containing phosphatase-2, dephosphorylating STAT-5 (24).
This pathway can inhibit responses to IL-2 and TCR ligation
providing a potent mode for attenuating multiple T cell effector
functions (19, 25). The same receptor can attenuate NK cell cytokine production and cytotoxic activity (26, 27), and promote the
generation of regulatory T cells (Treg) (28). Adenosine also
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from
extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence
modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 59AMPphosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity
were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total
ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosomegenerated adenosine can trigger a cAMP response in adenosine A2A receptor-positive but not A2A receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or
ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28
cross-linking could be inhibited by exogenously added 59AMP in a CD73-dependent manner. However, 59AMP converted to
adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated
through the adenosine A2A receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular
adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment. The Journal of
Immunology, 2011, 187: 000–000.
2
CANCER EXOSOMES GENERATE IMMUNE-SUPPRESSING ADENOSINE
impairs maturation and function of dendritic cells, but these effects are mediated principally through the A2B receptor (29).
We recently showed that CD73 is expressed by nanometer-sized
vesicles called exosomes, secreted by human bladder cancer cells
(30). In this report, we address the hypothesis that cancer cellderived exosomes may use CD73 or other expressed phosphatases
such as CD39 to participate in extracellular adenosine production.
Our data reveal ATP- and 59AMP-hydrolytic activities are associated with exosomes isolated from several cancer cell lines or
from malignant effusions of mesothelioma patients. This exosomally associated enzyme activity accounted for ∼20% of the total
ATP-hydrolytic activity within freshly obtained pleural fluid. Such
enzyme activity leads to formation of adenosine, which in turn
may contribute toward negative regulation of T cell function. We
propose this mechanism may have broad implications for attenuation of effector immune responses in cancer and in other settings.
Materials and Methods
Cell culture
Collection of pleural fluid
Pleural fluid was collected from patients with malignant pleural mesothelioma into sterile containers at the patients’ own homes. The collections
took 2–24 h depending on the volume present in the patient. Samples were
kept at room temperature during collection and during transport to the
laboratory, which took 20–30 min. Fluid was subjected to centrifugation
(300 3 g, 10 min, twice) to remove cells and the supernatants processed
for exosome purification as described later. In some experiments, cells and
thereafter exosomes were removed from the fluid by centrifugation, and
the impact of this on ATP-hydrolytic capacity of the pleural fluid was
measured. Ethical approval for obtaining patient-derived materials or
blood from healthy donors was obtained from the South East Wales Research Ethics Committee.
Exosome purification
Cell culture medium was subjected to serial centrifugation, removing cells
(300 3 g, 10 min) and removing noncellular debris (2000 3 g for 15 min).
The supernatant was then centrifuged at 10,000 3 g for 30 min, and the
supernatant was retained. This was underlayed with a 30% sucrose/D2O
cushion and subjected to ultracentrifugation at 100,000 3 g for 2 h. Sucrose and D2O was purchased from Sigma (Dorset, U.K.). The cushion was
collected and exosomes washed in PBS (32, 34, 35). The same process was
used for isolating exosomes from pleural fluid specimens. Exosome pellets
were resuspended in 100–150 ml PBS and frozen at 280oC. The quantity
of exosomes was determined by the micro BCA protein assay (Pierce/
Thermo Scientific, Cramlington, U.K.).
Determining exosome density
To quantify the density of exosomes, we used a protocol similar to that
previously described, based on ultracentrifugation on a linear sucrose
gradient (36). Briefly, cell culture medium was precleared of debris by 30
min centrifugation at 10,000 3 g. The supernatant was subjected to ultracentrifugation at 100,000 3 g to pellet exosomes, and the pellet was
Measuring ATP-hydrolytic activity in exosome preparations
The ApoGlow kit (Lonza), was used to measure ATP-hydrolysis activity
associated with exosomes. In brief, exosomes were incubated at room
temperature with typically 1000–4000 nM ATP (Sigma), and after incubations (5–60 min), Nucleotide Monitoring Reagent (containing luciferin)
was added. The plate was gently shaken for 1 min, and light was measured
(1 s, integrated) using a Wallac Victor-2 Multilabel Counter (PerkinElmer,
Cambridge, U.K.). Values were compared with a standard curve to quantify ATP levels. The sensitivity of the assay was 10–20 nM ATP. The CD39
inhibitor sodium metatungstate (POM-1) (38) (Tocris Bioscience, Bristol,
U.K.) was found to interfere with this luciferase assay, hence production of
inorganic phosphate from ATP was measured by the malachite green assay
(described later).
Immuno-affinity depletion of exosomes
To examine the effect CD39- or CD73-positive exosomes on ATP hydrolysis, we incubated purified exosomes (10 mg/sample) with primary Ab (10
mg/sample) (BioLegend, Cambridge, U.K.) in a final volume of 50 ml in
PBS. After overnight incubation, rolling at 4˚C, 1 ml PBS was added and
exosomes pelleted at 100,000 3 g for 40 min. Pellets were resuspended in
10 ml PBS, prior to adding 30 ml protein G-coated Dynal beads (Invitrogen, Paisley, U.K.). Samples were shaken at room temperature for 3 h,
and beads were pelleted using a magnet. The ATP-hydrolytic activity associated with the bead-free supernatants or in association with the beads
was measured as above after 60-min incubation with ATP substrate (2000
nM). As a positive control, an Ab against CD9 was used (R&D Systems,
Abingdon, U.K.). As a control for nonspecific binding, we used the same
dose of IgG1 isotype-matched Ab (BioLegend), or no Ab.
Measuring 59AMP-hydrolytic activity in exosome preparations
The malachite green colorimetric assay was used to quantify the production
of inorganic phosphate from ATP or 59AMP substrate (Sigma) using the
SensoLyte kit (AnaSpec, Fremont, CA). Exosomes that had been previously prepared and frozen in PBS required washing to remove this
phosphate that would significantly interfere with the assay. This was done
by adding excess volume of MES buffer (0.025 M MES, 125 mM NaCl)
and re-pelleting exosomes by ultracentrifugation. 59AMP diluted in MES
buffer at doses up to 500 mM was added to specified exosome doses, and
after incubation at room temperature for 30 min, the copper-based colorimetric reagent was added and absorbance measured at 660 nm after 5-min
gentle shaking. Values were compared with a standard curve to quantify
phosphate levels. The sensitivity of the assay was 0.39–0.78 mM phosphate. The CD73 inhibitor a,b-methylene-ADP (APCP; Sigma) was used
in some assays.
Measuring cAMP production in response to adenosine
An adenosine A2A receptor-overexpressing HEK293 cell line (CB-80200261) was obtained from Codex Biosolutions (Montgomery Village, MD)
and confirmed by real-time quantitative PCR to express ∼70-fold the A2A
receptor mRNA levels of the parent cell (data not shown). The cells were
cultured to 70–80% confluence in DMEM/10% FBS in 6-well plates and
incubated with 50 mM of a cAMP phosphodiesterase inhibitor [4-(3butoxy-4-methoxy-benzyl)imidazolidin-2-one; Sigma] 30 min prior to addition of adenosine or other stimuli. After 20 min incubation at 37oC, the
cells were lysed on ice and cAMP levels determined using the reagents of
and the manufacturer’s protocol for the Parameter cAMP immunoassay kit
(R&D Systems). Similarly, the Jurkat T cell line (ECACC, Salisbury, U.K.)
was also used in some cAMP assays.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Human cancer cell lines, as a source of exosomes, included bladder-HT1376
and colorectal-CACO2 (from Cancer Research-UK, London, U.K.), prostate-DU145 and PC3 (from American Type Culture Collection, Teddington, U.K.), and the breast cancer line MCF7 (from ECACC, Salisbury,
U.K.). All purchased cells were obtained during 2008–2009 and maintained for ,6 mo in culture. Lines were authenticated by the supplier cell
bank by combinations of cytogenetic, isoenzyme, and DNA-profile analyses. A mesothelioma cell line (Meso) was developed in the department
from pleural effusions (31, 32). The HT1376 cells were maintained in
DMEM supplemented with nonessential amino acids, penicillin, streptomycin, and 5% FBS (which had been depleted of exosomes by overnight
ultracentrifugation at 100,000 3 g). All other cells were maintained in
RPMI 1640 with the same supplements excluding nonessential amino
acids. The cells were seeded into bioreactor flasks (Integra Biosciences,
SLS, Nottingham, U.K.) and maintained at high-density culture for exosome production as described (33). Cells were confirmed negative for
mycoplasma contamination by monthly screening (Mycoalert; Lonza,
Slough, U.K.).
resuspended in 300 ml PBS. The preparation was overlaid on a continuous
sucrose gradient (0.2 M sucrose to 2.5 M sucrose). Specimens were
centrifuged at 4oC, overnight at 210,000 3 g, using an MLS-50 rotor in an
Optima-Max ultracentrifuge (Beckman Coulter). Fractions of 300 ml were
collected (from the top of the tube), and the refractive index (at 20oC) was
measured using an automatic refractometer (J57WR-SV; Rudolph Research
Analytical, Hackettstown, NJ). This instrument is subject to weekly 2-point
calibration (with H2O and calibration standard certified as 1.56999 6
0.0001 at 20oC by Cargille Laboratories, Cedar Grove, NJ). The density
(g/ml) of each fraction was calculated from the published table in the
Beckman Ultracentrifugation catalogue as described (37). Each fraction
was washed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.025
M MES, 125 mM NaCl), and subjected to 150,000 3 g ultracentrifugation.
After removing all supernatant, pelleted material was resuspended in 30 ml
MES buffer. This was split 1/3 for hydrolysis activity assays and 2/3 for
Western blotting as described (30).
The Journal of Immunology
3
T cell purification and analyses
PBMCs of healthy donors were isolated by centrifugation on a Histopaque
gradient (Sigma). Flow cytometric analysis of PBMCs was performed with
a FACSCanto cytometer running FACSDiva version 6.1.2 software (Becton
Dickinson, Oxford, U.K.). Compensation was performed for each Ab
panel using FACSComp Beads (Becton Dickinson). Conjugated Abs used
included CD39–allophycocyanin (BioLegend), Foxp3–FITC, CD24–PE–
Cy5, CD3–FITC (eBioscience, Hatfield, U.K.), CD73–PE, CD8–PE–Cy7,
and CD4–allophycocyanin–Cy7 (Becton Dickinson). CD3+ T cells were
purified using immuno-magnetic beads (Miltenyi Biotec, Surrey, U.K.),
according to the manufacturer’s protocols. For functional analyses, purified T cells (105 cells/well) were activated using CD3/CD28 Ab-coated
beads (Invitrogen), at a bead to cell ratio of 2:1. Doses of 59AMP (0 to 50
mM) were added at the same time. For some treatments, the 59AMP
substrate was pretreated with exosomes (at concentrations of 100–150 mg/
ml) for 1 h and the exosomes removed by ultracentrifugation (150,000 3
g, 1 h) in an Optima-Max ultracentrifuge in a TLA110 rotor. The supernatant above the exosome pellet was carefully removed and used as
“exosome-modified 59AMP.” At day 3, proliferation was assessed by
pulsing cells with [3H]thymidine as described (32), and cytokines (IL-2
and TNF-a) were quantified using DuoSet ELISA, following the manufacturer’s protocol (R&D Systems). The adenosine A2A receptor antagonist SCH 58261 (Sigma) was used in some assays.
Comparisons between treatments was made using one-way ANOVA with
Tukey’s multiple comparison test or using two-way ANOVA with Bonferroni posttests. The p values ,0.05 were considered significant. These
were calculated using Prism-4 (version 4.03) graphing and statistical
software (GraphPad, San Diego, CA).
Results
Cancer exosomes exhibit ATP-hydrolytic activity, which is
partly CD39 dependent
For our first approach, we analyzed exosomes from HT1376
bladder cancer cells and took advantage of the natural property of
exosomal vesicles to float at characteristic densities when subjected
to ultracentrifugation on continuous sucrose gradients (0.2–2.5 M
sucrose range). Classically, exosomes float at densities of ∼1.1–
1.2g/ml, and this property is a useful analytical tool for discriminating exosomal from non-exosomal factors secreted by cancer
cells. Fractions collected from the gradients were subjected to
refractometry to quantify the density, and the capacity of each
fraction to hydrolyze ATP was measured using a luciferase-based
ATP assay. Fractions at densities of ,1.1 g/ml showed no loss in
the starting level of ATP after 30 min incubation. However, there
was almost a total loss in ATP, reaching the detection limits of the
assay, with fractions of 1.1–1.2 g/ml density. Fractions of densi
ties .1.2 g/ml contained some ATP-hydrolytic activity, but this
diminished with increasing density (Fig. 1A). The presence of
TSG101 protein, commonly used as a marker to indicate a multivesicular body origin of vesicles (i.e., exosomes), coincided in
fractions harboring ATP-hydrolytic function. Similarly, CD39 was
also detected within the fractions bearing ATP-hydrolytic capacity
(Fig. 1A, Western blot).
We next examined if this activity was present in exosomes
isolated from other cell types. Exosomes were purified using the
30% sucrose/D2O cushion method, and after quantification by
protein assay, the ATP-hydrolytic activity was measured as described above as a function of exosome dose. Whereas hydrolysis
of ATP was clearly demonstrable in exosomes from various cell
types, there were considerable differences in the level of activity
among the cellular sources (Fig. 1B). The breast cancer cell line
MCF7 demonstrated very strong hydrolysis of ATP, with almost
complete elimination (99% decrease) of ATP levels after 30-min
incubation using only 5 mg of exosomes/well. In contrast, exosomes isolated from a mesothelioma cell line showed weak ATP-
FIGURE 1. Cancer exosomes can hydrolyze exogenous ATP. A, The
pellet formed from 100,000 3 g centrifugation of HT1376 cell culture
medium was overlaid on a continuous sucrose gradient and subjected to
further ultracentrifugation for 20 h. The density of collected fractions was
determined, prior to incubation for 30 min at room temperature with ATP
(4000 nM). The levels of ATP remaining were quantified and plotted
against fraction density. A proportion of collected fractions was analyzed
by immunoblot for TSG101 and CD39, revealing the fractions that contain
CD39-positive exosomes (A, inset panels). The data are representative of
four such gradient isolations. B, Exosomes (5–20 mg/well) purified by
sucrose/D2O cushion from assorted cancer cell types were incubated with
ATP (2000 nM) for 30 min at room temperature. Graph represents percent
hydrolysis of substrate against exosome dose. C, To examine the role of
CD39 in ATP hydrolysis, exosomes (1 or 10 mg/well) were incubated with
ATP (50 mM) 30 min at room temperature with increasing doses of POM-1
(0–50 mM). Levels of inorganic phosphate produced were quantified using
the malachite green assay. Symbols represent mean 6 SEM of triplicates.
*p , 0.05, ***p , 0.001 (one-way ANOVA with Tukey’s multiple
comparison test). D, To determine the association between CD39-positive
exosomes and ATP-hydrolytic capacity, HT1376 exosomes (10 mg/sample)
were incubated with Abs (10 mg) against CD39, CD73, or CD9 as indicated and subjected to immuno-affinity depletion using protein G Dynal
beads. The ATP-hydrolytic activity associated with the supernatant (left) or
bead-associated (right) fractions was measured as above. Anti-CD9 was
used as a positive control for efficient immuno-depletion of exosomes, and
an irrelevant isotype-matched control Ab or no Ab was used to indicate
degree of nonspecific binding to beads. Bars show mean 6 SD of duplicates.
hydrolytic activity, with only a 2% decrease in ATP levels under
the same conditions. CD39 was present in the fractions containing
peak hydrolytic activity (Fig. 1A), suggesting it may play a role in
this reaction. We briefly examined the relative levels of CD39 in
cell lysates and exosomes by Western blot (Supplemental Fig. 1).
In the three cell lines examined, a band for CD39 was detectable
within cell lysates. In comparison, exosomes revealed detectable
but relatively weak staining for CD39. Given this weak staining, it
was not readily possible to correlate the level of ATP hydrolysis
with CD39 expression levels by exosomes, and because cell surface CD39 measured by flow cytometry (Supplemental Fig. 1,
bars) was broadly comparable, this could not account for the
differences seen in exosomal ATP-hydrolytic activity (Fig. 1B). To
address possible involvement of CD39 in ATP hydrolysis, sucrose
cushion-purified exosomes were assessed for ATP hydrolysis in
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Statistics
4
CANCER EXOSOMES GENERATE IMMUNE-SUPPRESSING ADENOSINE
Cancer exosomes exhibit 59AMP-hydrolytic activity, which is
CD73 dependent
We also performed similar experiments to investigate whether
exosomes exhibit 59AMP-hydrolytic activity. Fractions from continuous sucrose gradients were assessed for their capacity to
produce inorganic phosphate from exogenously added 59AMP
substrate. This was measured colorimetrically using the malachite
green assay after 30-min incubation of each fraction with 59AMP.
This yielded similar results as for ATP hydrolysis, with the
59AMP-hydrolytic activity focused to fractions of classical exosomal density, with little detectable at densities ,1.1 g/ml or
.1.22 g/ml (Fig. 2A).
We also observed differences in hydrolysis of 59AMP between
different exosome sources (Fig. 2B). Those exosomes that exhibited strong ATP hydrolysis (e.g., CACO2) did not necessarily
harbor strong 59AMP activity, and similarly DU145 exosomes,
which were potent at 59AMP hydrolysis, were among the poorest
at ATP hydrolysis (Figs. 1B, 2B). Nevertheless, all cancer exosomes tested to date exhibit some level of ATP and 59AMP hydrolytic activity.
We investigated if CD73 was the principal molecule mediating
59AMP hydrolysis. HT1376 exosomes were incubated with increasing doses of 59AMP in the absence or presence of the CD73specific inhibitor APCP. The lowest dose of APCP (10 mM) was
sufficient to attenuate phosphate generation by exosomes by
.75% (at 500 mM 59AMP dose), and increasing the dose of
APCP 20-fold only slightly inhibited this further (83% inhibition
at 500 mM 59AMP) (Fig. 2C). The data show CD73 to be responsible principally for the exosomal 59AMP-hydrolytic activity.
FIGURE 2. Cancer exosomes can hydrolyze exogenous 59AMP. A, The
sucrose gradient method was used to examine hydrolysis of exogenously
added 59AMP (100 mM). Levels of inorganic phosphate were quantified
after 30 min at room temperature and plotted against fraction density. B,
Exosomes (5–20 mg/well) purified by sucrose/D2O cushion from assorted
cancer cell types were incubated with 59AMP (50 mM) for 30 min at room
temperature. Graph represents percent hydrolysis of substrate against
exosome dose. C, The capacity of HT1376 exosomes to hydrolyze 59AMP
in the presence of the CD73 inhibitor APCP was almost entirely abrogated,
demonstrating CD73 as a principal mediator of 59AMP hydrolysis by
exosomes. Symbols represent mean 6 SEM of triplicates. ***p , 0.001 at
each inhibitor dose (two-way ANOVA with Bonferroni post tests).
In summary, we show that cancer exosomes exhibit striking ATP
and 59AMP hydrolytic activities involving exosomal CD39 and
CD73, respectively.
Exosomes in malignant effusions contribute to ATP-hydrolytic
function
To examine the possible physiological relevance of ATP hydrolysis
by exosomes, we performed some analyses of pleural fluid taken
from patients with pleural malignant mesothelioma. This fluid is
a tumor effusion previously described by us and others as containing exosomes (31, 39), and although it does not represent the
tumor microenvironment per se, it does reflect an in vivo tumorconditioned environment.
We first isolated exosomes from pleural fluid of three donors and
examined the ATP- hydrolytic activity of such exosomes, comparing directly with exosomes from the HT1376 cell line (Fig. 3A).
Data reveal exosomes isolated from this ex vivo source do exhibit
ATP-hydrolytic activity that is generally comparable with if not
more potent than that of HT1376-derived exosomes. The physiological levels of exosomes within this fluid are ∼3 to 4 mg/ml
(Supplemental Fig. 2A). Although this is lower than the exosome
doses used in these assays, we can confirm that ATP substrate is
significantly hydrolyzed given sufficient time of incubation with
low-dose exosomes (Supplemental Fig. 2B).
We next examined what proportion of the total ATP-hydrolytic
activity within the pleural fluid is attributable to the exosomes
therein. A fresh pleural fluid specimen was subject to differential centrifugation. Native (untouched) fluid exhibited strong ATP
hydrolysis, eliminating ∼90% of available substrate (Fig. 3B).
However, by removing the mixed cellular component of the fluid
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
the presence of increasing doses of POM-1, an inhibitor of CD39
(38). For this assay, we measured phosphate production, using the
malachite green assay, as POM-1 had some luciferase-inhibitory
effect. The assay revealed POM-1 inhibition of exosomal ATP
hydrolysis (up to 50% at 50 mM POM-1) indicating CD39 is at
least partly responsible for the hydrolytic activity (Fig. 1C). We
also attempted to examine the effect of eliminating CD39-positive
exosomes on ATP-hydrolytic activity, using immunoaffinity depletion with protein G beads. Using an Ab against the tetraspanin
CD9, which is very strongly expressed by carcinoma exosomes
(A. Clayton and J. Webber, unpublished observations), we achieved ∼90% reduction of ATP hydrolysis in the supernatant,
whereas the corresponding bead-associated material revealed
strong ATP hydrolysis (of .98%), consistent with an efficient
immunoprecipitation of exosomes. In contrast, this level of efficiency was not achieved using several different Abs against CD73
or CD39, an issue related to either Ab affinity or more likely
due to relatively low levels of expression of these molecules on
the exosome surface that make this approach comparatively inefficient. Nevertheless, immuno-depletion with either of these Abs
resulted in reduced ATP hydrolysis (of ∼30% and 20%, respectively), whereas isotype-matched control Ab or control samples
lacking Ab showed no loss in ATP-hydrolytic function (Fig. 1D,
left). There was some general nonspecific binding of exosomes to
the protein G beads, but the ATP-hydrolytic activity related to the
beads was at least twice this background level when using CD39
or CD73 Ab (Fig. 1D, right). The data show ATP hydrolysis
is associated with CD9-, CD73-, and CD39-positive exosomes.
Furthermore, targeting CD39 by chemical inhibition or immunodepletion methods is not sufficient to eliminate fully this ATPhydrolytic activity. This highlights the possibility that additional
exosomal constituents may also exhibit ATP-hydrolytic activity in
a CD39-independent manner.
The Journal of Immunology
5
FIGURE 3. Exosomes in malignant effusions contribute to ATP hydrolytic activity. A, Pleural fluid exosomes (10 mg/well) from three donors
were purified by sucrose cushion and incubated with ATP (2000 nM) for
30 min at room temperature. For comparison, HT13786 exosomes (10 mg/
well) were also used in this assay. Graph represents percent hydrolysis of
substrate. Bars show mean 6 SEM of triplicates. B, The ATP hydrolytic
activity of fresh pleural fluid was measured before and after centrifugation
to remove cells and thereafter to remove exosomes. Bars show mean 6
SEM of triplicates.
Cancer exosomes convert ATP to adenosine and trigger
intracellular cAMP production
We next examined whether exosomes were capable of converting
exogenous ATP to form adenosine in a sequential manner. To
demonstrate this, we used an HEK293 cell line that overexpresses
the high-affinity adenosine A2A receptor, and we measured an
elevation in the intracellular production of cAMP in cell lysates
after exposure to exosomes and ATP.
The cells responded efficiently to the addition of exogenous
adenosine, used as a positive control, producing readily measurable
intracellular cAMP levels within 20 min of stimulation. In contrast,
adding exogenous ATP at the same doses had no effect on cAMP
levels at all but the highest dose, in which there was a small increase
(from 14.5 pM at 250 nM ATP to 39 pM at 500 nM ATP). At the
same doses, however, ATP became a potent stimulator of a cAMP
response in the presence of exosomes (111 pM at 500 nM ATP),
whereas exosomes alone had only a negligible effect on cAMP (8.8
pM untreated versus 12.9 pM exosomes at 0 nM ATP) (Fig. 4A).
The assay performed on the parent cell, which does not express
A2A receptor, revealed no dose-dependent changes in cAMP with
any treatment (Fig. 4B), confirming this as an A2A receptor-driven
response. To confirm cAMP can also change within T cells, we
performed similar experiments with Jurkat cells, which also express A2A receptor. Addition of ATP substrate (500 nM or 2 mM)
in the absence of exosomes did not significantly elevate cAMP
levels (Fig. 4C). In the presence of exosomes, the cAMP response
was enhanced, but this was absolutely substrate dependent, as
exosomes alone had no cAMP-elevating effect. The data are
consistent with a conversion of exogenous ATP to adenosine,
catalyzed by exosomes, and that exosome-generated adenosine
triggers a cAMP-signaling response in A2A receptor-positive cells.
FIGURE 4. Exosomes form adenosine from ATP and stimulate cAMP
signaling. A and B, Adenosine A2A receptor-overexpressing HEK293 cells
(A) or the untransfected parent cell (B) were incubated for 20 min at 37oC
with doses of ATP (0 to 500 nM), or ATP with 120 mg/ml CACO2 exosomes, or with adenosine as a positive control. Cells were lysed on ice, and
levels of intracellular cAMP were measured. Symbols represent mean 6
SD of duplicates. ***p , 0.001 (two-way ANOVA with Bonferroni
posttests). C, Similarly, Jurkat cells were treated with ATP (at 500 nm or
2000 nM) in the absence or presence of 100 mg/ml HT1376 exosomes or
with 2000 nM adenosine as a positive control, and cAMP levels were
assessed as above. Bar shows mean 6 SEM of triplicates. **p , 0.01,
***p , 0.001 (one-way ANOVA with Tukey’s multiple comparison test).
A2AR, A2Areceptor.
Cancer exosomes inhibit T cell functions by adenosine
production
We next examined if exosomally produced adenosine had a functional impact on T cells. We used multicolor flow cytometry to
examine the constitutive expression of CD39 and CD73 by human
PBMCs (Fig. 5A). The data show that unlike mice, the expression
of CD39 and CD73 is not particularly limited to human Treg.
Approximately 20% of T cells are positive for these enzymes.
While the CD4+ subset had an equal proportion of CD39 and
CD73 single-positive cells, there was a clear bias in the CD8+
subset for CD73 single-positive cells. Similarly, there was a bias
within the Treg population for CD39 single-positive cells. The
proportion of double-positive cells in any population was ,2%
(average of five healthy donors) (Fig. 5B, bar graph). Circulating
T cells in healthy donors therefore do express significant levels
of nucleotidase that could participate in extracellular adenosine
production.
To test this, purified CD3+ T cells were incubated with increasing doses of 59AMP in the absence or presence of the CD73
inhibitor APCP. T cells were activated by adding anti-CD3/CD28
Ab-coated beads, and proliferation and cytokine (IL-2 and TNF-a)
production was measured at day 3. The T cell response was attenuated in a dose-dependent manner when adding 59AMP as
a substrate. As expected, incubations in the presence of 10 mM
APCP abrogated this response, restoring full activation (Fig. 5C).
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
(by centrifugation twice at 300 3 g), the activity remaining within
the supernatant was drastically reduced to ∼20% ATP hydrolysis,
indicating that it is cells present within this environment that drive
the majority of hydrolytic activity. The residual activity present
in the cell-free supernatant was entirely eliminated after ultracentrifugation at 100,000 3 g to pellet exosomes (Fig. 3B). Taken
together, the data demonstrate that although exosomes are not
the principal source of enzymes driving ATP hydrolysis, they are
nevertheless a factor contributing toward the total activity within
human pleural effusions.
6
CANCER EXOSOMES GENERATE IMMUNE-SUPPRESSING ADENOSINE
Discussion
FIGURE 5. Exosomes impair T cell functions by adenosine production.
A and B, PBMCs from a healthy donor were analyzed by multicolor flow
cytometry for expression of CD39 and CD73. The dot plots reveal broad
distribution of these nucleotidases constitutively among CD8+ and CD4+
T cell subsets, with no distinctive restriction of these enzymes to the Treg
population. The proportions (%) of CD39- or CD73-positive cells are indicated (A). Bar graph shows the average proportions of single- and
double-positive cells from six donors (B). C, Purified T cells, activated by
CD3/CD28 cross-linking, showed attenuated proliferation and IL-2 and
TNF-a secretion by day 3 in the presence of 59AMP (0–50 mM) (black
bars, left). Addition of the CD73 inhibitor APCP at 10 mM abrogated this
effect (gray bars, left). HT1376 exosomes (100 mg/ml) were added to
59AMP (0–50 mM) and incubated for 1 h at room temperature. Exosomes
were removed by ultracentrifugation (150,000 3 g, 1 h). The supernatants
(exosome-modified substrate) were added to T cells as above in the absence (black bars, right) or presence of APCP (10 mM) (gray bars, right).
Bars represent mean 6 SEM of triplicates. *p , 0.05, **p , 0.01, ***p ,
0.001 (one-way ANOVA with Tukey’s multiple comparison test). D,
Similarly, exosome-modified 59AMP substrate was added to CD3/CD28
activated T cells in the absence or presence of the A2A receptor antagonist
SCH58261 (10 mM). Levels of proliferation and IL-2 at day 3 are shown
relative to 0 mM AMP treatment. Symbols represent mean 6 SEM of four
replicates. *p , 0.05, **p , 0.01, ***p , 0.001 (one-way ANOVA, with
Tukey’s multiple comparison test).
We present in this report a demonstration that exosomes secreted
by cancer cells are capable of dephosphorylating exogenous ATP
and 59AMP to form adenosine. These hydrolytic activities are due
in part to expression of functional CD39 and CD73 by exosomes.
This mechanism may contribute to rising adenosine levels within
the microenvironment and hence participate in attenuating T cell
function. The study highlights these enzymes as relevant factors
for exosome-mediated immune evasion in cancer.
Although some reports describe cancer exosomes having
immune-activating functions, there are also accumulating data
pointing to varied suppressive effects (40). Documented mechanisms include induction of T cell apoptosis (41), TGF-b–mediated
impaired cytolytic functions and Treg induction (32), downregulation of activating receptors like NKG2D (31), and negative
effects also on APCs (42). To date, these mechanisms appear to
require delivery of receptor ligands, growth factors, or microRNA
in a contact- and/or uptake-dependent manner to immune cells.
This report, therefore, presents a novel exosome mechanism that
does not require direct contact with immune cells. We argue that
the expression of catalytically functional membrane-associated
enzymes, such as CD73, may be a particularly important and
fundamental feature of exosomes in general, in which their dissemination into the microenvironment provides a vast surface
area for membrane-tethered enzymes to mediate their functions.
Hence, our report describes such a role for exosomal CD39/CD73
in adenosine generation in the cancerous microenvironment with
negative consequences for immune function.
Blockade of CD73 activity (with APCP) showed strong inhibition, suggesting CD73 accounts for ∼75% of the ability of
exosomes to hydrolyze AMP. It was not possible to impair this
hydrolysis further by increasing the dose of inhibitor. Similarly,
chemical inhibition of CD39 (with POM-1) or CD39 immunodepletion did not fully abrogate ATP hydrolysis. This suggests that
there may be other exosomally associated factors that contribute
to the hydrolysis of these substrates. It is important to note that
several proteomics studies have highlighted that a host of proteins
with NTP-binding and NTP-hydrolyzing domains are expressed
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The data show that human T cells have a constitutive ability to
form adenosine from 59AMP, which impairs their function, and
that CD73 is essential for this to occur. In the same experiments,
we treated 59AMP substrate solutions with cancer exosomes and
incubated for 1 h to allow substrate hydrolysis to occur and for
adenosine to be formed. These samples were then subjected to
robust ultracentrifugation (150,00 3 g for 1 h) to pellet and
remove the exosomes. This step ensured that the assays were
measuring the effect of exosomes on the substrate, rather than
measuring the direct effect of exosomes on T cell functions. The
resultant supernatants were added to T cells in the same manner,
in the absence or presence of APCP, and proliferative/cytokine
responses were measured. We observed similar levels of attenuated function with exosome-treated substrates in the absence of
APCP. By treating T cells with this CD73 inhibitor, however, the
inhibitory effects of the exosome-pretreated substrate remained
(Fig. 5C), which is consistent with a conversion of 59AMP by
exosomes to form adenosine, which attenuates T cell function
independently of T cell CD73. In addition, when exosome-modified 59AMP was added in the presence of an A2A receptor antagonist, T cell IL-2 production was restored, and proliferation
was also restored at all but the highest substrate dose (Fig. 5D).
The data show exosome-generated adenosine can negatively regulate T cell functions.
The Journal of Immunology
these in vitro experiments was higher than that found in the tumorassociated pleural fluid of mesothelioma patients, this approach
allowed us to achieve rapid and almost complete (.95%, data not
shown) substrate conversion to adenosine in ∼1 h of incubation.
This allowed us to add exosome-converted substrate as rapidly as
possible to T cells. We do not know whether the microenvironmental doses of exosomes will ever reach such levels in vivo,
nevertheless physiological exosome doses are capable of ∼60%
substrate conversion in 24 h. Hence in vivo, lower exosome doses,
but present perhaps continuously within this microenvironment,
would likely provide a sufficient source of prolonged ectoenzyme
activity, contributing to rising adenosine levels.
At present, it is difficult to estimate the relative importance
of exosomes compared with other cells or factors contributing
to adenosine production in the tumor microenvironment in vivo.
Using pleural fluid taken freshly from patients with pleural malignant mesothelioma offered an opportunity to begin addressing
this question. Centrifugation to remove the mixed population of
tumor cells and leukocytes present within such tumor effusions
revealed that the majority (80%) of ATP-hydrolytic activity was
associated with these cellular components. Nevertheless, the remainder could be accounted for by eliminating exosomes. At least
in this ex vivo system, therefore, exosomes contribute ∼20% of
the ATP-hydrolytic activity within this tumor-associated environment. We do not yet know if this remains true within the immediate pericellular space of the tumor, as evaluating exosome dose
and function within tumor interstitial fluid remains a significant
challenge.
In summary, these findings implicate the secretion of exosomes
by cancer cells as a mechanism contributing to rising adenosine
levels in the tumor microenvironment. Exosomes may therefore
contribute toward adenosine-mediated attenuation of T cell
functions, even in the absence of direct T cell–exosome interactions. Abrogation of exosome secretion by cancer cells or targeting their ectonucleotidase activities may prove therapeutically
attractive.
Acknowledgments
We thank Dr. J. Welton and Mrs. L. Court for assistance in cell culture maintenance, exosome purifications, and analyses.
Disclosures
The authors have no financial conflicts of interest.
References
1. Naganuma, M., E. B. Wiznerowicz, C. M. Lappas, J. Linden, M. T. Worthington,
and P. B. Ernst. 2006. Cutting edge: critical role for A2A adenosine receptors in
the T cell-mediated regulation of colitis. J. Immunol. 177: 2765–2769.
2. Németh, Z. H., D. Bleich, B. Csóka, P. Pacher, J. G. Mabley, L. Himer,
E. S. Vizi, E. A. Deitch, C. Szabó, B. N. Cronstein, and G. Haskó. 2007.
Adenosine receptor activation ameliorates type 1 diabetes. FASEB J. 21: 2379–
2388.
3. Fan, M., and S. Jamal Mustafa. 2006. Role of adenosine in airway inflammation
in an allergic mouse model of asthma. Int. Immunopharmacol. 6: 36–45.
4. Csóka, B., Z. H. Németh, P. Rosenberger, H. K. Eltzschig, Z. Spolarics,
P. Pacher, Z. Selmeczy, B. Koscsó, L. Himer, E. S. Vizi, et al. 2010. A2B
adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation. J. Immunol. 185: 542–550.
5. Day, Y. J., L. Huang, H. Ye, L. Li, J. Linden, and M. D. Okusa. 2006. Renal
ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma. J. Immunol. 176: 3108–3114.
6. Day, Y. J., M. A. Marshall, L. Huang, M. J. McDuffie, M. D. Okusa, and
J. Linden. 2004. Protection from ischemic liver injury by activation of A2A
adenosine receptors during reperfusion: inhibition of chemokine induction. Am.
J. Physiol. Gastrointest. Liver Physiol. 286: G285–G293.
7. Ohta, A., E. Gorelik, S. J. Prasad, F. Ronchese, D. Lukashev, M. K. Wong,
X. Huang, S. Caldwell, K. Liu, P. Smith, et al. 2006. A2A adenosine receptor
protects tumors from antitumor T cells. Proc. Natl. Acad. Sci. USA 103: 13132–
13137.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
by exosomes of diverse origin (43). It is possible, therefore, that
some of these or other yet unidentified exosomal constituents
account for the remaining hydrolytic activities.
Signaling downstream of the A2A and A2B receptors involves
activation of adenylyl cyclase leading to production of cAMP.
This signaling response was used to demonstrate that exosomes
are capable of sequentially hydrolyzing ATP and thereafter
59AMP to form adenosine. Adding ATP or exosomes alone to
A2A-overexpressing HEK293 cells did not stimulate cAMP production, but when combined they were almost as effective as
matched doses of adenosine in driving cAMP signaling. Similarly,
elevated cAMP could also be triggered by cotreatment with ATP
and exosomes in a T cell line (Jurkat). Extracellular adenosine
generated by cancer exosomes can therefore drive cAMP signaling
in A2A receptor-positive cells.
Adenosine levels within cancerous interstitial fluid measured in
murine tumor models reach micromolar concentrations, particularly in hypoxic regions, and are some 10- to 20-fold the levels
found in noncancerous tissue (44). Such doses are broadly consistent with those used in this and other studies (44, 45) and have
been shown to impart suppressive effects on effector T cells. We
also examined the effects of adenosine generation on T cell
function and showed that 59AMP as substrate was effective at
attenuating T cell proliferative responses and the production of
inflammatory cytokines. This, however, was true even in the absence of any cancer exosome contribution and was dependent
on constitutive CD73 expression by T cells. Chemically blocking
CD73 hydrolytic function abrogated this inhibition. Most recent
studies of T cell adenosine production have focused on the CD4+
CD25hiFoxp3+ natural Treg population. These are double-positive
for CD39 and CD73 in mice (12) and according to one report also
in human (45). Thus, they can drive the conversion of these
substrates to adenosine, as a mechanism of immune suppression
(12). The broad distribution of these nucleotidases by other lymphocyte subsets in humans is known (46) but has only recently
been examined in terms of functional importance (47). Moncrieffe et al. (47) isolated CD39+CD4+Foxp32 T cells from arthritic patients and showed ATP-hydrolytic capacity of this nonTreg population. But these cells did not inhibit proliferation,
probably because of the high levels of IFN-g and IL-17 produced
by these cells (47). Of interest, the patients showed decreased
levels of CD73 expression by CD4+ T cells taken from inflamed
joints compared with circulating cells. This suggests that low
T cell CD73 expression levels support a proinflammatory environment. We also show in this article that CD39 is broadly
expressed by CD4+ and to some degree CD8+ T cells and reveal
a bias of CD8+ T cells for CD73 expression, in agreement with
past studies (48). Few double-positive Treg were present in healthy donors, and numerically, Treg represent only a small proportion of enzyme-positive T cells. Exosomes produced by cancer
cells, however, are double-positive for CD39 and CD73 and are
therefore capable of adenosine production from ATP indepen
dently of contributions to substrate hydrolysis from T cells.
It was difficult to measure the impact of exosomally generated
adenosine on T cell function because of the endogenous capacity of
CD73+ T cells to hydrolyze 59AMP. Blocking T cell CD73 was
effective at attenuating adenosine production from 59AMP, and
this in turn blocked adenosine-driven hyporesponsiveness. Under
such blocking conditions, adding exosome-modified-59AMP substrate inhibited T cell function consistent with this being an
exosome-generated, rather than T cell-generated, adenosine effect.
Furthermore, by removing exosomes from this substrate, the data
underline this immune-suppressive mechanism to be independent
of exosome–T cell contact. Although the exosome dose used in
7
8
CANCER EXOSOMES GENERATE IMMUNE-SUPPRESSING ADENOSINE
28. Zarek, P. E., C. T. Huang, E. R. Lutz, J. Kowalski, M. R. Horton, J. Linden,
C. G. Drake, and J. D. Powell. 2008. A2A receptor signaling promotes peripheral
tolerance by inducing T-cell anergy and the generation of adaptive regulatory
T cells. Blood 111: 251–259.
29. Wilson, J. M., W. G. Ross, O. N. Agbai, R. Frazier, R. A. Figler, J. Rieger,
J. Linden, and P. B. Ernst. 2009. The A2B adenosine receptor impairs the
maturation and immunogenicity of dendritic cells. J. Immunol. 182: 4616–4623.
30. Welton, J. L., S. Khanna, P. J. Giles, P. Brennan, I. A. Brewis, J. Staffurth,
M. D. Mason, and A. Clayton. 2010. Proteomics analysis of bladder cancer
exosomes. Mol. Cell. Proteomics 9: 1324–1338.
31. Clayton, A., J. P. Mitchell, J. Court, S. Linnane, M. D. Mason, and Z. Tabi. 2008.
Human tumor-derived exosomes down-modulate NKG2D expression. J. Immunol. 180: 7249–7258.
32. Clayton, A., J. P. Mitchell, J. Court, M. D. Mason, and Z. Tabi. 2007. Human
tumor-derived exosomes selectively impair lymphocyte responses to interleukin2. Cancer Res. 67: 7458–7466.
33. Mitchell, J. P., J. Court, M. D. Mason, Z. Tabi, and A. Clayton. 2008. Increased
exosome production from tumour cell cultures using the Integra CELLine
Culture System. J. Immunol. Methods 335: 98–105.
34. Andre, F., N. E. Schartz, M. Movassagh, C. Flament, P. Pautier, P. Morice,
C. Pomel, C. Lhomme, B. Escudier, T. Le Chevalier, et al. 2002. Malignant
effusions and immunogenic tumour-derived exosomes. Lancet 360: 295–305.
35. Lamparski, H. G., A. Metha-Damani, J. Y. Yao, S. Patel, D. H. Hsu, C. Ruegg,
and J. B. Le Pecq. 2002. Production and characterization of clinical grade
exosomes derived from dendritic cells. J. Immunol. Methods 270: 211–226.
36. Théry, C., A. Clayton, S. Amigorena, and G. Raposo. 2006. Isolation and
characterization of exosomes from cell culture supernatants and biological fluids.
Curr. Protoc. Cell Biol. Chapter 3, Unit 3.22.
37. Raposo, G., H. W. Nijman, W. Stoorvogel, R. Liejendekker, C. V. Harding, C. J.
M. Melief, and H. J. Geuze. 1996. B lymphocytes secrete antigen-presenting
vesicles. J. Exp. Med. 183: 1161–1172.
38. Reutershan, J., I. Vollmer, S. Stark, R. Wagner, K. C. Ngamsri, and
H. K. Eltzschig. 2009. Adenosine and inflammation: CD39 and CD73 are critical
mediators in LPS-induced PMN trafficking into the lungs. FASEB J. 23: 473–
482.
39. Bard, M. P., J. P. Hegmans, A. Hemmes, T. M. Luider, R. Willemsen,
L.-A. A. Severijnen, J. P. van Meerbeeck, S. A. Burgers, H. C. Hoogsteden,
and B. N. Lambrecht. 2004. Proteomic analysis of exosomes isolated from
human malignant pleural effusions. Am. J. Respir. Cell Mol. Biol. 31: 114–121.
40. Théry, C., M. Ostrowski, and E. Segura. 2009. Membrane vesicles as conveyors
of immune responses. Nat. Rev. Immunol. 9: 581–593.
41. Taylor, D. D., and C. Gerçel-Taylor. 2005. Tumour-derived exosomes and their
role in cancer-associated T-cell signalling defects. Br. J. Cancer 92: 305–311.
42. Yu, S., C. Liu, K. Su, J. Wang, Y. Liu, L. Zhang, C. Li, Y. Cong, R. Kimberly,
W. E. Grizzle, et al. 2007. Tumor exosomes inhibit differentiation of bone
marrow dendritic cells. J. Immunol. 178: 6867–6875.
43. Mathivanan, S., and R. J. Simpson. 2009. ExoCarta: a compendium of exosomal
proteins and RNA. Proteomics 9: 4997–5000.
44. Blay, J., T. D. White, and D. W. Hoskin. 1997. The extracellular fluid of solid
carcinomas contains immunosuppressive concentrations of adenosine. Cancer
Res. 57: 2602–2605.
45. Mandapathil, M., B. Hilldorfer, M. J. Szczepanski, M. Czystowska, M. Szajnik,
J. Ren, S. Lang, E. K. Jackson, E. Gorelik, and T. L. Whiteside. 2010. Generation
and accumulation of immunosuppressive adenosine by human CD4
+CD25highFOXP3+ regulatory T cells. J. Biol. Chem. 285: 7176–7186.
46. Kansas, G. S., G. S. Wood, and T. F. Tedder. 1991. Expression, distribution, and
biochemistry of human CD39. Role in activation-associated homotypic adhesion
of lymphocytes. J. Immunol. 146: 2235–2244.
47. Moncrieffe, H., K. Nistala, Y. Kamhieh, J. Evans, A. Eddaoudi, S. Eaton, and
L. R. Wedderburn. 2010. High expression of the ectonucleotidase CD39 on
T cells from the inflamed site identifies two distinct populations, one regulatory
and one memory T cell population. J. Immunol. 185: 134–143.
48. Thomson, L. F., J. M. Ruedi, A. Glass, G. Moldenhauer, P. Moller, M. G. Low,
M. R. Klemens, M. Massaia, and A. H. Lucas. 1990. Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositolanchored lymphocyte differentiation antigen ecto-59-nucleotidase (CD73). Tissue Antigens 35: 9–19.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
8. Hoskin, D. W., J. S. Mader, S. J. Furlong, D. M. Conrad, and J. Blay. 2008. Inhibition of T cell and natural killer cell function by adenosine and its contribution
to immune evasion by tumor cells (Review). [Review] Int. J. Oncol. 32: 527–535.
9. Haskó, G., J. Linden, B. Cronstein, and P. Pacher. 2008. Adenosine receptors:
therapeutic aspects for inflammatory and immune diseases. Nat. Rev. Drug
Discov. 7: 759–770.
10. Thorn, J. A., and S. M. Jarvis. 1996. Adenosine transporters. Gen. Pharmacol.
27: 613–620.
11. Havre, P. A., M. Abe, Y. Urasaki, K. Ohnuma, C. Morimoto, and N. H. Dang.
2008. The role of CD26/dipeptidyl peptidase IV in cancer. Front. Biosci. 13:
1634–1645.
12. Deaglio, S., K. M. Dwyer, W. Gao, D. Friedman, A. Usheva, A. Erat, J. F. Chen,
K. Enjyoji, J. Linden, M. Oukka, et al. 2007. Adenosine generation catalyzed by
CD39 and CD73 expressed on regulatory T cells mediates immune suppression.
J. Exp. Med. 204: 1257–1265.
13. Borsellino, G., M. Kleinewietfeld, D. Di Mitri, A. Sternjak, A. Diamantini,
R. Giometto, S. Höpner, D. Centonze, G. Bernardi, M. L. Dell’Acqua, et al.
2007. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of
extracellular ATP and immune suppression. Blood 110: 1225–1232.
14. Stella, J., L. Bavaresco, E. Braganhol, L. Rockenbach, P. F. Farias, M. R. Wink,
A. A. Azambuja, C. H. Barrios, F. B. Morrone, and A. M. Oliveira Battastini.
2010. Differential ectonucleotidase expression in human bladder cancer cell
lines. Urol. Oncol. 28: 260–267.
15. Jin, D., J. Fan, L. Wang, L. F. Thompson, A. Liu, B. J. Daniel, T. Shin,
T. J. Curiel, and B. Zhang. 2010. CD73 on tumor cells impairs antitumor T-cell
responses: a novel mechanism of tumor-induced immune suppression. Cancer
Res. 70: 2245–2255.
16. Zhi, X., S. Chen, P. Zhou, Z. Shao, L. Wang, Z. Ou, and L. Yin. 2007. RNA
interference of ecto-59-nucleotidase (CD73) inhibits human breast cancer cell
growth and invasion. Clin. Exp. Metastasis 24: 439–448.
17. Stagg, J., U. Divisekera, N. McLaughlin, J. Sharkey, S. Pommey, D. Denoyer,
K. M. Dwyer, and M. J. Smyth. 2010. Anti-CD73 antibody therapy inhibits
breast tumor growth and metastasis. Proc. Natl. Acad. Sci. USA 107: 1547–1552.
18. Ohta, A., and M. Sitkovsky. 2001. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage.
Nature 414: 916–920.
19. Erdmann, A. A., Z. G. Gao, U. Jung, J. Foley, T. Borenstein, K. A. Jacobson, and
D. H. Fowler. 2005. Activation of Th1 and Tc1 cell adenosine A2A receptors
directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo. Blood
105: 4707–4714.
20. Sipka, S., I. Kovács, S. Szántó, G. Szegedi, L. Brugós, G. Bruckner, and
A. József Szentmiklósi. 2005. Adenosine inhibits the release of interleukin-1beta
in activated human peripheral mononuclear cells. Cytokine 31: 258–263.
21. Lappas, C. M., J. M. Rieger, and J. Linden. 2005. A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells. J. Immunol.
174: 1073–1080.
22. Jeffe, F., K. A. Stegmann, F. Broelsch, M. P. Manns, M. Cornberg, and
H. Wedemeyer. 2009. Adenosine and IFN-alpha synergistically increase IFNgamma production of human NK cells. J. Leukoc. Biol. 85: 452–461.
23. Koshiba, M., D. L. Rosin, N. Hayashi, J. Linden, and M. V. Sitkovsky. 1999.
Patterns of A2A extracellular adenosine receptor expression in different functional subsets of human peripheral T cells. Flow cytometry studies with antiA2A receptor monoclonal antibodies. Mol. Pharmacol. 55: 614–624.
24. Zhang, H., D. M. Conrad, J. J. Butler, C. Zhao, J. Blay, and D. W. Hoskin. 2004.
Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 39,59-monophosphate and phosphatases. J. Immunol. 173: 932–944.
25. Raskovalova, T., A. Lokshin, X. Huang, Y. Su, M. Mandic, H. M. Zarour,
E. K. Jackson, and E. Gorelik. 2007. Inhibition of cytokine production and cytotoxic activity of human antimelanoma specific CD8+ and CD4+ T lymphocytes by
adenosine-protein kinase A type I signaling. Cancer Res. 67: 5949–5956.
26. Priebe, T., C. D. Platsoucas, and J. A. Nelson. 1990. Adenosine receptors and
modulation of natural killer cell activity by purine nucleosides. Cancer Res. 50:
4328–4331.
27. Raskovalova, T., X. Huang, M. Sitkovsky, L. C. Zacharia, E. K. Jackson, and
E. Gorelik. 2005. Gs protein-coupled adenosine receptor signaling and lytic
function of activated NK cells. J. Immunol. 175: 4383–4391.