REPRINTED BY PERMISSION OF THE AMERICAN ASOCIATION OF IMMUNOLOGISTS,

INC. FROM "UMBILICAL CORD-DERIVED MESENCHYMAL STROMAL CELLS MODULATE

MONOCYTE FUNCTION TO SUPPRESS T CELL PROLIFERATION” BY CUTLER AJ. ET AL.
THE JOURNAL OF IMMUNOLOGY VOL. 185 PP. 6617-23 COPYRIGHT 2010.

Umbilical Cord-Derived Mesenchymal

Stromal Cells Modulate Monocyte Function to
Suppress T Cell Proliferation

This information is current as Antony J. Cutler, Vasanti Limbani, John Girdlestone and
of December 15, 2010 Cristina V. Navarrete

J Immunol 2010;185;6617-6623; Prepublished online 27

October 2010;
doi:10.4049/jimmunol.1002239
http://www.jimmunol.org/content/185/11/6617

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 The Journal of Immunology

Umbilical Cord-Derived Mesenchymal Stromal Cells

Modulate Monocyte Function to Suppress T Cell Proliferation

Antony J. Cutler,* Vasanti Limbani,* John Girdlestone,*,† and Cristina V. Navarrete*,†

Mesenchymal stromal cells (MSCs) may be derived from a variety of tissues, with human umbilical cord (UC) providing an abun-
dant and noninvasive source. Human UC-MSCs share similar in vitro immunosuppressive properties as MSCs obtained from bone
marrow and cord blood. However, the mechanisms and cellular interactions used by MSCs to control immune responses remain to
be fully elucidated. In this paper, we report that suppression of mitogen-induced T cell proliferation by human UC-, bone marrow-,
and cord blood-MSCs required monocytes. Removal of monocytes but not B cells from human adult PBMCs (PBMNCs) reduced the
immunosuppressive effects of MSCs on T cell proliferation. There was rapid modulation of a number of cell surface molecules on
monocytes when PBMCs or alloantigen-activated PBMNCs were cultured with UC-MSCs. Indomethacin treatment significantly
inhibited the ability of UC-MSCs to suppress T cell proliferation, indicating an important role for PGE2. Monocytes purified from
UC-MSC coculture had significantly reduced accessory cell and allostimulatory function when tested in subsequent T cell pro-
liferation assays, an effect mediated in part by UC-MSC PGE2 production and enhanced by PBMNC alloactivation. Therefore, we

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identify monocytes as an essential intermediary through which UC-MSCs mediate their suppressive effects on T cell prolifer-
ation. The Journal of Immunology, 2010, 185: 6617–6623.

M
esenchymal stromal cells (MSCs) have a profound
immunosuppressive capability in vitro (1) and are
currently being assessed as a novel cellular therapeutic
anti-inflammatory agent in numerous clinical trials. MSCs are
being considered as an adjunct to conventional therapy or a stand-
alone therapy to treat a range of autoimmune diseases, acute and
chronic kidney rejection posttransplant, and acute and chronic
graft-versus-host disease (GVHD) and to aid tissue repair/wound
healing (2, 3). Expanded MSCs have been shown to have no
toxicity or adverse events when infused (4), and the most prom-
ising use of MSCs to date is in the treatment of patients with
steroid-resistant GVHD following hematopoietic stem cell trans-
plantation (5). Most clinical trials currently use MSCs derived
from bone marrow (BM); however, MSCs may also be derived
from umbilical cord blood (UCB), adipose tissue, placenta, dental
pulp, and umbilical cord (UC) (reviewed in Ref. 6).
The mechanisms by which MSCs may ameliorate GVHD are
unclear, but experimental evidence suggests that MSCs act on
many of the leukocyte subsets involved directly or indirectly in the
generation of an antihost immune response posthematopoietic stem
cell transplantation. MSCs reduce the proliferative capacity of naive
and memory T cells, B cells and effector function of gdT cells,
NK cells, and neutrophils. MSCs also block and modulate the in
vitro maturation and function of monocyte-derived dendritic cells
*Histocompatibility and Immunogenetics Research Group, National Health Service
Blood and Transplant; and †Division of Infection and Immunity, University College
London, London, United Kingdom
Received for publication July 8, 2010. Accepted for publication September 30, 2010.
This work was supported by Grant C043 from the National Institute for Health
Research UK.
Address correspondence and reprint requests to Dr. Cristina V. Navarrete, Histocom-
patibility and Immunogenetics Research Group, National Health Service Blood and
Transplant, London NW9 5BG, U.K. E-mail address: Cristina.Navarrete@nhsbt.nhs.uk
Abbreviations used in this paper: BM, bone marrow; CB, cord blood; DC, dendritic
cell; DC-SIGN, dendritic cell-specific intercellular adhesion molecule-3–grabbing non-
integrin; GVHD, graft-versus-host disease; moDC, monocyte-derived dendritic cell;
MSC, mesenchymal stromal cell; UC, umbilical cord; UCB, umbilical cord blood.
Copyright ! 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
(moDCs) (reviewed in Ref. 2). MSCs secrete a plethora of growth
factors, cytokines, and immunomodulatory mediators. PGE2, NO,
HLA-G, IDO, IL-10, TGF-b, hepatocyte growth factor, IL-6, and
insulin-like growth factor-1 have all been implicated in MSC in-
duced immunosuppression (reviewed in Ref. 7).
Acute GVHD is initiated by host-derived dendritic cells (DCs),
activated by the inflammatory environment induced by condition-
ing regimes. Donor T cells recognize mismatched MHC molecules
on the host tissues and cells including DCs, become activated,
proliferate, and differentiate into mature effectors cells to drive
pathogenic responses (8). Following myeloablative conditioning or
reduced intensity conditioning of the patient, host DCs are rapidly
replaced by donor-derived DCs in the blood (9) and the skin (10).
Donor-derived DCs may then indirectly present host-derived Ag
and exacerbate the graft-versus-host response (11). Because mono-
cytes are able to develop into DCs under inflammatory conditions
(12), it is conceivable that suppression of GVHD by MSCs is related
to their ability to influence the maturation and function of DCs
(13, 14).
We and others have determined that UC-derived MSCs sup-
press T cell proliferation (15, 16). In this paper, we extend these
observations and explore the cellular interactions used by MSCs
for optimal suppression of T cell proliferation. We show that
monocytes are required for the optimal suppression of T cell
proliferation induced by TCR cross-linking by UCB-, BM-, and
UC-MSCs. Within 24 h of coculture and prior to their differen-
tiation into DCs, monocyte phenotype and function are modulated
by interaction with MSCs. Accessory function and the allosti-
mulatory capacity of monocytes are downregulated by MSCs via
a mechanism that is in part driven by UC-MSC–derived PGE2.
Thus, MSCs appear to act indirectly on T cells via monocytes,
which act as an essential intermediary cell allowing optimal sup-
pression of T cell proliferation by MSCs.
Materials and Methods
Cell lines and PBMCs
Human UC-derived and cord blood (CB)-derived MSCs, generated as pre-
viously described (16, 17), were maintained in culture in low glucose-DMEM

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002239
6618 UC-MSCs MODULATE MONOCYTES TO SUPPRESS T CELL PROLIFERATION

supplemented with 10% FBS, penicillin, streptomycin, and L-glutamine
(Invitrogen, Carlsbad, CA). Human BM-MSCs were purchased from Lonza,
Basel, Switzerland and maintained in culture in Mesencult medium with
10% supplements (StemCell Technologies, Grenoble, France). Upon reach-
ing confluence cells were released from the plate using trypsin-EDTA
(Sigma-Aldrich, St. Louis, MO), counted, and replated. UC-MSCs were
used from passages 3 to 15 with similar results obtained throughout. Human
adult PBMCs (PBMNCs) of buffy coat samples chosen at random were
provided by National Health Service Blood and Transplant (Colindale, U.K).
PBMNCs were prepared by density centrifugation over Lymphoprep (Axis-
Shield, Oslo, Norway), and HLA typing was performed by the Histocom-
patibility and Immunogenetics Department, National Health Service Blood
and Transplant, Colindale Center. UC-MSCs were obtained from third-party
donors and HLA mismatched with the PBMNCs used in these experiments.
Flow cytometry
Cells in PBS, 0.05% sodium azide, 2.5% FBS and 1% Fc block (Miltenyi
Biotec, Bergisch Gladbach, Germany) were stained with CD14 specific Ab
(Immunotools, Friesoythe, Germany) to identify monocytes and counter-
stained using a selection of CD73-, CD123-, CD206-, DC-specific in-
tercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN)–, and
HLA-DR–specific Abs (all BD Biosciences, Oxford, U.K.). Isotype con-
trols and unstained cells were used for all fluorochromes (BD Bio-
sciences). Stained cells were then washed and resuspended in PBS, and
Inhibition and measurement of UC-MSC PGE2 production
UC-MSCs were incubated with 5–10 mM indomethacin (Sigma-Aldrich) or
an equivalent volume of carrier (DMSO) (Sigma-Aldrich) in low glucose-
DMEM + 10% FBS and penicillin/streptomycin for 24 h. Indomethacin-
treated or control UC-MSCs were subsequently harvested, washed, and cul-
tured with nonactivated or alloantigen-activated PBMNCs at a ratio of 1:2.5
in RPMI 1640 medium-10% AB serum for 24 h. PGE2 levels were measured
in tissue culture supernatant by ELISA following the manufacturer’s in-
structions (Assay Designs, Enzo Life Sciences, Farmingdale, NY).
Statistical analysis
Analysis of statistics was carried out using SPSS (version 15) software by
using unpaired or paired student’s t tests where appropriate. All data are
presented as mean 6 SD, and p , 0.05 was considered significant.
Results
Monocytes are required for optimal suppression of T cell
proliferation by MSCs
We have previously shown that UC-MSCs reduce the proliferation
of T cells in vitro (16) and therefore sought to examine and define
the cellular interactions required for MSCs to exert their sup-

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data were acquired using a FACSort flow cytometer and CellQuest soft- pressive effect. Addition of monocytes has previously been shown
ware (BD Biosciences). Data were analyzed using WinMDI software (The to restore the ability of BM-MSCs to suppress proliferation of
Scripps Research Institute, La Jolla, CA). purified CD4+ T cells in response to superantigen (18). We con-
Cell purification firmed that addition of monocytes to cocultures of CD4+ T cells
T + NK cells, monocytes, and B cells were removed from PBMNCs, using
anti-CD2, CD14, or CD19 microbeads (Miltenyi Biotec), respectively.
Untouched CD4+ T cells and monocytes were isolated using the CD4+
T cell isolation kit II or monocyte isolation kit II, respectively (Miltenyi
Biotec). All cells were purified using an autoMACS, according to the
manufacturer’s instructions (Miltenyi Biotec).

Monocyte conditioning and purification

Monocytes were purified from nonactivated or alloantigen-activated PBMNC-
conditioning cultures as follows. Nonactivated PBMNC cultures (5 3 104
PBMNC 6 2 3 104 UC-MSCs) and alloantigen-activated PBMNC cultures
(2.5 3 104 CD4+ T cell and 2.5 3 104 CD2-depleted allogeneic PBMNCs 6
2 3 104 UC-MSCs) were cultured in RPMI 1640 medium (Invitrogen) sup-
plemented with 10% human AB serum (Lonza, Basel, Switzerland) and
penicillin/streptomycin for 24 h. After 24 h, PBMNCs or alloantigen-activated
PBMNC MSC cocultures were harvested and initially centrifuged over a 28%
Percoll (Sigma-Aldrich) density gradient to remove MSCs. All cultures were
further centrifuged over a 35% Percoll density gradient to enrich for mono-
cytes. The resulting interface was harvested and washed and untouched
CD14+ cells isolated using the monocyte isolation kit II (Miltenyi Biotec).
All cell sorts were carried out using an autoMACS.
Cell proliferation
Alloantigen stimulation. This was perfomed using the MLR assay as fol-
lows: freshly isolated CD4+ T cells (2.5 3 106/ml in PBS) were incubated
with 1.25 mM CFSE (Sigma-Aldrich) at room temperature. After 5 min,
30.25 volume of FBS was added, and the cells were incubated for an FIGURE 1. MSCs require the presence of monocytes to suppress T cell
additional 10 min. CFSE-labeled cells were washed twice in PBS and proliferation. A, PBMNCs (black bar), CD19-depleted PBMNCs (gray
recounted prior to culture. CFSE-labeled CD4+ T cells (5 3 104) were hatched bar) or CD14-depleted PBMNCs (gray bar) from four independent
incubated with allogeneic highly purified monocytes from conditioning
blood donors were incubated with activation beads in the presence or
cultures (5 3 103) at a ratio of 10:1. After 7 d of culture, proliferating
and activated T cells were identified using CD3 Ab (Immunotools), CD25 absence of UC-MSCs. Proliferation assessed by [3H] uptake after 72 h of
(Serotec, Kidlington, U.K.), and CFSE dye dilution. Monocytes were culture. The degree of suppression of T cell proliferation induced by UC-
excluded from the analysis using CD14 Ab (Immunotools). Stained cells MSCs is expressed as a percentage relative to T cell proliferation in the
were then washed, resuspended, and acquired using the FACSort flow absence of UC-MSCs. Data are shown as mean values 6 SD. Statistical
cytometer as described previously. significance of differences between CD14-depleted PBMNCs versus
PHA and bead stimulations. PBMNCs or purified cell populations plated at PBMNCs and CD19-depleted PBMNCs were determined using paired
5 3 104 cells/well in RPMI 1640 medium + 10% AB serum (Lonza) in Student t test. ****p = 0.0001. B–D, PBMNCs (black bar) or CD14-de-
U-bottomed 96-well plates (Nunc, Naperville, IL) were stimulated with pleted PBMNCs (gray bar) (n = 4) were incubated with activation beads
anti-CD2, -CD3, -CD28, T cell activation beads (1.75 3 104; Miltenyi with or without MSCs from UCB (B), BM (C), or UC (D) (n = 2 MSC lines
Biotec), or PHA (1 mg/ml; Sigma-Aldrich) with or without MSCs (2 3 per source) and proliferation assessed by [3H] uptake after 72 h of culture.
104 cells/well). Some assays were carried out in the presence or absence
The degree of suppression of T cell proliferation induced by MSC
of indomethacin (5–10 mg/ml) (Sigma-Aldrich). Tritiated [3H]thymidine
(37 kBq/well; Amersham Biosciences, Little Chalfont, Buckinghamshire, expressed as a percentage relative to T cell proliferation in the absence of
U.K.) was added after 2 d of culture, and the cells were harvested 16–18 h MSCs. The data are representative of two independent experiments and
later. Thymidine incorporation was measured in a beta counter (PerkinElmer, shown as mean values 6 SD. Statistical significance of differences were
Wellesley, MA). determined using paired Student t test.
The Journal of Immunology 6619

FIGURE 2. UC-MSCs induce pheno-

typic and physical changes in the mono-
cyte population. Flow cytometric analysis
of monocyte phenotype in culture. Non-
activated PBMNC (A, B) or alloantigen
activated (CD4+ T cells: CD2-depleted
APCs in a 1:1 ratio) (C) were cultured
with (n, n = 4) or without (N, n = 6) UC-
MSCs for 24 h or 7 d. The characteristics
and surface phenotype of cells gated on
CD14 expression were analyzed. Statis-
tical significance of differences in geo-
metric mean channel fluorescence was

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determined using paired Student t test.
*p , 0.05; **p , 0.01; and ***p ,
0.001. The data are representative of four
independent experiments and shown as
mean values 6 SD.

and MSCs reconstituted the optimal suppressive action of MSCs
on T cell proliferation (data not shown) and extended these
observations to demonstrate that depletion of monocytes from
PBMNCs completely abolished the capacity of UC-MSCs to
suppress T cell proliferation induced by direct activation of T cells
by anti-CD2, -CD3, and -CD28 stimulation. Depletion of B cells
had no effect on the ability of MSCs to suppress T cell pro-
liferation (Fig. 1A). Importantly, MSCs derived from UCB (Fig.
1B) and BM (Fig. 1C) in addition to those derived from UC (Fig.
1D) all required monocytes to suppress proliferation of T cells
in vitro.
Coculture of PBMNCs and UC-MSCs induces rapid
phenotypic changes in the monocyte population
BM-MSCs alter the phenotype of moDCs differentiated in vitro
with GM-CSF and IL-4 (13, 14, 19) and that of mature macro-
phages (20). We therefore followed the phenotype of monocytes
cocultured for 24 h and 7 d in vitro with third party UC-MSCs
in nonactivated (PBMNC 6 UC-MSC) or alloantigen-activated
(CD4+ T cell and CD2-depleted allogeneic PBMNC 6 UC-MSC)
PBMNC cell culture conditions. UC-MSCs were found to rap-
idly induce physical and phenotypic changes in the monocyte
population within 8 h (data not shown). UC-MSC coculture
increased the size and granularity of monocytes only in non-
activated PBMNC cocultures (Fig. 2A). UC-MSC coculture in-
creased surface expression of CD73, CD206, and DC-SIGN to
a significant degree on CD14+ cells from both culture conditions
(Fig. 2B, 2C). HLA-DR expression was decreased on monocytes
by UC-MSCs compared with those in medium alone by day 7 in
culture, but notably HLA-DR expression was equivalent after 24 h
in all culture conditions (Fig. 2B, 2C). CD14 expression initially
increased after 24 h but then diminished by day 7 to levels that
were lower or equivalent to control cultures of nonactivated or
alloantigen-activated culture, respectively (Fig. 2B, 2C). CD123
expression on monocytes was maintained by day 7 where non-
activated PBMNCs were cultured with UC-MSCs but not in me-
dium alone (Fig. 2B), whereas no significant difference was
observed on monocytes from alloantigen-activated PBMNC/UC-
MSC coculture (Fig. 2C). Importantly, similar alterations in mono-
cyte phenotype were seen with MSCs derived from multiple UC
tissue donors, BM, and CB (data not shown). The majority of the
changes observed with UC-MSC coculture could be replicated by
using UC-MSC–conditioned medium or culturing UC-MSCs and
PBMNCs across a Transwell (data not shown). However, DC-SIGN
and CD123 expression were unaffected, indicating that additional
signals other than soluble factors produced by MSCs may be re-
quired for induction of all of the phenotypic changes observed in
monocytes.
MSC coculture reduces the accessory and allostimulatory
function of monocytes
Monocytes are a major component of the cellular influx into an
inflammatory environment (21). They are also able to differentiate
into inflammatory DCs or macrophages with both pro- and anti-
inflammatory properties dependent on environmental stimuli (22–
24). To investigate whether monocyte function as well as pheno-
type was modulated following UC-MSC exposure, we analyzed
the capacity of monocytes to support T cell proliferation in response
6620 UC-MSCs MODULATE MONOCYTES TO SUPPRESS T CELL PROLIFERATION
to PHA stimulation, because highly purified CD4+ T cells only re-
spond to PHA in the presence of accessory cells such as monocytes
(25). Monocytes (.85% CD14+ with ,0.5% UC-MSC contami-
nation) purified from nonactivated PBMNCs following 24 h of
coculture with UC-MSCs had profoundly reduced accessory cell
function in subsequent PHA stimulation assays when compared
with those cultured in medium alone (Fig. 3A). Monocytes purified
from highly pure CD14+ and UC-MSC coculture also had reduced
accessory function compared with those cultured in medium alone
(Fig. 3B), indicating that other mononuclear cell populations are not
required for the MSC effect on monocytes. UC-MSC–modulated
monocytes were further assessed for their capacity to induce pro-
liferation of allogeneic CD4+ T cells. CD14+ cells purified from
nonactivated PBMNC and UC-MSC coculture had a reduced ability
to induce proliferation of allogeneic T cells (Fig. 3C). In addition,
monocytes purified from highly pure CD14+ and UC-MSC co-
culture exhibited reduced allostimulatory capacity in two of three
experiments (data not shown). The data suggest that UC-MSCs may
interact directly with monocytes to rapidly impair their accessory
functions without any requirement for a third-party leukocyte subset.
Inhibition of PGE2 production releases T cells from
UC-MSC–induced suppression
PGE2 has previously been identified as one of many MSC-derived
soluble factors proposed to modulate immune function (2). PGE2
FIGURE 3. Monocytes from PBMNC/UC-MSC coculture have reduced
accessory cell and allostimulatory function. A, CD4+ T cells were cultured
in medium alone (gray bar), with PHA (1 mg/ml) (hatched bar), with PHA
and CD14+ cells purified from a 24-h UC-MSC/nonactivated PBMNC
coculture (black bar), or with PHA and CD14+ cells purified from non-
activated PBMNC culture alone (white bar). Proliferation was measured by
[3H] uptake after 72 h of culture. The data shown are representative of five
independent experiments and shown as mean values 6 SD. B, CD4+ T cells
were cultured in medium alone (gray bar), with PHA (hatched bar), with
PHA and CD14+ cells purified from CD14+ cell/UC-MSC coculture after
24 h (black bar), or with PHA and CD14+ cells purified from CD14+ cells
in medium alone (white bar). Proliferation was measured by [3H] uptake
after 72 h of culture. The data shown are representative of two independent
experiments and shown as mean values 6 SD. C, CD14+ cells were purified
from nonactivated PBMNCs cultured in medium alone (white bar, n = 2) or
with UC-MSC (black bar, n = 2) for 24 h and cultured with allogeneic
CFSE-labeled CD4+ T cells. CD4+ T cell proliferation was assessed at 7
d by CFSE dye dilution and upregulation of CD25. The data are presented
as proliferation induced by CD14+ cells relative to controls where pro-
liferation is a reference at 100% and is shown as mean values 6 SD.
was constitutively produced by UC-MSCs and increased levels
were detected where nonactivated or alloantigen-activated PBMNCs
were cultured with UC-MSCs (Fig. 4A). As MSC-derived PGE2 may
modulate moDC maturation (26) and suppress T cell proliferation
(27), we therefore assessed whether UC-MSCs mediated their im-
munosuppressive effects via PGE2. In agreement with a previous
report (15, 15), we found that UC-MSCs were unable to inhibit PHA
induced proliferation of PBMNC in the presence of indomethacin
(a nonspecific cyclooxygenase-1/2 inhibitor) (Fig. 4B).
UC-MSCs reduce monocyte allostimulatory capacity—an
effect partially mediated by UC-MSC PGE2 production and
enhanced by activation
PGE2 has complex and often divergent actions on different cells
of the immune system (28). We therefore addressed whether UC-
MSC–derived PGE2 had a role in modulating monocyte allosti-
mulatory function. UC-MSCs were pretreated with indomethacin
for 24 h prior to coculture with nonactivated PBMNCs or alloantigen-
activated PBMNCs. Treatment abolished constitutive PGE2 produc-
tion by UC-MSCs but did not affect PGE2 production in the sub-
sequent culture (data not shown). Indomethacin treatment of UC-
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MSCs significantly reduced their ability to modulate monocyte allo-
stimulatory function compared with those purified from UC-MSCs
treated with carrier (DMSO) alone. Indeed, monocytes purified from
nonactivated PBMNC or alloantigen-activated PBMNC coculture
with indomethacin-pretreated UC-MSCs were able to stimulate al-
logeneic T cells to approximately a 2- and 4-fold greater extent, re-
spectively, than monocytes purified from nonactivated PBMNCs or
alloantigen-activated PBMNCs cultured with control UC-MSCs (Fig.
5A, 5B).
Inflammation has been proposed to “license” MSCs to suppress
immune function (29). We therefore compared the allostimulatory
FIGURE 4. PGE2 mediates UC-MSC suppression of T cell proliferation.
A, PGE2 was measured in the supernatant of PBMNCs (5 3 104) (spotted
bar, n = 2), MLR (2.5 3 104 CD4+ T cells + 2.5 3 104 CD2-depleted
APCs) (white bar, n = 3), PBMNC + UC-MSC (2 3 104) (black spotted
bar, n = 2), MLR + UC-MSC (black bar, n = 2), or UC-MSC alone (gray
bar) cultures after 24 h. B, A total of 5 3 104 PBMNCs were cultured with
PHA (1 mg/ml) (white bar), with PHA + 2 3 104 UC-MSC + DMSO
(black bar) or with PHA + 2 3 104 UC-MSC + 5 mM indomethacin
(hatched bar). Proliferation was measured by [3H] uptake after 72 h of
culture. The data shown are representative of five independent experiments
and shown as mean values 6 SD.
The Journal of Immunology
FIGURE 5. UC-MSC–derived PGE2 in part diminishes the allostimu-
latory capacity of monocytes. CD14+ cells were purified from (A) non-
activated PBMNCs cultured in medium alone (white bar, n = 8),
nonactivated PBMNC + UC-MSC coculture (black bar, n = 8) or non-
activated PBMNC + indomethacin-pretreated UC-MSC coculture (hatched
black bar, n = 3), (B) alloantigen-activated PBMNCs in medium alone
(white bar, n = 5), alloantigen-activated PBMNC + UC-MSC coculture
(black bar, n = 5), or alloantigen-activated PBMNC + indomethacin-pre-
treated UC-MSC coculture (hatched black bar, n = 4), or (C) nonactivated
PBMNC (n = 8), nonactivated PBMNC + UC-MSC (n = 2), or PBMNC +
225 nM PGE2 (n = 8) after 24 h of culture. Purified CD14+ cells were
subsequently cultured with allogeneic CFSE-labeled CD4+ T cells at
a ratio of 1:10, and the ability of purified monocytes to stimulate the
proliferation of allogeneic CD4+ T cells was assessed by CFSE dye di-
lution and upregulation of CD25 after 7 d. The data are presented as
proliferation relative to medium controls where proliferation is a reference
at 100% and is shown as mean values 6 SD. Statistical significance was
determined using the unpaired Student t test where *p , 0.05.
capacity of monocytes purified from a nonactivated PBMNC or
alloantigen-activated PBMNC coculture with UC-MSCs. The abil-
ity of monocytes to stimulate an alloresponse from CD4+ T cells
was more profoundly affected when purified from alloantigen-
activated PBMNC and UC-MSC coculture compared with those
purified from nonactivated PBMNC and UC-MSC culture. Mono-
cytes stimulated allogeneic T cells on average 5-fold versus a 2-fold
lesser extent, respectively (compare Fig. 5A, 5B). Importantly,
HLA-DR expression was equivalent on monocytes purified from
all cultures at 24 h (Fig. 2B, 2C). Furthermore, pretreatment of
UC-MSCs with indomethacin only allowed recovery of monocyte
allostimulatory capacity to ∼50% of control values when purified
from cocultures (Fig. 5B). Whereas monocytes purified from non-
activated PBMNC/indomethacin-treated UC-MSC cultures recovered
allostimulatory capacity to ∼80% of controls (Fig. 5A). These data
raise the possibility that inflammation may induce additional factors
in MSCs and/or PBMNCs other than PGE2 to suppress T cell pro-
liferation.
The effects of indomethacin indicated a role for PGE2 pro-
duction by UC-MSCs in modulating the allostimulatory ability of
monocytes. We therefore assessed whether PGE2 acted alone or
required other factors to mediate this effect. PBMNCs were cul-
tured with PGE2 at levels observed in nonactivated PBMNC and
UC-MSC coculture. Monocytes purified following 24 h of culture
were then used as stimulator cells for allogeneic CD4+ T cells.
Monocytes from PGE2 cultures exhibited an increased capacity to
stimulate allogeneic T cells compared with those from control
cultures (Fig. 5C). Therefore, our data suggest that additional fac-
tors in concert with UC-MSC–derived PGE2 act on the monocyte
6621
population to modulate their function and suppress T cell prolife-
ration.
Discussion
MSCs have been shown to modulate the actions of a diverse range
of immune cells and a number of soluble factors derived from, or
induced by MSCs have been proposed to mediate the anti-
inflammatory or suppressive effects of MSCs (2). Given the in-
creasing use of MSCs as a cellular therapy product in many
clinical trials it is of critical importance to elucidate the cellular
interactions required for MSCs to suppress immune responses and
to understand whether MSCs act directly on effector cells or via
a third party. In this study, we demonstrate that MSCs from di-
verse sources are dependent on monocytes to suppress T cell
proliferation induced by TCR cross-linking and that UC-MSCs act
via an indomethacin-sensitive agent PGE2. MSCs derived from
UC tissue rapidly alter monocyte phenotype, affect accessory cell
function, and fundamentally reduce their ability to activate and
induce the proliferation of allogeneic T cells. The modulatory
effect of UC-MSCs on monocytes was enhanced in an activated
Downloaded from www.jimmunol.org on December 15, 2010
environment and was in part dependent on constitutive UC-MSC
PGE2 production. Importantly, the modulatory effect of UC-MSC–
derived PGE2 was effective only during initial contact between
UC-MSCs and monocytes contained within nonactivated PBMNCs
or alloantigen-activated PBMNCs.
Our data clearly implicate monocytes as a key intermediary in
UC-MSC–induced suppression of T cell proliferation. This may
be a key interaction in vivo as peripheral blood and splenic
monocytes are rapidly recruited to inflammatory sites (30, 31) and
are able to differentiate into both inflammatory DCs and macro-
phages (21, 22). Once differentiated, both of these mononuclear
phagocytic cell populations play pivotal roles in promoting im-
mune responses and resolving inflammation depending on the
environmental cues (22, 32). BM-MSCs have been shown to alter
or block the maturation of monocytes into DCs (13, 14, 19, 26).
However, in contrast to our studies, monocytes and BM-MSCs in
earlier reports were cultured in the presence of GM-CSF and IL-4
over 5–6 d in vitro to induce DC differentiation. Our data suggest
that monocyte function is directly and stably modulated by
UC-MSCs within 24 h of coculture (Figs. 3, 5) and that MSCs
from disparate sources all act through monocytes to reduce T cell
proliferation (Fig. 1B–D). Furthermore, the allostimulatory ca-
pacity of highly purified CD14+ monocytes was greatly reduced
following culture with UC-MSCs (data not shown). The data re-
inforce the notion that MSCs from a variety of sources primarily
and directly act through monocytes to reduce T cell proliferation.
In agreement, BM-MSCs have previously been shown to interact
with monocytes to modulate immune responses through soluble
mediators (33) and to reduce monocyte accessory function in
superantigen-stimulated cultures (18). It is of note that BM-MSCs
may in addition reduce mature DC function (14) and alter mac-
rophage phenotype and function (20).
The phenotypic changes observed in the monocyte population
suggest that UC-MSCs induce macrophages rather than a DC
subset. We found no evidence for induction of a CD14lo/neg CD1a,
CD1c, or CD83+ DC population or modulation of CD80 or CD86
on the CD14 positive population (data not shown). Although
originally described as a DC-specific marker, DC-SIGN may be
expressed by macrophages (34). CD123 (IL-3Ra) expression is
associated with a plasmacytoid DC phenotype and was maintained
following culture with UC-MSCs; however, plasmacytoid DCs do
not coexpress CD14 (35). Indeed, CD14 expression was signifi-
cantly increased initially in the presence of UC-MSCs but then
declined over time dependent on the activation status of the cultures
6622 UC-MSCs MODULATE MONOCYTES TO SUPPRESS T CELL PROLIFERATION
(Fig. 2). In agreement, monocytes or CD14+ precursors cultured with
BM-MSC in DC differentiation conditions maintained expression of
CD14 (13, 14) and were suggested to exhibit a macrophage-like
morphology (13). IL-6 may play a key role in driving monocyte
differentiation into macrophages (36). IL-6 is produced by UC-MSCs
(data not shown) and in concert with M-CSF may (13, 14) or may not
(26) mediate BM-MSC modulation of moDC differentiation. How-
ever, in our hands, neutralization of IL-6 and M-CSF had no effect on
the ability of UC-MSCs to modulate monocyte phenotype (data not
shown). Finally, expression of CD73, a component of the immuno-
suppressive adenosine pathway, is upregulated in the presence of UC-
MSC (Fig. 2B, 2C) and PGE2 (data not shown) and is induced on
mature activated macrophages (37).
Macrophage and monocyte populations are heterogeneous with
respect to the activatory and tissue environment guiding their
phenotype and function (23). UC-MSC upregulated expression of
CD206 on CD14+ cells (Fig. 2B, 2C) a molecule frequently asso-
ciated with an alternatively activated macrophage phenotype (38).
CD206 was also induced on mature macrophages by BM-MSCs
(20). Alternatively activated macrophage defined by their induction
by IL-4/13 signaling may induce tissue remodeling, regulate im-
mune responses (24) and inhibit T cell proliferation in vitro (39).
Indeed, DC-SIGN expression another molecule induced by UC-
MSCs (Fig. 2) may also be induced by IL-4 (40). However, IL-4
was not detected following UC-MSC and PBMC cocultures (data
not shown), and therefore from our data, we would not classify the
induced macrophage population as alternatively activated.
PGE2 can exert opposing effects on the immune system (28).
Depending on the context, PGE2 may either be pro- (41) or anti-
inflammatory (42). BM-MSC–derived PGE2 has been proposed to
block DC differentiation (26) and induce monocyte IL-10 pro-
duction (43). We and others (15) have shown that PGE2 pro-
duction may play a key role in UC-MSC–induced suppression of
T cell proliferation (Fig. 4). PGE2 is constitutively produced by
BM-MSCs (27) and monocytes (44). Therefore to study the role
of UC-MSC–derived PGE2 in modulating monocyte function,
UC-MSCs were pretreated with indomethacin, thus reversibly
blocking constitutive UC-MSC PGE2 production (data not shown),
prior to culture with immune cells. However, comparable amounts
of PGE2 were detected in subsequent cultures with nonactivated,
alloantigen-activated PBMNCs or purified CD14+ cells whether
UC-MSCs were pretreated with indomethacin or not (data not
shown). The data indicating, as previously described (27), a strong
induction of de novo cyclooxygenase activity when MSCs are
cultured with leukocytes. Despite this observation, monocytes pu-
rified from coculture with indomethacin-treated UC-MSC–stimu-
lated allogeneic CD4+ T cells to proliferate to a significant degree
above those from control UC-MSC cultures (Fig. 5). PGE2 treat-
ment of PBMNCs reproduced some but not all of the phenotypic
changes induced by UC-MSCs on monocytes (data not shown).
However, treatment of PBMNCs with PGE2 in isolation in some
instances enhanced the allostimulatory capacity of monocytes (Fig.
5C). Therefore, the absence of PGE2 production by UC-MSCs at the
initial stages of priming may therefore allow other mediators to “re-
wire” monocytes to be either nonresponsive to PGE2 or alter their
response to PGE2.
BM-MSCs have shown promise in the treatment of GVHD but
only in patients with active disease (5, 45). MSCs share with
monocytes the ability to home to sites of inflammation (46) and
may enhance recruitment of macrophages to sites of injury (47).
However, depending upon the stimulus, the suppressive action of
MSCs may be enhanced or diminished by inflammation. Trig-
gering through TLRs inhibited MSC-suppressive function (48),
whereas IFN-g enhanced the suppressive ability of BM-MSCs
(49, 50). The allostimulatory capacity of monocytes purified from
a MLR was significantly reduced compared with those purified
from PBMNCs following coculture with UC-MSC (Fig. 5A, 5B).
Our data support the idea that MSC suppression is enhanced in an
allogeneic environment. As discussed, IFN-g, PGE2, and IL-10 all
may play a role in MSC-induced suppression. However, in our
studies, induction of PGE2 and IL-10 secretion were equivalent in
either nonactivated PBMNC or alloantigen-activated PBMNC
coculture with UC-MSCs, and neutralizing IL-10 in these cultures
did not affect the ability of UC-MSCs to modulate monocyte
allostimulatory function (data not shown). Secretion of IFN-g,
TNF-a, IL-6, and IL-1b were all induced following PBMNC
culture with UC-MSCs but were not enhanced by stimulation (data
not shown). Therefore, as-yet unidentified factors are induced dur-
ing UC-MSC and MLR coculture, which are able to further reduce
monocyte allostimulatory capacity and to enhance the suppressive
capacity of UC-MSCs.
In conclusion, our data demonstrate that UC-MSCs require
monocytes to suppress mitogen-stimulated T cell proliferation.
Monocyte phenotype and accessory and allostimulatory functions
Downloaded from www.jimmunol.org on December 15, 2010
were stably and profoundly modulated following interaction with
UC-MSCs. UC-MSC PGE2 production, an indomethacin-sensitive
agent, appeared to play a key role in the suppression of T cell
proliferation and in part the modulation of monocyte function.
Importantly, the influence of UC-MSC–derived PGE2 on mono-
cyte function was limited to the initial phase of contact. Finally,
PGE2 treatment of PBMNCs alone could not reproduce a similar
modulation of monocyte function. Therefore UC-MSC–derived PGE2
acts at the initial contact between MSC and monocyte and in concert
with other factors to modulate monocyte function and suppress T cell
proliferation.
Acknowledgments
We thank the National Health Service Blood and Transplant for the provi-
sion of adult buffy coats and the Histocompatibility and Immunogenetics
Department, Colindale, for assistance.
Disclosures
The authors have no financial conflicts of interest.
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