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doi:10.1182/blood-2004-04-1559
Prepublished online October 19, 2004;
2005 105: 1815-1822
Sudeepta Aggarwal and Mark F. Pittenger
responses
Human mesenchymal stem cells modulate allogeneic immune cell
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TRANSPLANTATION
Human mesenchymal stem cells modulate allogeneic immune cell responses
Sudeepta Aggarwal and Mark F. Pittenger
Mesenchymal stem cells (MSCs) are mul-
tipotent cells found in several adult tis-
sues. Transplanted allogeneic MSCs can
be detected in recipients at extended time
points, indicating a lack of immune recog-
nition and clearance. As well, a role for
bone marrow–derived MSCs in reducing
the incidence and severity of graft-versus-
host disease (GVHD) during allogeneic
transplantation has recently been re-
ported; however, the mechanisms remain
to be investigated. We examined the im-
munomodulatory functions of human
MSCs (hMSCs) by coculturing them with
purified subpopulations of immune cells
and report here that hMSCs altered the
cytokine secretion profile of dendritic cells
(DCs), naive and effector T cells (T helper
1[T
H
1] and T
H
2), and natural killer (NK)
cells to induce a more anti-inflammatory
or tolerant phenotype. Specifically, the
hMSCs caused mature DCs type 1 (DC1)
to decrease tumor necrosis factor ␣
(TNF-␣) secretion and mature DC2 to in-
crease interleukin-10 (IL-10) secretion;
hMSCs caused T
H
1 cells to decrease inter-
feron ␥ (IFN-␥) and caused the T
H
2 cells to
increase secretion of IL-4; hMSCs caused
an increase in the proportion of regula-
tory T cells (T
Regs
) present; and hMSCs
decreased secretion of IFN-␥ from the NK
cells. Mechanistically, the hMSCs pro-
duced elevated prostaglandin E2 (PGE
2
)
in co-cultures, and inhibitors of PGE
2
production mitigated hMSC-mediated im-
mune modulation. These data offer in-
sight into the interactions between alloge-
neic MSCs and immune cells and provide
mechanisms likely involved with the in vivo
MSC-mediated induction of tolerance that
could be therapeutic for reduction of GVHD,
rejection, and modulation of inflammation.
(Blood. 2005;105:1815-1822)
© 2005 by The American Society of Hematology
Introduction
The isolation of stem cell populations has burgeoned in the last 10
years, opening many new opportunities to evaluate the stem cells
and their use in tissue regeneration. Hematopoietic stem cells
(HSCs) were identified after a long search for cells that would
allow survival following radiation exposure. During this period,
several studies demonstrated that bone marrow would form new
bone when transplanted to an ectopic site.
1
The isolation and
culture of cells from bone marrow that could form this ectopic bone
were first demonstrated by Friedenstein et al
2
using the guinea pig
as a model. Later, several groups published similar data for rat and
rabbit cells harvested from bone marrow and other tissues.
2-4
Similar to the hematopoietic stem cell and its lineages, the concept
emerged that there may be a mesenchymal stem cell (MSC), a
single cell capable of forming bone, cartilage, and other mesenchy-
mal tissues.
3,4
Haynesworth et al
5
developed a reliable in vivo
bone-forming assay and were able to isolate and culture human
MSCs in therapeutic quantities.
Subsequently, in vitro experiments demonstrated that clonal
human MSCs are able to differentiate into various lineages
including osteoblasts, chondrocytes, and adipocytes.
6,7
In vitro and
in vivo studies have also indicated the capability of MSCs to
differentiate into muscle,
8
neural precursors,
9,10
cardiomyo-
cytes,
11-13
and possibly other cell types.
14,15
In addition, MSCs have
been shown to provide cytokine and growth factor support for
expansion of hematopoietic and embryonic stem cells.
16-19
Numer-
ous studies with a variety of animal models have shown that MSCs
may be useful in the repair or regeneration of myocardial tis-
sues,
13,20,21
damaged bone,
22-25
tendon,
26
cartilage,
27
and menis-
cus.
28
Perhaps one of the most remarkable and least understood
findings is the ability of MSCs to migrate to sites of tissue
injury.
13,29-31
Several clinical studies using autologous whole bone
marrow, presumably containing MSCs, HSCs, and/or endothelial
progenitor cells have also been reported for patients with myocar-
dial infarcts.
32-34
Importantly, encouraging results have been re-
ported for ex vivo–cultured MSCs in early clinical use including
engraftment of autologous
35,36
or allogeneic
37
(also Lazarus et al,
manuscript submitted, August 2004) bone marrow transplants,
allogeneic MSCs for the collagen I genetic disease osteogenesis
imperfecta,
38
and recently for the treatment of graft-versus-host
disease (GVHD).
39
Human MSCs (hMSCs) can be isolated from several, perhaps
most, tissues, although bone marrow is most often used. Human
MSCs express intermediate levels of human leukocyte antigen
(HLA) major histocompatibility complex (MHC) class I, and they
can be induced to express MHC class II antigen
40-43
and Fas ligand
by interferon ␥ (IFN-␥) treatment. MSCs do not express costimula-
tory molecules B7-1, B7-2, CD40, and CD40 ligand and probably,
therefore, do not activate alloreactive T cells.
41,43,44
In addition,
MSCs differentiated into various mesenchymal lineages do not
appear to alter their interaction with T cells.
45,46
MSCs isolated
from humans and other mammalian species including baboon,
canine, caprine, and rodents do not elicit a proliferative response
From Osiris Therapeutics, Baltimore, MD.
Submitted April 26, 2004; accepted September 26, 2004. Prepublished online
as Blood First Edition Paper, October 19, 2004; DOI 10.1182/blood-2004-04-
1559.
Supported in part by Defense Advanced Research Projects Agency (DARPA)
grant no. N66001-02-C-8068.
S.A. and M.F.P. are currently employed at Osiris Therapeutics, Inc, which is
developing cellular therapeutics based on human mesenchymal stem cells.
The online version of the article contains a data supplement.
Reprints: Mark F. Pittenger, Osiris Therapeutics, 2001 Aliceanna St, Baltimore,
MD 21231; e-mail: mpittenger@osiristx.com.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
1815BLOOD, 15 FEBRUARY 2005
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For personal use only. by guest on June 3, 2013. bloodjournal.hematologylibrary.orgFrom
from allogeneic lymphocytes.
24,47-50
In fact, MSCs suppress prolif
-
eration of allogenic T cells in an MHC-independent manner.
40,41,44
Few studies have investigated the interaction of MSCs with
specific immune cell subtypes.
51-53
In order to understand the mechanisms involved in the observed
MSC-mediated immunosuppressive action, we used a step-wise
approach in which we isolated various immune cell populations,
cocultured them with hMSCs, and then examined the alterations in
cytokine secretion profile by these cells. We observed that each
interaction resulted in altered phenotypic expression of factors by
the immune cells. Furthermore, as most of the observed MSC-
mediated T-cell suppressive activity in vitro has been attributed to
soluble mediators, we evaluated several factors and identified the
role of one of the factors, prostaglandin E2 (PGE
2
), produced by
MSCs in their immunomodulatory activity.
Materials and methods
Materials
Recombinant cytokines and antibodies against cytokines or CD antigens
were from BD Biosciences, San Diego, CA, unless noted otherwise. The
enzyme-linked immunosorbent assay (ELISA) kits to detect the cytokines
interleukin-6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), or
PGE
2
were purchased from R&D Systems, Minneapolis, MN.
Methods
Culture of the human MSCs. Bone marrow aspirates drawn from the iliac
crest of donors were obtained from Poietics Technologies (Gaithersburg,
MD) following informed consent. Human MSCs were isolated using
previously described methods.
6,54
Briefly, the bone marrow aspirate (10
mL) was combined with Dulbecco phosphate-buffered saline (DPBS; 40
mL) and centrifuged at 900g for 10 minutes at 20°C. The cells were then
resuspended and gently layered onto a Percoll cushion (density, 1.073
g/mL; Invitrogen, Carlsbad, CA) at 1 to 3 ⫻ 10
8
nucleated cells/25 mL. The
low-density hMSC-enriched mononuclear fraction was collected, washed
with 25 mL DPBS, and centrifuged to collect the cells. Cells were
resuspended in hMSC complete culture medium (Dulbecco modified Eagle
medium with low glucose [Invitrogen], 10% fetal bovine serum [FBS] from
preselected lots [JRH BioSciences, Lenexa, KS], antibiotic/antimycotic
[Invitrogen], and glutamax [Invitrogen]) and plated at 3 ⫻ 10
7
cells/185
cm
2
. The cultures were maintained at 37°C in a humidified atmosphere
containing 95% air and 5% CO
2
and subcultured prior to confluency. The
hMSCs expanded in culture showed positive surface staining for CD73
(SH-3), CD105 (SH-2), and CD44, but lacked CD14, CD34, and CD45
surface expression. The hMSCs were routinely frozen in medium contain-
ing 10% dimethyl sulfoxide (DMSO) in 90% FBS. The hMSCs retained the
capacity to differentiate into adipogenic, osteogenic, and chondrogenic
lineages (data not shown).
For in vitro experiments, frozen aliquots of hMSCs were thawed and
cultured in complete medium containing DMEM, 10% selected FBS, and
1% antibiotics (Invitrogen). Human MSCs grew as adherent cells and were
detached by incubation with trypsin (0.05% trypsin at 37°C for 3 minutes).
The donor population used in these experiments consisted of 9 donors
designated hMSC nos. 57, 59, 68, 71, 75, 101, 124, 151, and 218.
Isolation of the immune cell populations. Human peripheral blood
mononuclear cells (hPBMCs) were prepared from leucopheresis packs
(Cambrex, East Rutherford, NJ) by centrifugation on a Ficoll Hypaque
density gradient. Aliquots of the isolated hPBMCs were frozen and stored at
⫺140°C until further use. For in vitro experiments, frozen aliquots of the
hPBMCs were randomly chosen from the 5 unrelated donors, thawed, and used.
Dendritic cells type 1 (DC1)
Precursors of DC1 belong to the myeloid lineage of leukocytes and are
CD1c
⫹
. These cells were selected from hPBMCs using a 2-step magnetic
isolation method according to Dzionek et al.
55
First, the CD1c-expressing B
cells were depleted using CD19-labeled magnetic beads. Secondly, the
B-cell–depleted flow-through fraction was labeled with biotin-labeled
CD1c antibody (BDCA-1
⫹
; Miltenyi Biotech, Auburn, CA) and selected by
the antibiotin antibody-labeled micromagnetic beads. These labeled cells
were separated from the unlabeled cell fraction using magnetic columns
according to the manufacturer’s instructions (Miltenyi Biotech). The
isolated cell population was stained with the phycoerythrin (PE)–antibiotin
BDCA-1
⫹
antibody and was always more than 95% positive. For matura
-
tion of the DC1 population, recombinant human granulocyte-macrophage
colony-stimulating factor (rhGM-CSF, 1 ⫻ 10
3
IU/mL) and recombinant human
IL-4 (rhIL-4, 1 ⫻ 10
3
IU/mL) were added at the initiation of a 2-day culture.
56
Dendritic cells type 2 (DC2)
Precursors of DC2 belong to the plasmacytoid lineage of leukocytes and
express BDCA-4 antigen on their cell surface. These cells were isolated
directly from hPBMCs by immunomagnetic selection of BDCA-4
⫹
cells
(anti–BDCA-4; Miltenyi Biotech). The isolated cell population was stained
with PE–anti–BDCA-2 (another antigen coexpressed on BDCA-4
⫹
cells;
Miltenyi Biotech) and was always more than 80% positive. The DC2
population was matured by adding recombinant IL-3 (10 ng/mL) at the
initiation of a 2-day culture.
Naive T cells
For the isolation of naive T cells, the hPBMCs were magnetically labeled
with CD45RA microbeads (Miltenyi Biotech) and then loaded onto the
column in a magnetic field according to the manufacturer’s instructions.
The magnetically labeled CD45RA
⫹
cells were retained on the column and
were eluted as the positively selected cell fraction. An aliquot of the isolated
cell population was labeled with the fluorescein isothiocyanate (FITC)–
CD45RA antibody and was always more than 95% positive.
Natural killer (NK) cells
Natural killer cells were isolated by immunodepletion of the non-NK cells.
First, hPBMCs were magnetically labeled with a cocktail of biotin-
conjugated monoclonal antibodies (anti-CD3, -CD4, -CD14, -CD15, -CD19,
-CD36, -CD123, and 235a [glycophorin A]) and magnetic antibiotin
microbeads. Next, the labeled non-NK cells were retained in the magnetic
field, while the NK cells passed through. A small aliquot of the lineage-
negative flow-through population was stained with PE-conjugated CD56
antibody and was always more than 95% CD56
⫹
.
MSC–immune cell coculture
MSCs–dendritic cells. Human MSCs and the DC1 population isolated
from unrelated donors were cocultured (hMSC to DC1 ratio, 1:10) in the
presence of GM-CSF ⫹ IL-4. After 2 days, inflammatory bacterial lipopoly-
saccharide (LPS, 1 ng/mL) was added and the culture supernatant was then
analyzed for tumor necrosis factor ␣ (TNF-␣) levels after 16 hours. The
experiment was performed with 5 MSC and 5 unrelated DC1 donor pairs.
Human MSCs and the DC2 populations isolated from unrelated donors
were cocultured (hMSC to DC2 ratio, 1:1) in the presence of IL-3 for 2
days, following which LPS (1 ng/mL) was added and the levels of IL-10
were then examined after 16 hours. The hMSC to DC2 ratio used was 1:1
due to an inability to obtain sufficient DC2 from hPBMCs. The experiment
was performed with 5 unrelated hMSC-DC2 donor pairs.
MSCs–T cells. Human MSCs were plated into 12-well plates contain-
ing T cells (CD45RA
⫹
;2⫻ 10
5
cells/mL) from an unrelated donor (hMSC
to T-cell ratio, 1:10) in the presence of T helper 1 (T
H
1)–inducing conditions
(anti-CD3 [5 g/mL], anti-CD28 [1 g/mL], recombinant human IL-2
[rhIL-2, 4 ng/mL], rhIL-12 [1 g/mL], and anti–IL-4 [1 g/mL]) or
T
H
2-inducing conditions (anti-CD3 [5 g/mL], anti-CD28 [1 g/mL],
rhIL-2 [4 ng/mL], rhIL-4 [1 g/mL], and anti–IFN-␥ [1 g/mL]). After 48
hours, the nonadherent cells were harvested, washed extensively, and
stimulated with phytohemagglutinin (PHA, 2.5 g/mL) for another 16
hours, and the levels of IFN-␥ for T
H
1 and IL-4 for T
H
2 cocultures were
determined. The experiments were performed with 3 hMSC and 3 unrelated
1816 AGGARWAL and PITTENGER BLOOD, 15 FEBRUARY 2005
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hPBMC donor pairs. Data were expressed as percent change (mean ⫾ SD)
in IFN-␥ or IL-4 in cultures incubated with or without hMSCs.
For the regulatory T cells (T
Regs
), frozen aliquots of hMSCs were
thawed and expanded using standard culture conditions and used within
passage 6. The hMSCs were harvested, counted, and added into wells
(2 ⫻ 10
4
cells/mL) containing hPBMCs from an unrelated donor (hMSC to
PBMC ratio, 1:10) in the presence of rhIL-2 (300 U/mL). After 5 days of
coculture, nonadherent T cells were harvested and evaluated for the
proportion of T
Regs
present by flow cytometry using FITC-CD4 and
PE-CD25 antibodies.
57,58
MSCs–NK cells. Human MSCs were incubated with isolated NK cells
(hMSC to NK cell ratio, 1:1) from an unrelated hPBMC donor in the
presence of rhIL-2 (300 U/mL). Levels of IFN-␥ were measured in
coculture supernatants collected at 24 hours. The experiment was per-
formed with 3 unrelated hMSC and hPBMC donor combinations.
Proliferation assay
Human MSCs were plated in triplicates onto 96-well plates at 2 ⫻ 10
4
cells/mL in 100 L complete media and were allowed to adhere to the plate
for 1 to 2 hours. Human PBMCs, resuspended at 2 ⫻ 10
5
cells/mL were
added to wells (in 100 L volume) containing or lacking hMSCs in the
presence of the mitogen PHA (2.5 g/mL). The hMSC to hPBMC ratio was
1:10. Cocultures without PHA were used as controls. The culture was
continued and
3
H-thymidine (1 Ci [0.037 MBq]) was added 4 hours before
the end of the 72-hour culture. The cells were harvested and counted using a
1450 Microbeta TriLux apparatus (Perkin Elmer, Boston, MA). The T-cell
proliferation was represented as the incorporated radioactivity in counts per
minute (cpm). In selected experiments, the data were expressed as percent
change due to the variability in the baseline proliferative response to PHA
by hPBMCs from different donors. Percent change is defined as follows: %
change ⫽ [{(mean cpm of triplicate wells of hPBMC ⫹ PHA) ⫺ (mean
cpm of triplicate wells of hPBMC ⫹ hMSC ⫹ PHA)}/(mean cpm of
triplicate wells of hPBMC ⫹ PHA)] ⫻ 100.
PGE
2
determination
The levels for PGE
2
in the cell-culture supernatant were determined using
ELISA. For the PGE
2
inhibition experiments, the human MSCs were
resuspended in complete media in the presence or absence of PGE
2
inhibitors NS-398 (5 M; Cayman Chemicals, Ann Arbor, MI) or indometh-
acin (5 M; ICN Chemicals, Irvine, CA). These concentrations of PGE
2
inhibitors resulted in complete inhibition of PGE
2
secretion (⬍ 1-2 pg/mL)
and were chosen after performing a dose analysis (data not shown), and all
cells remained healthy at these concentrations. In order to examine if PGE
2
inhibitors had any effect on T-cell proliferation, the hMSCs were allowed to
adhere to the plate surface of a 96-well flat-bottom plate for 1 to 2 hours,
and hPBMCs from an unrelated donor (hMSC to PBMC ratio, 1:10), also
resuspended in PGE
2
inhibitor–containing culture media, were added in the
presence of the mitogen PHA (2.5 g/mL). Following 3 days of coculture,
3
H-thymidine was added during the last 4 hours, and the PBMCs were
harvested and
3
H-thymidine was counted using a 1450 Microbeta TriLux
apparatus. The experiment was performed with 3 unrelated hMSC-hPBMC
donor pairs.
In parallel experiments, the hMSCs were resuspended in complete
media containing or lacking the PGE
2
synthesis inhibitor NS-398 and were
plated in a 12-well plate (2 ⫻ 10
4
cells/mL). Isolated DC1 or CD45RA
⫹
T
cells from an unrelated donor were resuspended in media containing a PGE
2
inhibitor and added to the hMSC cultures under TNF-␣–producing (LPS;
1 ng/mL) or IFN-␥–producing (PHA; 5 g/mL) conditions. Following a
24-hour incubation, the levels of TNF-␣ from DC/hMSC cocultures and
levels of IFN-␥ from T-cell/hMSC cocultures were measured and calculated
as the percent change in cytokine levels between cultures with or without
PGE
2
inhibitor NS-398. The DCs or T cells treated with NS-398, without
the added hMSCs, were used as controls.
Statistical analysis
Data were presented as the mean ⫾ the standard deviation from 3 or more
experiments. The statistical significance was assessed by 2-tailed Student t test.
Results
MSC–immune cell interaction
Previous reports have demonstrated that MSCs are able to suppress
an ongoing immune response by inhibiting the T-cell proliferation
stimulated in a mixed-lymphocyte culture or by incubation with
mitogens.
40-44
Therefore, we first examined the hMSC-mediated
immunosuppressive effect of the hMSCs. All of the studies were
performed using HLA-unmatched donor populations of hMSCs
and hPBMCs. Human MSCs from 5 donors were cocultured with
hPBMCs isolated from 3 unrelated donors. Results from these
experiments are summarized in Table 1. In all the experiments, the
presence of hMSCs with hPBMCs resulted in a statistically
significant (P ⬍ .0001) decrease in PHA-induced proliferation.
Although the PHA-induced proliferation rate was different for each
donor hPBMC, the inhibition in the presence of hMSCs was
50% to 60%.
MSC-DC interaction. Purified populations of the DC1 were
cocultured with hMSCs as described in “Materials and methods.”
For DC1/hMSC cocultures, there was a significant (P ⬍ .0001)
decrease in TNF-␣ levels secreted. The absolute levels of LPS-
induced TNF-␣ differed for each donor DC1, however, the average
decrease in the cytokine levels in the presence of hMSCs from 5
independent experiments was 50 ⫾ 5% (Figure 1A). Treatment of
the hMSCs alone with GM-CSF, IL-4, or LPS did not result in any
detectable TNF-␣ production. As DC1 were matured using rIL-4,
which can interfere with IL-10 determination, levels of IL-10
secreted were not measured following DC1 activation. A similar
degree of hMSC-mediated TNF-␣ inhibition was seen within 16
hours when the hMSCs were cocultured with LPS-activated
monocytes (data not shown).
Table 1. Inhibition of PHA-stimulated PBMC proliferation by human MSCs
No.
MSCs
PBMC no. 1001 PBMC no. 1002 PBMC no. 11-30338
PBMCs ⴙ PHA PBMCs ⴙ PHA ⴙ MSCs PBMCs ⴙ PHA PBMCs ⴙ PHA ⴙ MSCs PBMCs ⴙ PHA PBMCs ⴙ PHA ⴙ MSCs
51 25 044 ⫾ 2849 9 717 ⫾ 1379 12 188 ⫾ 2105 5564 ⫾ 594 34 799 ⫾ 3312 10 474 ⫾ 3954
57 25 044 ⫾ 2849 12 727 ⫾ 958 12 188 ⫾ 2105 5434 ⫾ 4039 34 799 ⫾ 3312 15 159 ⫾ 3359
78 25 044 ⫾ 2849 11 236 ⫾ 1250 12 188 ⫾ 2105 7458 ⫾ 973 34 799 ⫾ 3312 11 649 ⫾ 2319
101 25 044 ⫾ 2849 12 070 ⫾ 2207 12 188 ⫾ 2105 6841 ⫾ 3334 34 799 ⫾ 3312 10 556 ⫾ 4461
151 25 044 ⫾ 2849 8 519 ⫾ 1359 12 188 ⫾ 2105 4172 ⫾ 582 34 799 ⫾ 3312 9521 ⫾ 597
Human MSCs from 5 different donors were plated onto a 96-well plate (2 ⫻ 10
4
cells/mL) in a 100-L volume. Following adherence of MSCs, PBMCs from an unrelated
donor were added in the presence of PHA (hMSC to PBMC ratio, 1:10).
3
H-thymidine incorporation was measured at 72 hours of coculture (set in triplicates) and data were
expressed as mean cpm ⫾ SD. The experiment was performed with 2 additional hPBMC donors. The cpm for MSC alone or hPBMC alone was less than 1500 in all
experiments.
hMSCs MODULATE ALLOGENEIC IMMUNE CELL RESPONSES 1817BLOOD, 15 FEBRUARY 2005
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The activated DC2 secreted moderate levels of IL-10 in culture
upon LPS stimulation. Figure 1B shows the percent increase in
IL-10 levels from 5 experiments when hMSCs were cocultured
with the activated DC2. The average increase in the IL-10 levels
(mean ⫾ SD) in the presence of hMSCs was 140 ⫾ 15% and was
statistically significant (P ⬍ .001). The treatment of the hMSCs
alone with IL-3 or LPS did not result in a significant increase in
IL-10 production. Changes in the TNF-␣ levels were not measured
in the activated DC2 because the preparation may contain approxi-
mately 10% DC1
⫹
, and therefore, may result in false positives
upon LPS stimulation.
MSC–T-cell interaction. Naive T cells were activated to differ-
entiate into T
H
1 effector cells in the presence or absence of hMSCs.
The effector T cells undergoing T
H
1 differentiation secreted
moderate levels of IFN-␥. However, when the hMSCs were present
during the T-cell differentiation, there was a significant (P ⬍ .0001)
decrease in the amount of IFN-␥ produced. As the baseline levels of
IFN-␥ produced by each donor’s cells differed, the data were
expressed as the percent change (mean ⫾ SD) in IFN-␥ in cultures
with or without hMSCs. The results in Figure 2A show that the
decrease in IFN-␥ levels in the presence of hMSCs from 3
independent experiments was 60 ⫾ 5%.
Naive T cells were also activated to differentiate into T
H
2
effector cells in the presence or absence of hMSCs. The effector T
cells undergoing T
H
2 differentiation secreted moderate levels of
IL-4. However, when hMSCs were present during the differentia-
tion, there was a significant (P ⬍ .0001) increase in the amount of
IL-4 produced. As the baseline levels of IL-4 produced by each
donor differed, the data were expressed as the percent change
(mean ⫾ SD) in secreted IL-4 in cultures with or without hMSCs.
Results (Figure 2B) showed that the average increase in IL-4 levels
in the presence of hMSCs from 3 independent experiments was
500 ⫾ 45%.
Figure 2C shows the paired comparison of the percentage of
CD4
⫹
CD25
⫹
regulatory T cells detected from 5 different experi
-
ments in the presence or absence of hMSCs. There was a significant
(P ⬍ .0001) increase in the T
Reg
population when the PBMCs were
cocultured with hMSCs (also see Supplemental Figures, available
on the Blood website; click on the Supplemental Figures link at the
top of the online article). Figure 2D shows a representative flow
cytometry scatter plot from 1 experiment.
MSC–NK cell interaction. Purified NK cells in culture will
secrete IFN-␥ when stimulated with IL-2. The results in Figure 3
show that when hMSCs were cocultured with IL-2–stimulated NK
cells, there was a statistically significant (P ⬍ .0001) decrease in
IFN-␥ levels produced. The amount of IFN-␥ produced by NK cells
upon IL-2 stimulation differed for each donor, however, the
average decrease in IFN-␥ secretion in the presence of hMSCs was
80 ⫾ 10%. The presence of IL-2 did not result in any detectable
IFN-␥ secretion from hMSCs alone.
PGE
2
as MSC-derived immune modulator
Previous reports from our colleagues have indicated that MSCs can
modify T-cell functions by soluble factor(s).
41,44,73
Therefore, we
attempted to identify molecules involved in the hMSC modulation
of immune cell activities. We observed that hMSCs in culture
secrete several factors including IL-6, IL-8, PGE
2
, and vascular
endothelial growth factor (VEGF). When PBMCs from unrelated
donors were added to hMSCs (hMSC to PBMC ratio, 1:10), the
levels of each factor significantly (P ⬍ .001) increased several fold
(Figure 4A). The experiments were repeated with 2 or more
independent hMSC/PBMC donor pairs and the data were presented
as the measured mean cytokine levels (pg/mL ⫾ SD). This
enhancement of PGE
2
production was reproducible also when the
hMSCs were incubated with the proinflammatory recombinant
cytokines TNF-␣ or IFN-␥ as shown in Figure 4B. The contribu-
tions of each cell type to the increase in secreted PGE
2
and VEGF
are under investigation.
As PGE
2
has been shown to modulate a wide variety of immune
functions in vitro,
59
we examined whether inhibiting production of
PGE
2
leads to reversal of any of the hMSC-mediated immunomodu
-
latory effects. The data depicted in Figure 5 demonstrate that, in the
presence of PGE
2
synthesis inhibitors, there was a more than 70%
Figure 1. MSCs alter cytokine secretion from DC1 and DC2. Human
MSCs were cocultured with (A) mature monocytic dendritic cells (DC1) or
(B) mature plasmacytoid dendritic cells (DC2) in the presence or absence
of inflammatory stimulus LPS (1 ng/mL). When hMSCs were present,
there was a more than 50% decrease in the secretion of TNF-␣ by
activated DC1 (TNF-␣ levels secreted by LPS-treated DC1 ⫽ 100%). The
coculture of hMSCs with DC2 consistently increased the secretion of IL-10
by more than 100% (IL-10 levels secreted by LPS-treated DC1 ⫽ 100%).
The graphs represent the cytokine levels (mean percent change ⫾ SD)
from 5 independent experiments in the presence or absence of hMSCs.
Figure 2. MSCs interact with T cells and induce a T
H
1toT
H
2 shift. Human MSCs were cocultured with hPBMCs from unrelated donors under (A) T
H
1-, (B) T
H
2-, or
(C) T
Reg
-inducing conditions as described. In the presence of hMSCs, there was a more than 50% decrease in IFN-␥ secreted from T
H
1 cells compared with controls without
hMSCs present (IFN-␥ levels secreted by T
H
1 cells alone ⫽ 100%). For T
H
2 cultures, there was a more than 500% increase in IL-4 produced when hMSCs were present
(control T
H
2 cells ⫽ 100%). Bars indicate the change in cytokine levels (mean % change ⫾ SD) in cultures incubated with or without hMSCs from 3 independent experiments.
For T
Reg
cultures, the proportion of CD4
⫹
CD25
⫹
cells from 5 experiments is plotted. Results indicated that when hMSCs were present, there was a consistent increase in the
proportion of CD4
⫹
CD25
⫹
T
Regs
. (D) A representative flow diagram from one experiment in which T
Regs
were generated with or without hMSCs is shown.
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increase in
3
H-thymidine incorporation and the difference was
statistically significant (P ⬍ .001). These stimulated levels were
similar to the levels achieved when hMSCs were not present,
suggesting that PGE
2
synthesis inhibitors negated the hMSC-
mediated immunosuppressive effects on T cells. Furthermore, in
the presence of a PGE
2
inhibitor, there was a statistically significant
increase in TNF-␣ and IFN-␥ from the activated DCs and T cells,
respectively (P ⬍ .001 and P ⬍ .005, respectively), indicating that
hMSC-secreted PGE
2
plays an important role in hMSC-mediated
immunomodulatory effects. The results from 3 independent experi-
ments are summarized in Table 2.
Discussion
In this paper, we have examined interactions between culture-
expanded hMSCs and different immune cells in order to better
understand the mechanisms of MSC-mediated immune modula-
tion. This is the first report showing that hMSCs interact with each
of the isolated cells of the immune system and that hMSCs are
capable of altering the outcome of the immune cell response by
inhibiting 2 of the most important proinflammatory cytokines (ie
TNF-␣ and IFN-␥) and by increasing expression of suppressive
cytokines, including IL-10. Mechanistically, we have shown that
inhibitors of PGE
2
synthesis mitigated the overall hMSC suppres
-
sive effects, suggesting that PGE
2
may be responsible for much of
the hMSC-mediated immunomodulatory effects in vitro.
The inhibition of TNF-␣ secretion by DCs has been shown to
inhibit their maturation, migration to lymph nodes, and ability to
stimulate allo-T cells by altering the expression of several receptors
and coreceptors necessary for antigen capture and processing.
60-62
We have not examined the effects of hMSCs on DC surface
expression, although recently, the effects of hMSCs on differentia-
tion, maturation, and function of monocyte-derived DCs were
reported by Zhang et al.
53
Using a similar in vitro MSC/DC
coculture system, these authors have shown that hMSCs inhibit the
up-regulation of several of the maturation markers on DCs,
resulting in their decreased capacity to activate allo-reactive T
cells. Our results are in agreement with this report, and further
strengthen the hypothesis that MSCs, due to their ability to inhibit
TNF-␣ secretion by DCs, may lead to an immunologic tolerance state.
The ability of MSCs to interact with HLA-unrelated immune
cells and modulate their response has important implications in
transplantation biology. Recipients of allogeneic transplants often
experience acute GVHD due to alloreactive T cells present in the
allograft.
63-66
Graft-versus-host disease involves a pathophysiology
that includes host tissue damage, increased secretion of proinflam-
matory cytokines (TNF-␣, IFN-␥, IL-1, IL-2, IL-12), and the
activation of DCs and macrophages, NK cells, and cytotoxic T
cells.
67
All of these events are very important components of
GVHD management, wherein inhibition of proinflammatory cyto-
kines has been shown to be beneficial in resolution of the severity
and incidence of GVHD.
68,69
Our in vitro data suggest that hMSCs,
in their ability to inhibit IFN-␥ and increase IL-4 secretion, may
orchestrate a shift from the prominence of proinflammatory T
H
1
cells toward an increase in anti-inflammatory T
H
2 cells, beneficial
for GVHD management. In fact, recently, Le Blanc et al
39
reported
that by using haploidentical hMSCs, they were able to treat a
patient suffering with acute GVHD. Our results may offer one
explanation for the clinical outcome observed by the authors.
Several studies have now shown that hMSCs in culture can
mediate suppression of T-cell proliferation by a secreted factor.
Among the favored explanations is the secretion of immunosuppres-
sive cytokines TGF- and/or IL-10 by MSCs.
41,44
However,
blocking these factors with antibodies does not completely reverse
the MSC-mediated immunosuppression.
41,44
Another potential
mechanism to explain the MSC-mediated immunosuppression is a
veto cell–like activity.
42
However, veto cells suppress cytotoxic T
lymphocyte (CTL) precursor functions against antigens present on
their own cell surface, but not against third-party allogeneic cells.
This suppression mechanism contrasts with the extensive data with
Figure 4. MSCs secrete important factors, the levels of which increase
upon PBMC coculture. Human MSCs and human PBMCs isolated from
unrelated donors were cocultured (MSC to PBMC ratio, 1:10) for 24 hours, and
cell supernatants were collected and analyzed for various secreted factors by
ELISA. (A) Human MSCs secreted factors IL-6, IL-8, VEGF, and PGE
2
, and the
levels of each of these factors increased more than 3-fold upon hPBMC
coculture (IL-6: 6-fold; IL-8: 6-fold; VEGF: 3-fold; and PGE
2
: 10-fold). Bars
represent cytokine levels (mean pg/mL ⫾ SD) from 2 independent hMSC and
1 hPBMC donors. (B) Human MSCs from 3 independent donors were cultured
in the presence of TNF-␣ and IFN-␥ for 24 hours, following which PGE
2
levels in
the supernatant were determined using ELISA. The results shown are from
duplicate cultures performed in parallel (mean ⫾ SD).
Figure 3. MSCs inhibit IFN-␥ secretion from purified NK cells. Natural killer cells
were purified and cocultured with hMSCs for different times in the presence of rhIL-2.
The levels of IFN-␥ in the supernatant were quantified in the presence or absence of
hMSCs after 24 hours of coculture. The bars show the percent change in IFN-␥
secreted compared with IL-2–stimulated NK cells ( ⫽ 100%). When hMSCs were
present, there was a more than 80% decrease in levels of IFN-␥ secreted by
IL-2–stimulated NK cells. Results from 3 independent hMSC/NK experiments are
plotted. Error bars represent mean ⫾ SD.
Figure 5. MSCs’ immunomodulatory effects are mediated in vitro via secretion
of PGE
2
. Human MSCs and PBMCs from unrelated donors were cocultured in media
containing PGE
2
inhibitors indomethacin (Indo) or NS-398 for 3 days in the presence
of PHA (2.5 g/mL).
3
H-thymidine incorporation was measured as an indication of
PBMC proliferation and data were presented as percent change in incorporated
3
H-thymidine in presence or absence of inhibitors (hPBMCs stimulated with PHA in
absence of MSCs ⫽ 100%). Bars represent percent change in
3
H-thymidine (mean ⫾
SD) from 3 independent experiments.
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MSCs wherein T-cell proliferation is inhibited across strain (allo-)
and across species (xeno-).
40,41,44,49
Therefore, the veto cell–like
activity may play an important role in the low immunogenecity of
MSCs, but certainly does not explain the suppression of T-cell
proliferation by the MSCs. More recently, the role of trypto-
phan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO)–
mediated tryptophan degradation has been suggested to play a role
in the MSC-mediated immunosuppression.
70
In that report, Meisel
et al have shown that MSCs, in the presence of IFN-␥, express IDO
activity that in turn degrades essential tryptophan in the media, and
hence, may lead to inhibition of T-cell proliferation.
70
Although
tryptophan degradation may contribute to the inhibition of T-cell
proliferation at later time points (the authors show appearance of
IDO expression at 5 days of MSC ⫹ IFN-␥ culture), this is in
disagreement with the inhibition kinetics of T cells by MSCs
(within 2 days in the case of PHA stimulation). Furthermore, a
previous report
44
and our own work demonstrate that MSC/PBMC
coculture does not lead to T-cell apoptosis, normally a definite
outcome of tryptophan depletion from the media.
We have shown that hMSCs can secrete PGE
2
in quantities that
may be sufficient to be involved in MSC-mediated immunomodula-
tion. Spontaneous and continuous production of cyclooxygenase-2
(COX-2)–dependent PGE
2
has been observed in murine small
intestine lamina propria cells
71
and cells lining the airway mucus
membranes,
72
probably as a mechanism to down-regulate an
inappropriate inflammatory response to nonpathogenic antigens
residing in the environmental flora of these organs. Tse et al
45
also
reported low levels of PGE
2
secretion by MSCs, but they did not
see significant reversal in T-cell proliferation when using PGE
2
synthesis inhibitors. This disagreement with the results reported
Figure 6. Proposed mechanisms of action of MSCs. We propose that MSCs mediate their immunomodulatory effects by interacting with cells from both the innate (DC,
pathways 2-4; NK cell, pathway 6) and adaptive immunity systems (T cell, pathways 1 and 5). MSC inhibition of TNF-␣ secretion and promotion of IL-10 secretion may affect DC
maturation state and their functional properties, resulting in skewing the immune response toward in an anti-inflammatory/tolerant phenotype. Alternatively, when MSCs are
present an inflammatory microenvironment, they inhibit IFN-␥ secretion from T
H
1 and NK cells and increase IL-4 secretion from T
H
2 cells, thereby promoting a T
H
1 3 T
H
2 shift.
It is likely that MSCs also mediate their immunomodulatory actions by direct cell-cell contact as well as by secreted factors. Several MSC cell-surface molecules and secreted
molecules are depicted. CCL indicates chemokine ligand; TCR, T-cell receptor.
Table 2. Cytokine secretion and effect of PGE
2
inhibitor NS-398 on immune cells
Cytokine secretion
DCs, TNF-␣, pg/mL* T cells, IFN-␥, pg/mL†
Without MSCs With MSCs Without MSCs With MSCs
Inhibitor ⫺⫹⫺⫹⫺ ⫹⫺ ⫹
Experiment no. 1 789 ⫾ 42 880 ⫾ 46 460 ⫾ 44 624 ⫾ 92 1750 ⫾ 32 1600 ⫾ 58 695 ⫾ 100 1390 ⫾ 121
Experiment no. 2 1630 ⫾ 55 1600 ⫾ 57 289 ⫾ 12 1289 ⫾ 57 2287 ⫾ 60 2800 ⫾ 75 1235 ⫾ 80 2764 ⫾ 85
Experiment no. 3 458 ⫾ 30 524 ⫾ 38 144 ⫾ 64 369 ⫾ 48 3196 ⫾ 101 ND 1657 ⫾ 34 2677 ⫾ 74
ND indicates not done; ⫺, no inhibitor; ⫹,5M NS-398.
*Human MSCs and mature DCs were cocultured with or without PGE
2
inhibitor NS-398 in the presence of activating stimulus LPS. The TNF-␣ levels in the supernatant
were determined by ELISA. Inhibition of PGE
2
always resulted in an increase in TNF-␣ levels.
†Human MSCs and T cells were cocultured with or without PGE
2
inhibitor NS-398 in the presence of mitogen PHA and levels of IFN-␥ were determined by ELISA. Results
show that inhibition of PGE
2
always resulted in an increase in IFN-␥ levels.
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herein may be due to differences in experimental design. We
observed that hMSCs exhibit a bell-shaped time-dependent curve
of PGE
2
secretion (ie, after an initial increase there is a decrease in
levels of PGE
2
after 4 to 5 days in culture [data not shown]).
Therefore, an inability to observe any significant effect of PGE
2
inhibitors by Tse et al
45
may be due to performing their evaluations
at 5 days.
As shown here, hMSCs, when cocultured with immune cells
resulted in increased PGE
2
and VEGF secretion. This can be
explained only by a synergistic rather than an additive effect,
suggesting MSC–immune cell cooperation at sites of inflammation.
Although the exact contributions of each cell source to the
increased levels of secreted factors are not known, our preliminary
experiments in which human VEGF secretion was measured
following rat MSCs (rMSCs)–human PBMCs coculture, and vice
versa, showed that the hPBMCs, following coculture with rMSCs,
increased secretion of human VEGF (data not shown). What other
PGE
2
-secreting cells are also capable of mediating this effect
remains to be explored.
To the question of whether differentiated MSCs induce a T-cell
proliferative response, we detected comparable amounts of PGE
2
in
supernatants derived from cultures under control and adipocyte
differentiation conditions (data not shown), and therefore, expect
comparable levels of inhibition of T-cell proliferation. Le Blanc et
al found that differentiated MSCs had immunologic properties
similar to the undifferentiated MSCs.
46
We have detected alloge
-
neic MSCs in vivo in several animal models at extended time
periods (months) wherein the allo-MSCs expressed markers of
differentiated cell types, suggesting differentiated MSCs are not
rejected. However, some caution should be exercised in predicting
the in vivo role of MSC-mediated PGE
2
secretion, as it could be an
in vitro phenomenon. Development of an animal model is under
way to investigate the in vivo effects further.
To summarize, through the interactions of hMSCs with the
various immune cells, it appears that hMSCs inhibit or limit
inflammatory responses and promote the mitigating and anti-
inflammatory pathways. A proposed model of these interactions is
shown in Figure 6, which illustrates that hMSCs alter the outcome
of the immune response by inhibiting inflammatory DC1 signaling
(pathway 2) and promoting anti-inflammatory DC2 signaling
(pathway 4). Also, when immature effector T cells are present,
hMSCs may interact with them directly and inhibit the develop-
ment of proinflammatory T
H
1 and NK signaling (pathway 1 and 6,
respectively) and promote anti-inflammatory T
H
2 (pathway 5)
and/or suppressive T
Reg
signaling (pathway 3). The results imply
that when hMSCs are present in an inflammatory environment
(such as that artificially created by activating DCs, macrophages,
NK cells, or T cells using various stimuli), they may alter the
outcome of the on-going immune response by altering the cytokine
secretion profile of DC subsets (DC1 and DC2) and T-cell subsets
(T
H
1, T
H
2, or T
Regs
), thereby resulting in a shift from a proinflam
-
matory environment toward an anti-inflammatory or tolerant cell
environment. Such a model of the hMSC–immune cell interaction
is consistent with the experiments reported here.
In conclusion, while the complete mechanism of immune
modulation by MSCs will require further investigations, the
studies presented here provide a working model to understand
MSC-mediated immunomodulation that may be an active com-
ponent in inflammation modulation, tolerance induction, and
reduction of transplantation complications such as rejection
and GVHD.
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VOLUME 105, NUMBER 4
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