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Mesenchymal stromal cells promote or suppress the proliferation of Tlymphocytes from cord blood and peripheral blood: the importance of low cellratio and role of interleukin-6Mehdi Najar a; Redouane Rouas a; Gordana Raicevic a; Hichame Id Boufker a; Philippe Lewalle a; NathalieMeuleman a; Dominique Bron a; Michel Toungouz a; Philippe Martiat a; Laurence Lagneaux a
a Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels,Belgium
First Published on: 28 June 2009
To cite this Article Najar, Mehdi, Rouas, Redouane, Raicevic, Gordana, Id Boufker, Hichame, Lewalle, Philippe, Meuleman, Nathalie,Bron, Dominique, Toungouz, Michel, Martiat, Philippe and Lagneaux, Laurence(2009)'Mesenchymal stromal cells promote orsuppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role ofinterleukin-6',Cytotherapy,99999:1,
To link to this Article: DOI: 10.1080/14653240903079377
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Mesenchymal stromal cells promote or suppressthe proliferation of T lymphocytes from cord bloodand peripheral blood: the importance of low cell
ratio and role of interleukin-6
Mehdi Najar*, Redouane Rouas*, Gordana Raicevic, Hichame Id Boufker,
Philippe Lewalle, Nathalie Meuleman, Dominique Bron, Michel Toungouz,
Philippe Martiat and Laurence Lagneaux
Laboratory of Experimental Hematology, Institut Jules Bordet, Universite Libre de Bruxelles (ULB), Brussels, Belgium
Background aims
Mesenchymal stromal cells (MSC) have been shown to possess
immunomodulatory functions and proposed as a tool for managing or
preventing graft-versus-host disease (GvHD) as well as promoting
clinical transplantation tolerance. We investigated the capacity of
human bone marrow (BM) MSC to modulate the proliferation of T
cells obtained from peripheral blood (PB) and umbilical cord blood
(CB). We addressed the importance of the MSC:T-cell ratio,
requirement for cell contact and impact of soluble factors on the
MSC-mediated effects. We also analyzed whether regulatory T cells
could be modulated by MSC in co-cultures.
Methods
The effect of different MSC concentrations on T-cell proliferation
induced by allogeneic, mitogenic or CD3/CD28 stimulation was
analyzed using bromodeoxyuridine (BrdU) incorporation and
carboxyfluorescein diacetate�succinimidyl ester (CFDA-SE) labeling.
The level of regulatory T cells was assessed using quantitative real-
time polymerase chain reaction (PCR) and flow cytometry analysis.
Results
MSC induced a dose- and contact-dependent inhibition of T-cell
proliferation but lymphocytes from CB and PB were differentially
affected. At low concentrations, MSC supported both CB and PB T-cell
proliferation, rather than inhibiting their proliferation. This
supportive effect was contact independent and soluble factors such
interleukin-6 (IL-6) appeared to be involved. Interestingly, among the
expanded T-cell population in both CB and PB, regulatory T cells were
increased and were a part of the new cells promoted by MSC at low
doses.
Conclusions
MSC represent an attractive tool for reducing the lymphocyte response
by inhibiting T-cell activation and proliferation as well as promoting
tolerance by maintaining and promoting the expansion of regulatory
cells. Nevertheless, the dual ability of MSC to either sustain or
suppress T-cell proliferation according to conditions should be
considered in the context of clinical applications.
Keywords
Allogeneic T cells, immunosupportive, immunosuppressive, interleukin-
6, mesenchymal stromal cells.
IntroductionMesenchymal stromal cells (MSC) are multipotent pro-
genitors that can be isolated from various adult and fetal
tissues [1�3]. They are self-renewing cells that are capable
of supporting hematopoiesis and differentiating along
multiple mesenchymal and non-mesenchymal lineages,
including osteocytes, chondrocytes, adipocytes, myocytes
and cells of the central nervous system [4,5]. Because of
Correspondence to: Professor Philippe Martiat, MD, PhD, Universite Libre de Bruxelles, Institut Jules Bordet, Laboratoire d’Hematologie
Experimentale, Blvd de Waterloo no. 121�1000 Bruxelles, Belgium. E-mail: pmartiat@ulb.ac.be
*The two first authors contributed equally to this work.
Cytotherapy (2009) Vol. 00, No. , 1�14
– 2009 ISCT DOI: 10.1080/14653240903079377
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
their differentiation capacities, MSC have emerged as
promising tools for tissue repair and regenerative medi-
cine, as well as cell and gene therapy. MSC are not
inherently immunogenic because they do not constitu-
tively express major histocompatibility complex (MHC)
class II antigens or co-stimulatory molecules. Additionally,
MSC have been shown to possess immunomodulatory
properties. These characteristics have generated clinical
interest in using MSC to improve the efficiency of
hematopoietic stem cell transplantation (HSCT), manage
graft-versus-host disease (GvHD) and modulate autoim-
mune disorders [6].
In vitro, MSC exert immunosuppressive effects through
the regulation of different immune cells by several
mechanisms. It is well established that MSC can suppress
T-lymphocyte proliferative responses induced by dendritic
cells (DC) [7], alloantigens and mitogens [8], CD3/CD28
agonists and cognate peptide [9]. The inhibitory effects of
MSC are dose-dependent [8] and MSC target equally any
T-lymphocyte subset (CD4�, CD8�, CD2� and CD3�
subpopulations). MSC act on unstimulated T cells by
preventing their activation. However, when T cells are
already stimulated, MSC reduce the expression levels of
their activation markers. Nevertheless, there is still con-
flicting data in the literature regarding the mechanisms by
which MSC modulate immune cells. These potential
mechanisms include both direct cell�cell contact as well
as the production of immunoregulatory factors. Several
soluble factors produced by MSC have been reported to
mediate the suppression of T-cell proliferation. These
factors include prostaglandin E2 (PGE2), hepatic growth
factor (HGF), transforming growth factor-b (TGF-b),
interferon-g (IFN-g), interleukin (IL)-10, leukemia inhi-
bitory factor (LIF), human leukocyte antigen-G (HLA-G)
and indoleamine 2,3-dioxygenase (IDO) [10]. MSC differ-
entiation into various mesenchymal lineages does not alter
their interaction with T cells, and the exposure of MSC to
IFN-g increases their inhibitory effect [11,12].
MSC promote the survival and inhibit the proliferation
and maturation of B cells by arresting them in the G0/G1
phase of the cell cycle [13]. In addition, MSC have been
reported to induce both stimulation and impairment of
immunoglobulin production by B lymphocytes without
affecting co-stimulatory molecule expression and cytokine
production [14,15]. It has been observed that the functions
of natural killer (NK) cells, the major effectors of innate
immunity, are also affected by MSC. MSC alter the
phenotype of NK cells, suppress cytokine-induced pro-
liferation of freshly isolated NK cells and prevent the
induction of effector functions [16]. The differentiation,
maturation and function of DC are altered by MSC [17].
Moreover, MSC that are co-cultured with DC inhibit the
alloreactivity of T cells and induce the generation of
alloantigen-specific regulatory T cells [18]. MSC induce a
cytokine profile shift in the T-helper (Th)1/Th2 balance
towards the anti-inflammatory Th2 phenotype [19] and
contribute to the expansion of FoxP3� regulatory T cells
[20]. All these findings demonstrate that MSC act
as pleiotropic immune regulators to suppress immune
responses through the production of multiple soluble
factors and/or direct cell�cell contact in order to affect
all the actors of immune responses: T cells, NK cells, B
cells and DC.
The aim of this study was to compare the capacity of
human bone marrow (BM) MSC to modulate the
proliferation of T-cell subsets obtained from adult periph-
eral blood (PB) and umbilical cord blood (CB); the latter is
considered a source of naive T cells. We investigated the
importance of the MSC:T-cell ratio, requirement of cell
contact, impact of soluble factors and effect on regulatory
T cells during co-culture.
MethodsHuman MSC culture and expansion
BM was harvested from the sternum or iliac crest of 10
healthy volunteers after informed consent. The mean age
of the donors was 3392 years (range 18�41 years).
Mononuclear cells (MNC) were isolated by density-
gradient centrifugation (LinfoSep, Biomedics, Madrid,
Spain), washed in Hank’s buffered salt solution (HBSS,
Lonza Europe, Verviers, Belgium) and seeded at 2�104
cells/cm2 in Dulbecco’s modified Eagle medium-low
glucose (DMEM-LG; Lonza) supplemented with 15%
fetal bovine serum (FBS; Sigma-Aldrich, Bornem,
Belgium), 2 mM L-glutamine and 50 U/mL penicillin
(both from Lonza). Cell cultures were incubated at 378C in
a 5% CO2 humidified atmosphere. After 48 h, non-
adherent cells were removed by washing and the medium
was changed twice a week. When subconfluency (80�90%)
was achieved, adherent cells were trypsinized (Lonza) and
expanded by replating at a lower density (200 cells/cm2).
MSC were immunophenotypically characterized by
flow cytometry using the following monoclonal antibodies
(MAb): anti-CD166�fluorescein isothiocyante (FITC;
2 M. Najar et al.
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DakoCytomation, Glostrup, Denmark), anti-CD45�FITC
and anti-HLA-DR�phycoerythrin (PE; Exalpha Biologi-
cals, Maynard, MA, USA), anti-CD34�PE and anti-
CD73�PE (BD Biosciences Pharmingen, San Diego, CA,
USA), anti-CD14�PE, anti-CD105�FITC and anti-
CD90�PE (R&D Systems, Minneapolis, MN, USA). A
colony-forming unit�fibroblast (CFU-F) assay was
performed after each passage to estimate the number of
mesenchymal progenitors in the culture. To study
their multilineage potential, MSC were cultured in the
appropriate induction medium to assess adipogenic,
osteogenic and chondrogenic differentiation, as described
previously [21].
Preparation of MSC conditioned media
We prepared conditioned media (CM) from MSC cultured
alone at a low concentration (105 cells/mL, which
corresponded to a 1:40 MSC:T-cell ratio culture) for 3
days. The supernatants were collected and frozen at �208Cuntil further use.
Purification of T-lymphocyte populations
PB samples were obtained from healthy donors after
informed consent. Umbilical CB samples were collected
after full-term delivery and after obtaining consent of the
informed mothers. MNC were obtained as described for
BM MNC. CD3� T lymphocytes were purified by
positive selection using the MACS system (Miltenyi
Biotec GmbH, Bergisch, Germany), according to the
manufacturer’s instructions. CD45RA� and CD45RO�
T cells were selected by combining negative selection to
eliminate CD3� T cells and positive selection to select
naive T cells (CD45RA�). The purity of the selected cells,
as determined by flow cytometry, was always above 95%.
CD4� CD25high CD127low regulatory T cells (Tregs)
were isolated from adult peripheral blood mononuclear
cells (PBMC) using Miltenyi Biotec’s CD4� CD25�
CD127dim/� regulatory T-cell isolation kit according to the
manufacturer’s instructions. CD4� CD25� T cells of the
negative fraction were considered further as Treg-depleted
T cells
T-cell activation
For mitogenic stimulation, cells were stimulated with 5
mg/mL phytohemagglutinin (PHA; Remel Europe, Dart-
ford, UK) and 20 U/mL IL-2 (Biotest AG, Dreieich,
Germany). T-cell proliferation was also induced by anti-
CD3/CD28-coated Dynabeads (Dynal, Biotech, Oslo,
Norway), as described by the manufacturer. For allogeneic
stimulation, we performed, in triplicates, mixed leukocyte
reactions (MLR) in 96-well plates. The MLR were
performed with irradiated (25 Gy) allogeneic PBMC to
stimulate the T cells, and multiple PBMC:T-cell ratios
(3:1, 1:1, 1:2) were tested.
MSC and T-cell co-cultures
MSC, obtained after one or two passages, were plated at
4�103 cells/cm2, which corresponded to 8�103 MSC/
mL in a flat-bottomed 24-well plate. After a short period of
adherence, allogeneic T lymphocytes purified from PB
and CB were incubated with the plated MSC for 5 days of
co-culture in RPMI-1640 medium supplemented with
10% FBS. We tested several MSC:T-cell ratios (from
1:80 to 1:1) to investigate their importance in the MSC-
mediated effects. To assess the role of cellular interactions,
we used a Transwell† system (Transwell Permeable
Supports, Life Sciences, Acton, MA, USA). In some
experiments, MSC CM (50% final volume) was added to
T-cell cultures in order to study the stimulatory effects of
MSC (n�7). We also assessed the impact of anti-IL-6 and
anti-stromal cell-derived factor-1a (anti-SDF-1a) neutra-
lizing antibodies (Ab) used at different concentrations (0.1,
1, 5 and 10 mg/mL) on the supportive effect exerted by
MSC on T-cell proliferation (n�7). These Ab were all
purchased from R&D Systems Europe, Abingdon, UK.
T-cell proliferation assay
Lymphocyte proliferation was assessed by bromodeoxyur-
idine (BrdU) incorporation or after carboxyfluorescein
diacetate�succinimidyl ester (CFDA-SE) labeling. For
BrdU incorporation (Roche Applied Science, Mannheim,
Germany), 5-day MLR cultures (n�9) were run in the
presence or absence of different concentrations of irra-
diated (25 Gy) MSC. On day 4 of the co-cultures, 50 mM
BrdU were added. The T-cell response was evaluated by
measuring BrdU incorporation in a colorimetric assay.
T-cell alloproliferation was expressed by the proliferation
index (PI), which is defined as the ratio between the optical
density (OD) of activated T-cell proliferation and the OD
of inactivated T cells, after eliminating the background.
For CFDA-SE labeling (CellTraceTM CFSE cell pro-
liferation kit; Invitrogen, Molecular Probes, Eugene, OR,
USA), 10 mM CFDA-SE dye was used to stain 107 T cells
before co-incubation with MSC. After 5 days of co-culture
Bifunctionality of MSC on T lymphocytes 3
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(n�8), CFSE fluorescence was analyzed by flow cytome-
try. The CFSE profile of the labeled cells was composed of
several distinctive peaks representing the number of cell
divisions that the proliferated lymphocytes had undergone
after activation. T-cell proliferation was expressed by the
mean generation number (MGN). CD3� T lymphocytes
were gated according to their forward- and side-scatter
features in order to exclude dead cells and cell debris;
5000�10,000 gated events were usually acquired. Samples
were run on a FACS Calibur (BD Biosciences) and
analyzed using CellQuest software (BD Biosciences).
In order to assess whether the MSC anti-proliferative
effect was associated with the inhibition of T-cell activa-
tion, we evaluated the impact of MSC on lymphocyte
activation marker expression, such as CD38 by flow
cytometry (n�7). An anti-CD38�PC5 Ab from Immuno-
tech Marseille, France was used for this assay.
T-cell viability assay
The impact of MSC on lymphocyte viability was assessed
using a trypan blue exclusion assay (Immunotech 7). T
cells, alone or with MSC at the indicated ratios, were
cultured in 24-well plates during 5 days. We also assessed
T-cell viability by using the BD Via-ProbeTM viability
staining solution (7-amino-actinomycin D; 7-AAD) com-
bined with CD45 labeling. In this case, T cells were first
stained with an anti-CD45�VioBlue (Miltenyi)-labeled
MAb to exclude MSC and then stained with 7-AAD
solution. Absolute volumetric cell counting was performed
with a MACSQUANT† flow cytometer.
Cytokine quantification assay
IL-6 and SDF-1a levels were measured in cell culture
supernatants (n�5) using enzyme-linked immunosorbent
assay (ELISA) techniques according to the manufacturer’s
instructions (Assay Designs, Ann Arbor, MI, USA, 6.01 pg/
mL sensitivity for IL-6; R&D Systems, 1�47 pg/mL
sensitivity for SDF-1a).
Detection of FoxP3� regulatory T cells
Detection of FoxP3 was performed using a quantitative
real-time polymerase chain reaction (qRT-PCR) for gene
expression and intracellular flow cytometry analysis for
protein expression. For qRT-PCR, total RNA was extracted
with Trizol reagent according to the manufacturer’s guide-
lines (Invitrogen) and first-strand cDNA was synthesized by
reverse transcription (Superscript first-strand synthesis
system for RT-PCR kit; Invitrogen). mRNA expression
was measured by qRT-PCR using a TaqMan Master mix kit
(PE Applied Biosystems, Foster City, CA, USA) on a 7900
HT sequence detection system (PE Applied Biosystems).
EF1-a mRNA was used as an internal control. The primers
and internal fluorescence TaqMan probes were designed
as follows: FoxP3 forward 5?-TTTCACCTACGC-
CACGCTCA-3?, reverse 5?-CCAGCTCATCCACGG-
TCCA-3? and probe 5?-FAM-CCACCTGGAA-
GAACGCCATCCGC-TAMRA-3?; EF1-a forward
5?-CTGAACCATCCAGGCCAAAT-3?, reverse 5?-GCCGTGTGGCAATCCAAT-3? and probe 5?-FAM-
AGCGCCGGCTATGCCCCTG-TAMRA-3?. The pro-
gram used for amplification was 10 min at 958C followed by
40 cycles of 15 s at 958C and 1 min at 608C.
FoxP3 intracellular staining was performed using the
Alexa Fluor† 647 anti-human FoxP3 flow kit (BioLegend,
San Diego, CA, USA), according to the manufacturer’s
instructions. Flow cytometry analysis was performed on a
FACScalibur flow cytometer using CellQuest software (BD
Biosciences).
Statistical analysis
Data are expressed as the mean9standard error of the
mean (SEM). Statistical comparisons were performed
using the paired Wilcoxon’s test. P-values lower than
0.05 were considered statistically significant.
ResultsGeneration and characterization of human MSC
MSC cultures were obtained from 20 mL BM aspirates
obtained from healthy donors. The mean duration of the
primoculture to reach subconfluence was 1392 days. After
one passage (P1), adherent cells displayed a fibroblast-like
morphology and were uniformly positive for CD73, CD90,
CD105 and CD166, but negative for CD14, CD34, CD45
and HLA-DR (Figure 1). The mean number of CFU-F
obtained from the BM samples was 4598/106 cells,
confirming the low level of MSC in BM. After P1, the
mean number of CFU-F was 90916�103, demonstrating
the expansion of MSC. The CFU-F efficiency remained
stable throughout the duration of the culture. For each new
MSC culture, the differentiation into osteocytes, adipo-
cytes and chondrocytes was performed after P1 and P2 to
confirm their multilineage potential (data not shown).
4 M. Najar et al.
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
MSC immunosuppressive effects on
activated T cells
MSC inhibit allogeneic PB T-cell proliferation in a BrdU assay
In a one-way MLR, MSC inhibited CD3� T-cell
alloproliferation in an MSC concentration-dependent
manner, as shown by reduced BrdU incorporation
(Figure 2). In the presence of strong allostimulation, an
MSC:CD3� ratio of 1:1 efficiently and significantly
inhibited T-cell proliferation, as the PI decreased from
9.6691.51 to 1.9490.59, representing 8098% inhibition
(PB0.04). For lower MSC:CD3� ratios, only the 1:10
ratio allowed significant inhibition (47.294.3%; PB0.04).
The magnitude of inhibition at the 1:10 ratio was smaller
than that observed for the 1:1 MSC:CD3� ratio. Inhibition
was still observed at a PBMC:CD3� ratio of 1:1, but
the degree of inhibition was smaller. Interestingly, in
some experiments where allostimulation was weaker
(PBMC:CD3� 1:2 ratio), only the high MSC:CD3� ratio
(1:1) induced significant T-cell inhibition. At lower
MSC:CD3� ratios, we observed an MSC-mediated im-
munostimulatory effect on the lymphocyte response.
Indeed, low numbers of MSC seemed to increase the T-
cell BrdU incorporation, and the PI increased from 8.069
2.15 to 10.0492.88 (the PI was �25% greater than the
control; PB0.04).
Lymphocyte proliferation induced by the PHA/IL-2
cocktail or CD3/CD28 agonists was measured in the
presence or absence of MSC. T-cell proliferation was
assessed by the BrdU incorporation assay after 5 days of
Figure 1. Flow cytometry characterization of human MSC. BM MSC at P1 were stained with specific MAb (black line) against CD14, CD34,
CD45, HLA-DR, CD73, CD90, CD105 and CD166. White lines indicate isotype-matched mouse IgG Ab control staining. Representative figure
(immunophenotyping was done for all donors).
Figure 2. MSC inhibit allogeneic T-cell proliferation. Different
PBMC:CD3� ratios were used to perform the MLR in the presence
or absence of various MSC concentrations. T-cell proliferation was
assessed by a BrdU incorporation assay after 5 days of culture. A
significant inhibition of the lymphocyte response was observed for high
MSC:T-cell ratios (*PB0.03). Data are expressed as the mean9
SEM PI of nine independent experiments.
Bifunctionality of MSC on T lymphocytes 5
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
co-culture. High MSC:T-cell ratios efficiently inhibited T-
cell proliferation (Table I).
MSC inhibit mitogenic PB T-cell proliferation in the CFSE assay
After 5 days of co-culture, MSC inhibited PHA/IL-2-
induced PB T-cell proliferation in a dose-dependent
manner, as characterized by a decrease of CFSE peak
generation number. In the presence of direct contact
between the two protagonists, MGN was significantly
(PB0.04) and strongly reduced at high MSC:CD3� ratios
(1:8 and 1:4) (Figure 3A). Indeed, the MGN decreased
from 6.390.31 to 3.1490.32 and 2.8690.24 for the 1:8
and 1:4 ratios, respectively. In the absence of direct contact
(using the Transwell system), the inhibition was less
effective than that observed in the case of direct contact,
and was only significant (PB0.04) for the 1:4 MSC:CD3�
ratio.
MSC inhibit mitogenic CB T-cell proliferation in CFSE assay
The inhibition of PHA/IL-2-activated CB CD3� cells by
MSC was slightly different (Figure 3B), as only direct cell
contact and high MSC:CD3� ratios (1:8 and 1:4) could
significantly (PB0.04) inhibit T-cell proliferation. In
contrast to PB CD3� cells, MSC failed to inhibit CB T-
cell proliferation when the Transwell system was used,
regardless of the MSC:CD3� ratios. Thus CB T cells
seemed less sensitive to MSC inhibition than PB CD3�
cells. Several phenotypical and functional differences have
been reported between PB and CB T lymphocytes [22�24]. The majority of CB T lymphocytes appeared to be
phenotypically immature, as the majority of CB CD3�
cells (96.2591%) were naive T lymphocytes expressing
the CD45RA isoform antigen. In contrast, PB presented a
similar proportion of CD45RA� (45.2295.28 %) and
CD45RO� (51.9296.75 %) CD3� cells (Table II). After
immunoselection, total PB CD3�, purified CD45RA�
and CD45RO� T-cell populations were co-cultured in
direct contact at a high MSC:T-cell ratio (1:4) in order to
investigate their sensitivity to immunosuppression. On
day 5, PHA/IL-2-induced proliferation of the three
T-cell subsets was reduced by MSC, but not completely
Table I. MSC inhibit activated T-cell proliferation.
Lymphocyte PI
T-cell activation Without MSC With MSC
PHA/IL-2 cocktail 6.7390.28 2.9890.39a
CD3/CD28 agonist 8.1490.19 2.8890.11a
T lymphocytes activated with either a PHA/IL2 cocktail or CD3/CD28
agonists were cultured in the presence of MSC at high concentrations
(MSC:T-cell 1:1). T-cell proliferation was assessed by BrdU assay after 5
days of co-culture. A significant inhibition of lymphocyte response was
observed in the presence of MSC, as shown by the reduction of the PI
(aPB0.05). Data are expressed as mean PI9SEM of seven
Figure 3. MSC inhibit mitogenic PB and CB T-cell proliferation. In
each experiment, CFSE-labeled PB and CB T cells were activated by
PHA/IL-2 and then co-cultured with different MSC concentrations
for 5 days in the presence or absence of direct contact. For adult PB
(A), T-cell inhibition, as shown by the reduction of mean generation
number, was dependent on the MSC:T-cell ratio. MSC in contact with
T cells induced significant inhibition of lymphocyte proliferation at
high cell ratios (1:4 and 1:8). When co-cultures were performed in the
absence of direct contact, only the 1:4 cell ratio allowed significant
inhibition to occur. For CB (B), T-cell inhibition by MSC, as shown by
the reduction of the mean generation number, occurred in a dose- and
contact-dependent manner. At high ratios, MSC in direct contact with
T cells significantly inhibited lymphocyte proliferation, but MSC failed
to exert such inhibition in Transwell experiments. Data are expressed
as the mean9SEM generation number from eight independent
experiments (*PB0.04).
6 M. Najar et al.
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abrogated as described previously. Meanwhile, the MSC
suppressive profile of naive T-cell (CD45RA�) prolifera-
tion was different, as the percentage of inhibition was
significantly (PB0.04) weaker than that observed for total
CD3� cells and purified CD45RO� cells (Figure 4).
MSC reduce CD38 expression on activated
T cells
Several cell-surface antigens, including CD38, have been
described as up-regulated on T cells following activation
with PHA [25]. CD38 expression on T cells from both PB
and CB was increased (59.496.33% and 7894.2%,
respectively) after PHA/IL-2 activation. However, the
expression of CD38 on activated T cells was significantly
(PB0.03) down-regulated by MSC (direct co-culture).
The decrease in lymphocyte CD38 expression was gradual
and dependent on the MSC:CD3� ratios (Figure 5). An
optimal reduction of CD38 cell-surface expression on T
cells was obtained with a high MSC:CD3� ratio (1:4).
MSC immunosupportive effects on activated
T cells
The T-cell response is enhanced at low MSC concentrations
After 5 days of co-culture, T-cell colony formation in
response to PHA/IL-2 was boosted in the presence of low
MSC concentrations or related CM. Using an inverted
microscope (200�), we observed this supportive
effect, which consisted of the appearance of wide colonies
(Figure 6A). Additionally, MSC sustained rather than
inhibited T-cell proliferation at low concentrations, result-
ing in the observation of an additional generation peak
(Figure 6A).
Indeed, MSC supported T-cell proliferation, as shown
by the significant (PB0.04) increase of MGN (Figure 6B)
that was observed either with (from 6.590.56 to 7.839
0.47) or without (from 5.4090.25 to 6.8090.37) contact
between both cells. These results suggest that contact is
Table II. Naive and memory T-cell distribution.
% CD45RA� cells % CD45RO� cells
CB 96.2591 0.36490.09
PB 45.2292.36 51.9293.02
T lymphocytes from PB and CB were assessed for the expression and
distribution of both CD45RA� (naive) and CD45RO� (memory) cells.
Data are presented as mean percentage of CD45RA�or CD45RO�
cells9SEM of five independent experiments evaluated by flow cytometry
Figure 4. MSC inhibition of PB lymphocyte subset proliferation.
T-cell populations (total CD3�, CD45RA� and CD45RO� cells)
were labeled with CFSE and their PHA/IL-2-induced proliferation
was assessed for MSC immunosuppressive activity. On day 5, MSC
co-cultured in direct contact with immunoselected T-cell subsets
differentially inhibited the lymphocyte response. The percentage of
CD45RA� cell inhibition by MSC was significantly weaker than that
observed for total CD3� cells and CD45RO� cells (*PB0.04).
Seven independent experiments were performed and data are expressed
as the mean9SEM percentage of T-cell inhibition.
Figure 5. MSC reduce CD38 expression on activated T cells. PHA/
IL-2-activated T lymphocytes from both PB and CB were co-cultured
(direct contact) with or without different concentrations of MSC for 5
days. In the co-cultures, MSC decreased CD38 expression on activated
T cells in a dose-dependent manner. Results were obtained by flow
cytometry and expressed as the percentage relative to the control.
Activated T cells alone (control) were considered to represent 100% of
the CD38 expression. Data are presented as the mean9SEM relative
percentage of CD38 expression from eight independent experiments.
Values were statistically significant (*PB0.03) compared with control
cultures (PHA/IL-2-activated T cells without MSC).
Bifunctionality of MSC on T lymphocytes 7
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
not required for the stimulation of T-cell proliferation by
MSC at low concentrations, and that soluble factors
secreted by MSC mediate this effect.
The viable T-cell number is increased at low MSC concentrations
Co-cultures of PHA/IL-2-activated T cells in the presence
of different MSC concentrations were incubated for 5 days
to assess the importance of the MSC:T-cell ratio (Figure 7)
on distinguishing MSC stimulatory and inhibitory effects.
At low MSC:CD3� ratios (1:80 and 1:40), a significant
(PB0.03) and strong increase in the number of viable
T cells was observed, both by trypan blue exclusion and
7-AAD staining. The number of T cells was�75% greater
than the control (culture without MSC), demonstrating
that MSC induced lymphocyte proliferation. However,
when the MSC:CD3� ratios were increased, a decreased
number of T cells was observed.
MSC CM exert immunostimulatory effects on
T-cell proliferation
To assess the role of soluble factors on the MSC
stimulatory effect, we tested the impact of CM obtained
from MSC cultured alone on T-cell proliferation. The
addition of such CM to PHA/IL-2-activated T cells
supported, rather than inhibited, lymphocyte proliferation.
Indeed, MGN was significantly (PB0.03) increased
compared with the control and rose from 590.2 to 6.49
0.24 (Figure 8). These experiments suggested that MSC
can constitutively support T-cell proliferation by produc-
tion of soluble factors.
IL-6 secretion by MSC is increased after
co-culture with T cells
As IL-6 and SDF-1a have been shown to be involved in
T-cell migration, survival and proliferation [26,27], we
Figure 6. MSC at low concentrations enhance the lymphocyte
response. (A) PHA/IL-2-activated T cells from PB were cultured
alone (control) or with MSC (1:40 ratio). After 5 days of co-culture
with a low concentration of MSC, lymphocyte colony formation, as
observed using an inverted microscope, was efficiently boosted. The
flow cytometry profile of the CFSE-labeled T cells showed an additional
peak generation. (B) CFSE-labeled PB T cells were activated by PHA/
IL-2 and co-cultured with low concentrations of MSC in the presence
or absence of direct contact. After 5 days of co-culture, the T-cell
generation number was significantly increased in a cell contact-
independent manner. Data are expressed as the mean9SEM
generation number from eight independent experiments (*PB0.04).
Figure 7. MSC at low concentrations increase the T-cell number.
PHA/IL-2-activated T lymphocytes from PB were cultured for 5 days
with different MSC concentrations, and viable T cells were counted
using trypan blue. At low cell ratios, the T-cell number was
significantly increased. However, at high cell ratios, this number was
strongly reduced. Seven independent experiments were performed and
the data are expressed as the mean9SEM cell number (�103)
(*PB0.03).
8 M. Najar et al.
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
used an ELISA to evaluate their production by MSC (n�5). MSC constitutively produced 2.2891.14 ng/mL IL-6
and 1.3390.5 ng/mL SDF-1a in cultures containing 105
MSC/mL. Co-cultures of MSC with PHA/IL-2-activated
T cells only increased IL-6 production in a contact-
independent manner, and SDF-1a production was not
affected. Indeed, IL-6 levels were increased by more than
18-fold in these co-cultures. Interestingly, increased IL-6
production was already optimal at the low cell ratio and
remained unchanged with higher MSC concentrations.
IL-6 is primarily responsible for the
immunosupportive effect of MSC
As observed previously, MSC cultured alone at low
concentrations or the corresponding CM failed to induce
T-cell inhibition but improved lymphocyte proliferation
by increasing both the cell number and MGN. MSC CM
also increased the total number of viable T cells, as
evaluated by trypan blue exclusion and 7-AAD staining.
Indeed, the initial cell number rose from 348943�103 to
9529159�103 cells. However, when increasing concen-
trations of anti-IL-6 neutralizing Ab were added to the
cultures, we observed a significant progressive decrease of
cell number (Figure 9) that finally reached 412971�103
counted cells when 10 mg/mL anti-IL-6 was used
(corresponding to the T-cell control value without
MSC-derived CM). These results demonstrated that
IL-6, which is constitutively produced by MSC, plays a
major role in this MSC-mediated immunostimulatory
effect on T-cell proliferation. On the other hand, the
addition of increasing doses of neutralizing Ab against
SDF-1a led to a similar gradual decrease of cell number,
but this decrease was less important compared with the
decrease observed with anti-IL-6 neutralizing Ab. More-
over, the T-cell number was increased when recombinant
IL-6 (10 ng/mL; R&D Systems) was added to activated T-
cell cultures at a rate similar to the one observed with
MSC CM. Neutralizing Ab used at the concentrations
described were not toxic to T cells when the viability was
assessed by trypan blue exclusion assay.
Nature of the T cells expanded in the presence of
MSC at low ratios
We evaluated the phenotype of T cells cultured with MSC
at two ratios: 1:40 (stimulation) and 1:4 (inhibition). The
stimulatory and inhibitory effects of MSC were evident on
both the CD4� and CD8� T-cell subsets, as their
percentages were identical in the presence or absence of
MSC. Although B1% of CB CD3� cells expressed the
CD45RO isoform, we observed a significant CD45RO
switch in the presence of 1:40 MSC, demonstrating the
Figure 8. MSC CM support T-cell proliferation. CM obtained from
MSC at low concentrations (105 cells/mL) were added to CFSE-
labeled T-lymphocytes. After 5 days, MSC CM significantly increased
the mean generation number of the proliferative T cells (*PB0.03).
Data are expressed as the mean9SEM generation number from seven
independent experiments.
Figure 9. Reversion of the MSC immunosupportive effect by
neutralizing IL-6 and SDF-1a. PHA/IL-2-activated T cells were
cultured for 5 days with CM obtained from MSC cultured at low
concentrations (105 cells/mL) in the presence or absence of different
concentrations of neutralizing Ab. A significant decrease of the T-cell
number was observed when increasing concentrations of anti-IL-6 and
anti-SDF-1a neutralizing Ab were added to the culture. Seven
independent experiments were performed and data are expressed as the
mean9SEM cell number (�103). Values were statistically significant
(*PB0.04) compared with control cultures (PHA/IL-2-activated T
cells with MSC CM but without neutralizing Ab).
Bifunctionality of MSC on T lymphocytes 9
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
generation of memory cells. The expansion of CD45RO�
T cells was also observed in the case of PB (data not
shown). To demonstrate whether regulatory T cells were
part of the newly expanded cells in the presence of MSC,
we analyzed FoxP3 expression after 5 days of culture. As
shown in Figure 10A, qRT-PCR showed a significant up-
regulation of relative FoxP3 mRNA in PHA/IL-2-acti-
vated T cells co-cultured with MSC, compared with
conditions where MSC were omitted. The ratio of
FoxP3/EF1-a for CB T cells was 16.8595.7 in the absence
of MSC versus 51.31914 and 38.73910 in the presence of
MSC at 1:40 and 1:4 ratios, respectively (PB0.01). The
FoxP3 mRNA up-regulation induced by MSC was also
observed in T cells obtained from PB (PB0.03), albeit
weaker. These results were confirmed by analyzing FoxP3
protein expression by intracellular flow cytometry (Figure
10B). In the presence of MSC, the percentage of CD3�
FoxP3� T cells from PB and CB increased. MSC, when
co-cultured with CD3� cells, recruit regulatory T cells
independently of the MSC:T-cell ratio. Thus these
regulatory T cells are a part of the new cells expanded
in the presence of MSC at low ratios.
Low-concentration MSC sustain mitogenic and
allogeneic proliferation of purified CD4�
CD25high CD127low Tregs and CD4� CD25� PB
T cells
Isolated CD4� CD25high CD127low Tregs and CD4�
CD25� PB T cells (Treg-depleted T cells) were stimulated
by either PHA/IL2 or allogeneic irradiated PBMC, in
triplicate, in the absence or presence of different MSC
concentrations. Cultures were run for 5 days and then
proliferation was assessed by BrdU incorporation. The T-
cell population proliferation was significantly (PB0.05)
inhibited and enhanced, respectively, for 1:4 and 1:40
MSC:T cell ratios, as shown in Table III.
DiscussionThe immunomodulatory effects of MSC were evaluated
on T cells purified from PB and CB that were activated by
mitogens (PHA), CD3/CD28 agonists or alloantigens
(MLR). Using BrdU incorporation and CFDA-SE labeling
Figure 10. MSC expand PB and CB FoxP3� regulatory T cells.
PHA/IL-2-activated CD3� T lymphocytes from both PB and CB
were co-cultured (direct contact) for 5 days with or without MSC (1/
40 and 1/4 cell ratios). (A) FoxP3 mRNA content of T cells in each of
the indicated conditions was determined by quantitative real-time
PCR. Data are expressed as the mean9SEM of normalized FoxP3
expression from 10 independent experiments (*PB0.03, **PB0.01).
(B) The percentage of FoxP3� T cells in culture was determined by
intracellular staining and analyzed by flow cytometry. Data are
expressed as the mean9SEM percentage of CD3� FoxP3� T cells
from five independent experiments (*PB0.03, **PB0.01).
Table III. Low concentrations of MSC sustain allogeneic
proliferation of purified CD4� CD25� CD127� Tregs and
CD4� CD25� PB T cells.
MSC:T-cell ratio Tregs CD4� CD25� T cells
1:4 83.7192.12 * 59.0996.59a
1:40 132.3798.36 * 129.6799.24a
Isolated CD4� CD25high CD127low Tregs and CD4� CD25� PB T cells
(Treg-depleted T cells) were stimulated by allogeneic irradiated PBMC, in
triplicate, in the absence or presence of different MSC concentrations.
Cultures were run for 5 days and then proliferation was assessed by BrdU
incorporation. Both T-cell populations’ proliferation was significantly
(aPB0.05) inhibited or enhanced, respectively, for 1:4 and 1:40 MSC:T-
cell ratios. Values were calculated after eliminating the respective
backgrounds, and T-cell proliferation in the absence of MSC was
considered 100%. Data are presented as mean relative percentage of
proliferation9SD of three independent experiments.
10 M. Najar et al.
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
techniques, we observed a dose-dependent inhibition of T-
cell proliferation by MSC, regardless of the stimulation
used. In our system, MSC-mediated inhibition was optimal
for high MSC:CD3� ratios (1:8 and 1:4). These observa-
tions are in agreement with those described in the
literature [8,28], demonstrating that the inhibitory effects
of MSC were stimuli-independent. Evaluating and doc-
umenting these effects in a defined system is of importance
because some groups have reported an optimal inhibition
at a 1:1 MSC:CD3� ratio [29] while others have observed
inhibition at a 1:1000 MSC:CD3� ratio [30]. These
differences are likely to be related to culture conditions:
the surface of the culture well-bottom determining the
final cell density in co-cultures for a same cell concentra-
tion, the responder cell type (total PBMC, splenocytes or
purified T cells) and MSC origin (murine or human) and
source (BM, CB, placental and adipose tissue) may all
affect the outcome of the experiment. Although direct
contact between the two populations allowed maximal
inhibition, at higher MSC:CD3� ratios a slight decrease in
T-cell proliferation was observed in the absence of direct
contact.
Recent clinical data have demonstrated the success of
allogeneic stem cell transplantation using HLA-mis-
matched unrelated human umbilical cord blood (UCB).
Although the incidence and severity of GvHD seem low,
the risks still exist and limited cell dose remains the main
setback [31,32]. MSC could improve both hematopoietic
engraftment and immune reconstitution after UCB trans-
plantation but also reduce and/or manage the GvHD
incidence. Indeed, co-transplantation of MSC and CB
stem cells has been reported recently [33]. In this way, we
aimed to explore the interactions of MSC with CB T
lymphocytes. Several differences, both phenotypical and
functional, have been described between T cells isolated
from PB and CB [22�24]. As we have reported, CB T cells,
which are mainly CD45RA� and considered to be naive
T cells [34], were less inhibited by MSC than total PB
T cells. This inhibition occurred only when MSC were
present at high concentrations and cultured in direct
contact with the T cells. In contrast with their CD45RO�
T-cell counterpart, purified CD45RA� T cells from adult
PB behaved like CB T cells in our system. This observa-
tion suggests that T-cell subpopulations can be differen-
tially affected by the immunomodulatory effects of MSC.
Li et al. [35] also investigated the effects of MSC on
umbilical CB- and PB-derived T lymphocytes. However,
they used MSC derived from human placenta and the
potential differences between the various sources of T cells
were not addressed.
Depending on their concentrations, we observed that
MSC possess two distinctive activities. Indeed, MSC are
able to support and suppress CB and PB T-cell responses.
This stimulatory activity only happened at low MSC
concentrations, did not require cell-to-cell contact, and
involved soluble factors. CM from MSC cultured alone
exerted immunostimulatory effects on T cells. Previous
studies have also reported that MSC at low concentrations
stimulate MLR, leading to an increase of T-cell prolifera-
tion in an MHC-independent manner [8]. Fang et al. [36],
who also explored the balance between the suppressive and
stimulatory effects of MSC on T-cell proliferation,
proposed that increasing levels of IL-2 and IFN-g in
MLR performed at low MSC:effector cell ratios were
implicated in this phenomenon. However, these cultures
were carried out using allogeneic PBMC, while we used
purified CD3� T cells and observed an optimal MSC
supportive effect at a lower MSC:T-cell ratio (1:40) than
the ratio (1:10) used in their experiments. Their observa-
tions appear to contradict the study of Rasmusson et al.
[37], which showed that MSC at low concentrations did
not significantly affect IL-2 levels in MLR. Moreover, Fang
et al. [36] suggested that the increased IFN-g secretion
associated with an up-regulation of MHC II molecules on
MSC could explain this stimulatory effect. There are
controversies about this observation, as many studies have
shown that MSC are unable to elicit allogeneic lympho-
cyte responses even after IFN-g stimulation and MHC II
up-regulation [9,11,38]. Furthermore, other groups have
described IFN-g as a key regulator of MSC immunosup-
pressive activity [19,39,40]. Additionally, cells isolated
from the amniotic mesenchymal tissue can induce either
inhibitory or stimulatory effects on allogeneic T cells. The
presence of cells expressing HLA-DR seems to be
implicated in the activation of T-cell proliferation [41].
In our study, we used human MSC that were HLA-DR�.
Furthermore, CM from these HLA-DR� and IFN-guntreated MSC allowed stimulation of lymphocyte growth.
All these observations support the view that the stimula-
tory effect of MSC appears to be IFN-g- and MHC II-
independent. Eddahri et al. [42] reported that IL-6
promotes the differentiation of naive T lymphocytes into
helper cells able to promote B-cell activation and Ab
secretion. As we observed, MSC constitutively produced
Bifunctionality of MSC on T lymphocytes 11
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
IL-6 and this production greatly increased, by 18-fold,
after co-culture with T cells in a contact-independent way.
Through the production of IL-6, MSC could modulate
interactions between B and T cells as well as B-cell
activation and Ab secretion. Traggiai et al. [43] recently
demonstrated that MSC modulate the B-cell response.
MSC promoted and enhanced both proliferation and
differentiation of B lymphocytes. Similarly, a stimulatory
effect of MSC on human B cells has been reported,
showing that MSC have the ability to stimulate or suppress
Ab secretion depending on the level of stimulus used to
trigger B cells [15]. At low cell ratios, MSC display a
stimulatory profile and IL-6 is in part responsible for
supporting T-cell proliferation. Meanwhile, despite the
presence of IL-6 in MSC�T-cell co-cultures at higher
ratios, the inhibitory mechanisms seem to be the dominant
MSC activity. The immunosuppressive potential of MSC
is not constitutive, but rather induced under specific
circumstances. The contrasting functions of MSC imply
the presence of a balance between the inhibitory and
stimulatory abilities of MSC. This balance is influenced by
the MSC:T-cell ratio, which critically affects the cellular
contact possibilities in the co-culture system. At high
MSC:T-cell ratios, which favor and enhance direct cellular
interactions, MSC become suppressive and acquire an
inhibitory profile responsible for T-cell inhibition.
As T-lymphocyte growth was supported after exposure
to low concentrations of MSC, we assessed the phenotype
of expanded T cells. Interestingly, we have shown that,
among the expanded T-cell populations in both CB and
PB, regulatory cells are increased and are a part of the new
cells promoted by MSC at low doses. MSC induced up-
regulation of the relative FoxP3� mRNA expression level
per lymphocyte and FoxP3� T-cell number, as demon-
strated by qRT-PCR and intracellular flow cytometry.
Regulatory T cells play a pivotal role in the control of self-
tolerance and autoimmune diseases. Tregs are also in-
volved in the regulation of T-cell homeostasis and the
modulation of immune responses to tumors, pathogens and
alloantigens. MSC supported the expansion of regulatory
T cells from both CB and adult PB. However, the up-
regulation of FoxP3 by MSC was lower in the case of PB
compared with CB, suggesting that MSC recruit regula-
tory T cells primarily in the CD45RA� subset. These
observations are in agreement with the study of Di Ianni
et al. [20], who reported that exposure of PB T cells to
MSC guarantees high Foxp3 expression and the main-
tenance of T-regulatory functions. Recently, Selmani et al.
[44] also reported a significant increase in the CD4�
CD25high FoxP3� population in MLR in the presence of
MSC used at a 1:2 ratio. In our study, the expansion of
regulatory T cells in both CB and PB occurred even for
low cell ratios (supporting effects) without inducing the
inhibition of T-cell proliferation. This observation suggests
that MSC recruit and promote preferentially regulatory T
cells independently of their concentrations. Meanwhile,
MSC support and inhibit the in vitro proliferation of
purified Treg and non-Treg CD4� T cells, according to
their concentration, showing that they can also limit the
growth of activated Tregs and support ‘non-Treg T-cell’
proliferation. BM transplants already contain MSC [45]
but at insufficient numbers, and an immunosuppressive
MSC effect is observed when additional MSC are co-
transferred; one can only speculate that this could reflect
the need for sufficient MSC local concentrations to bring
about immunosuppressive activity, although little is known
about human MSC distribution after transfusion. As the
MSC:T-cell ratio reflects the level of cellular interactions,
MSC that are not interacting with activated immune cells
seem to remain non-immunosuppressive and secrete
constitutively soluble factors promoting the ‘wellness’ of
many different cellular types from many tissues, including
lymphoid cells. The balance between the immunosuppres-
sive and immunosupportive properties of MSC seems to be
determined by its direct environment.
These results demonstrate that the MSC:T-cell ratio is
critically important for the immunomodulatory functions
of MSC, whatever the source of T cells used. Because of
their immunosuppressive properties and enhancement of
Tregs, MSC might be useful for modulating the immune
system. MSC represent an attractive tool for reducing the
lymphocyte response by inhibiting T-cell activation and
proliferation, as well as promoting tolerance by maintain-
ing and promoting the expansion of regulatory cells.
AcknowledgementsThis study received financial support from the ‘Fondation
Medic’, ‘Le Fonds National de la Recherche Scientifique’
(FNRS; grants 3.4.532.07 and 7.4.538.06), ‘Les Amis de
l’Institut Bordet’ and the ‘Fondation Lambeau-Marteau’.
Dr Laurence Lagneaux is Senior Research Associate of the
FNRS. The authors thanks M. Massy, C. De Bruyn and H.
Duvillier for their technical assistance in flow cytometry
analysis and B. Badran for molecular biology work.
12 M. Najar et al.
Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009
Declaration of interest: The authors report no conflicts of
interest. The authors alone are responsible for the content
and writing of the paper.
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