PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by: [Martiat, Philippe]On: 29 June 2009Access details: Access Details: [subscription number 912781698]Publisher Informa HealthcareInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

CytotherapyPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713656803

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

URL: http://dx.doi.org/10.1080/14653240903079377

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

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.

Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009

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

Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009

(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.

Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009

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.

References

1 Tondreau T, Meuleman N, Delforge A, Dejeneffe M, Leroy R,

Massy M et al. Mesenchymal stem cells derived from CD133-

positive cells in mobilized peripheral blood and cord blood:

proliferation, Oct4 expression, and plasticity. Stem Cells 2005;

23:1105�112.

2 Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ et al.

Multilineage cells from human adipose tissue: implications for

cell-based therapies. Tissue Eng 2001;7:211�28.

3 Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I,

Fisk NM et al. Identification of mesenchymal stem/progenitor

cells in human first-trimester fetal blood, liver, and bone marrow.

Blood 2001;98:2396�402.

4 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,

Mosca JD et al. Multilineage potential of adult human

mesenchymal stem cells. Science 1999;284:143�7.

5 Keating A. Mesenchymal stromal cells. Curr Opin Hematol

2006;13:419�25.

6 Jones BJ, McTaggart SJ. Immunosuppression by mesen-

chymal stromal cells: from culture to clinic. Exp Hematol

2008;36:733�41.

7 Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni

PD, Matteucci P et al. Human bone marrow stromal cells

suppress T-lymphocyte proliferation induced by cellular or

nonspecific mitogenic stimuli. Blood 2002;99:3838�43.

8 Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden

O. Mesenchymal stem cells inhibit and stimulate mixed

lymphocyte cultures and mitogenic responses independently

of the major histocompatibility complex. Scand J Immunol

2003;57:11�20.

9 Krampera M, Glennie S, Dyson J, Scott D, Laylor R, Simpson E

et al. Bone marrow mesenchymal stem cells inhibit the response

of naive and memory antigen-specific T cells to their cognate

peptide. Blood 2003;101:3722�9.

10 Nasef A, Ashammakhi N, Fouillard L. Immunomodulatory effect

of mesenchymal stromal cells: possible mechanisms. Regen Med

2008;3:531�46.

11 Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O.

HLA expression and immunologic properties of differentiated

and undifferentiated mesenchymal stem cells. Exp Hematol

2003;31:890�6.

12 Ryan JM, Barry F, Murphy JM, Mahon BP. Interferon-gamma

does not break, but promotes the immunosuppressive capacity of

adult human mesenchymal stem cells. Clin Exp Immunol 2007;149:

353�63.

13 Tabera S, Perez-Simon JA, Dıez-Campelo M, Sanchez-Abarca

LI, Blanco B, Lopez A et al. The effect of mesenchymal stem

cells on the viability, proliferation and differentiation of B-

lymphocytes. Haematologica 2008;93:1301�9.

14 Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V,

Cazzanti F et al. Human mesenchymal stem cells modulate B-

cell functions. Blood 2006;107:367�72.

15 Rasmusson I, Le Blanc K, Sundberg B et al. Mesenchymal stem

cells stimulate antibody secretion in human B cells. Scand

J Immunol 2007;65:336�43.

16 Spaggiari GM, Capobianco A, Abdelrazik H, Becchetti F,

Mingari MC, Moretta L. Mesenchymal stem cells inhibit natural

killer-cell proliferation, cytotoxicity, and cytokine production:

role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood

2008;111:1327�33.

17 Nauta AJ, Kruisselbrink AB, Lurvink E, Willemze R, Fibbe WE.

Mesenchymal stem cells inhibit generation and function of both

CD34�-derived and monocyte-derived dendritic cells. J

Immunol 2006;177:2080�7.

18 Li YP, Paczesny S, Lauret E, Poirault S, Bordigoni P, Mekhloufi

F et al. Human mesenchymal stem cells license adult CD34�

hemopoietic progenitor cells to differentiate into regulatory

dendritic cells through activation of the Notch pathway. J

Immunol 2008;180:1598�608.

19 Aggarwal S, Pittenger MF. Human mesenchymal stem cells

modulate allogeneic immune cell responses. Blood 2005;105:

1815�22.

20 Di Ianni M, Del Papa B, De Ioanni M, Moretti L, Bonifacio E,

Cecchini D et al. Mesenchymal cells recruit and regulate T

regulatory cells. Exp Hematol 2008;36:309�18.

21 Tondreau T, Lagneaux L, Dejeneffe M, Delforge A, Massy M,

Mortier C et al. Isolation of BM mesenchymal stem cells by

plastic adhesion or negative selection: phenotype, proliferation

kinetics and differentiation potential. Cytotherapy 2004;6:372�9.

22 D’Arena G, Musto P, Cascavilla N, Minervini MM, Di Giorgio

G, Maglione A et al. Inability of activated cord blood T

lymphocytes to perform Th1-like and Th2-like responses:

implications for transplantation. J Hematother Stem Cell Res

1999;8:381�5.

23 Garcia Vela JA, Delgado I, Bornstein R, Alvarez B, Auray MC,

Martin I et al. Comparative intracellular cytokine production by

in vitro stimulated T lymphocytes from human umbilical cord

blood (HUCB) and adult peripheral blood (APB). Anal Cell Pathol

2000;20:93�8.

24 Han P, McDonald T, Hodge G. Potential immaturity of the T-

cell and antigen-presenting cell interaction in cord blood with

particular emphasis on the CD40�CD40 ligand costimulatory

pathway. Immunology 2004;113:26�34.

25 Chadburn A, Inghirami G, Knowles DM. The kinetics and

temporal expression of T-cell activation-associated antigens

CD15 (LeuM1), CD30 (Ki-1), EMA, and CD11c (LeuM5) by

benign activated T cells. Hematol Pathol 1992;6:193�202.

26 Smol’nikova VV, Voznyuk AV, Potapnev MP. Cytokine-induced

differentiation and proliferation of human T lymphocytes in

vitro: effects of interleukin 2 and interleukin 6. Bull Exp Biol Med

2000;129:567�70.

27 Yonezawa A, Hori T, Sakaida H, Uchiyama T. SDF-1 has

costimulatory effects on human T cells: possible involvement of

MAPK (ERK2) activation. Microbiol Immunol 2000;44:135�41.

Bifunctionality of MSC on T lymphocytes 13

Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009

28 Djouad F, Plence P, Bony C, Tropel P, Apparailly F, Sany J et al.

Immunosuppressive effect of mesenchymal stem cells favors

tumor growth in allogeneic animals. Blood 2003;102:3837�44.

29 Plumas J, Chaperot L, Richard MJ, Molens JP, Bensa JC, Favrot

MC. Mesenchymal stem cells induce apoptosis of activated T

cells. Leukemia 2005;19:1597�604.

30 Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C

et al. Immunomodulatory effect of human adipose tissue-derived

adult stem cells: comparison with bone marrow mesenchymal

stem cells. Br J Haematol 2005;129:118�29.

31 Wang XN, Sviland L, Ademokun AJ, Dunn J, Cavanagh G,

Proctor SJ et al. Cellular alloreactivity of human cord blood cells

detected by T-cell frequency analysis and a human skin explant

model. Transplantation 1998;66:903�9.

32 Kurtzberg J. Update on umbilical cord blood transplantation.

Curr Opin Pediatr 2009;21:22�9.

33 Macmillan ML, Blazar BR, Defor TE, Wagner JE. Transplanta-

tion of ex-vivo culture-expanded parental haploidentical me-

senchymal stem cells to promote engraftment in pediatric

recipients of unrelated donor umbilical cord blood: results of a

phase I-II clinical trial. Bone Marrow Transplant 2009;43:447�54.

34 Harris DT, Schumacher MJ, Locascio J, Besencon FJ, Olson GB,

DeLuca D et al. Phenotypic and functional immaturity of human

umbilical cord blood T lymphocytes. Proc Natl Acad Sci USA

1992;89:10006�10.

35 Li C, Zhang W, Jiang X, Mao N. Human-placenta-derived

mesenchymal stem cells inhibit proliferation and function of

allogeneic immune cells. Cell Tissue Res 2007;330:437�46.

36 Fang L, Lange C, Engel M, Zander AR, Fehse B. Sensitive

balance of suppressing and activating effects of mesenchymal

stem cells on T-cell proliferation. Transplantation 2006;82:

1370�3.

37 Rasmusson I, Ringden O, Sundberg B, Le Blanc K. Mesench-

ymal stem cells inhibit lymphocyte proliferation by mitogens

and alloantigens by different mechanisms. Exp Cell Res

2005;305:33�41.

38 Tse WT, Pendleton JD, Beyer WM, Egalka MC, Guinan EC.

Suppression of allogeneic T-cell proliferation by human marrow

stromal cells: implications in transplantation. Transplantation

2003;75:389�97.

39 Krampera M, Cosmi L, Angeli R, Pasini A, Liotta F, Andreini A

et al. Role for interferon-gamma in the immunomodulatory

activity of human bone marrow mesenchymal stem cells. Stem

Cells 2006;24:386�98.

40 Chan JL, Tang KC, Patel AP, Bonilla LM, Pierobon N, Ponzio

NM et al. Antigen-presenting property of mesenchymal stem

cells occurs during a narrow window at low levels of interferon-

gamma. Blood 2006;107:4817�24.

41 Magatti M, De Munari S, Vertua E, Gibelli L, Wengler GS,

Parolini O. Human amnion mesenchyme harbors cells with

allogeneic T-cell suppression and stimulation capabilities. Stem

Cells 2008;26:182�92.

42 Eddahri F, Denanglaire S, Bureau F, Spolski R, Leonard WJ, Leo

O et al. Interleukin-6/STAT3 signaling regulates the ability of

naive T cells to acquire B-cell help capacities. Blood

2009;113:2426�33.

43 Traggiai E, Volpi S, Schena F, Gattorno M, Ferlito F, Moretta L

et al. Bone marrow-derived mesenchymal stem cells induce both

polyclonal expansion and differentiation of B cells isolated from

healthy donors and systemic lupus erythematosus patients. Stem

Cells 2008;26:562�9.

44 Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L et al.

Human leukocyte antigen-G5 secretion by human mesenchymal

stem cells is required to suppress T lymphocyte and natural

killer function and to induce CD4�CD25highFOXP3� regula-

tory T cells. Stem Cells 2008;26:212�22.

45 Barrett AJ, Le Blanc K. Prophylaxis of acute GVHD: manipulate

the graft or the environment? Best Pract Res Clin Haematol

2008;21:165�76.

14 M. Najar et al.

Downloaded By: [Martiat, Philippe] At: 08:01 29 June 2009