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Human mesenchymal stem cells shift CD8+ T cellstowards a suppressive phenotype by inducingtolerogenic monocytes

Irit Hof-Nahor1, Lucy Leshansky1, Shoham Shivtiel1, Liron Eldor2, Daniel Aberdam1, Joseph Itskovitz-Eldor1,3

and Sonia Berrih-Aknin4,*1INSERTECH, Bruce Rappaport Department of Medicine, Technion, Haifa, Israel2Department of Plastic Surgery, Rambam Health Care Campus, Haifa, Israel3Department of Obstetrics and Gynecology, Rambam Health Care Campus, Haifa, Israel4UMRS 974 - UPMC University, Paris 6/U974 - Inserm/UMR7215 - CNRS

*Author for correspondence (sonia.berrih-aknin@upmc.fr)

Accepted 20 May 2012Journal of Cell Science 125, 4640–4650� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.108860

SummaryThe mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been investigated under extremeconditions of strong T cell activation, which induces the rapid death of activated lymphocytes. The objective of this study was toinvestigate these mechanisms in the absence of additional polyclonal activation. In co-cultures of peripheral mononuclear blood cells

with human MSCs (hereafter referred to as hMSCs), we observed a striking decrease in the level of CD8 expression on CD8+ cells,together with decreased expression of CD28 and CD44, and impaired production of IFN-gamma and Granzyme B. This effect wasspecific to hMSCs, because it was not observed with several other cell lines. Downregulation of CD8 expression required CD14+

monocytes to be in direct contact with the CD8+ cells, whereas the effects of hMSCs on the CD14+ cells were essentially mediated bysoluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 andCD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. CD8+ cells that were preconditioned byMSCs had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of hMSCs in humanized NSG mice

showed similar trends, in particular decreased levels of CD44 and CD28 in human immune cells. Our study demonstrates a newimmunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic and/or suppressivephenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and

autoimmune diseases, but potentially detrimental in malignant diseases.

Key words: Human mesenchymal stem cells, Immunoregulation, CD8+ cells, Monocytes, Humanized mice

IntroductionMesenchymal stem cells (MSCs) are non-hematopoietic stem

cells, which were first identified in the bone marrow cavity by

Fridenshtein (Fridenshteın, 1982); their ability to differentiate into

various types of mesoderm tissues was then demonstrated

(Pittenger et al., 1999; Wakitani et al., 1995). MSCs can be

isolated from bone marrow, skeletal muscle, adipose tissue and

synovial membrane, as well as cord blood (da Silva Meirelles et al.,

2006) and are defined by using a combination of phenotypic

markers.

It is well established that human MSCs (hereafter referred to as

hMSCs) possess suppressive capabilities on several subsets of

immune cells, namely T and B cells, dendritic cells (DCs) and

natural killer (NK) cells, in response to polyclonal stimulation

(Uccelli et al., 2007). MSCs have a unique immunophenotype, with

a low expression of major histocompatibility complex (MHC) class

I and an absence of co-stimulatory molecules. Although several

groups have demonstrated that hMSCs have the capacity to inhibit

T cell proliferation (Benvenuto et al., 2007; Glennie et al., 2005),

controversy remains regarding the effect of hMSCs on T cell

activation. Le Blanc and colleagues (Le Blanc et al., 2004) and

Groh and colleagues (Groh et al., 2005) who conducted their

research on cultures of phytohaemagglutinin (PHA)-stimulated

lymphocytes and alloreactive T cells, reported that hMSCs prevent

the expression of CD25, CD38 and CD69 activation markers on T

lymphocytes. However, in a similar model, Aggarwal and

colleagues (Aggarwal and Pittenger, 2005) argued that hMSCs

actually induce a slight increase in the proportion of CD25+ T cells.

In addition, some studies have claimed that the inhibitory effect of

MSCs on T cells is confined to cellular proliferation rather than to

the effector function of T cells (Ramasamy et al., 2008).

CD8 is commonly used as a cytotoxic T cell marker, whereas its

downregulation has been suggested as one of the mechanisms for

peripheral tolerance (Rocha and von Boehmer, 1991; Schonrich et al.,

1991; Zhang et al., 1995). Xiao and colleagues (Xiao et al., 2007)

have argued that the downregulation of CD8 expression and the loss

of specific peptide–MHC binding during the immune response

following bacterial infection are subjected to detuning during normal

immune responses. Different CD8+ T cell subsets have been

identified based on the expression of cell surface markers, such as

the CD28 co-stimulatory molecule, which is necessary for the

initiation of most T cell responses. CD8+CD282 cells are defined as

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suppressor T cells that have been shown to down-modulate the

antigen-presenting cell (APC) function by inducing immunoglobulin-

like transcript 3 and 4 (ILT-3 and ILT-4) inhibitory receptors, leading

to the inhibition of CD4+ T-cell proliferation by antigen presenting

cells (APCs) (Chang et al., 2002).

The difficulty in achieving long-term allograft survival has been

attributed to the resistance of effector CD8+ cells (Trambley et al.,

1999) and/or memory T cells (Lakkis and Sayegh, 2003;

Valujskikh et al., 2002) to co-stimulatory blockade. Preclinical

studies reveal that hMSCs are capable of preventing graft rejection

in a rat model for cardiac allograft (Zhou et al., 2006) and in rat

kidney transplantation, as well as reducing the number of CD8+

cells in the infiltrates (De Martino et al., 2010). Injection with

hMSCs prolonged the survival of skin transplant in a baboon

model (Bartholomew et al., 2002) and extended heart allograft

survival when administered in a mouse model, with 33% of

recipients showing long-term tolerance (Casiraghi et al., 2008).

The mechanisms underlying the immunomodulatory effects of

MSCs are still poorly understood, and most studies have used strong

T cell activators that represent extreme situations and induce rapid

death of activated lymphocytes. The objective of this study was to

investigate the effect of hMSCs on peripheral mononuclear blood

cells without additional exogenous stimulation. Our findings

indicate that hMSCs shift the CD8+ cytotoxic cells towards a

suppressive phenotype, an effect that depends on CD14+

monocytes, whose phenotype is strikingly regulated by hMSC.

ResultshMSCs decrease expression of CD8 on CD8+ T cells

We evaluated whether hMSC lines that originate from adipose

tissue had the potency to modify the balance between the main T

cell subsets in direct co-cultures without additional stimulating

molecules. Kinetics analysis in living cells showed that CD8, but

not CD4 expression, was significantly decreased on day 6 of

incubation (P,0.001) and was prolonged over time, with .70%

reduction in the level of CD8 expression on day 11 (P,0.001)

(Fig. 1A). The viability of CD4+ and CD8+ T cells at the 7th day

of co-culture was similar (,80%). The absolute number of cells

Fig. 1. hMSCs decrease the expression of CD8 on

CD8+ cells. (A) 107 unstimulated PBMCs from healthy

donors were co-cultured in the presence or absence of

105 adipose-derived (AD5) hMSC line. The graph

represents the fluorescence geometric mean (GM) of

CD8 and CD4 expression on T cells when co-cultured

with the hMSC line, normalized to values in the absence

of hMSCs at the indicated time points (n55). (B) The

kinetics of the relative percentages of CD8+ and CD4+

T cells in the lymphocyte gate when PBMCs were co-

cultured with hMSCs, in comparison to values in the

absence of hMSCs are shown by a dashed line at the

indicated time points (n55). (C) A comparison between

the effect of the AD1, AD4 and AD5 hMSC cell lines on

CD8+ T cells for a period of 7 days. The results are

expressed by GM fluorescent levels normalized to

values in the absence of hMSCs. The dashed line

indicates the expression level of control CD8 (100%).

(D) The effect of hMSCs on CD4 expression. The

dashed line indicates the expression level of control

CD4 (100%). (E–G) Representative CD8 expression

profiles comparing CD8+ T-cell intensity expression

after seven days of direct co-culture with (E) hMSCs;

(F) human fibroblasts; and (G) the HaCat cell line.

Dashed lines indicate the expression level of control

CD8. Histograms are presented in duplicates. The

dashed bar placed at the end of the negative slope of

CD8 fluorescent intensity (E) indicates the cut-off value

for the determination of the percentage of CD8+ T cells.

(H) The inhibitory effect of hMSCs on CD8 intensity

expression is essentially mediated by soluble factors.

Co-culture experiments were performed either in direct

conditions, with transwell supports (TW) or with hMSC

conditioned medium (n55). Control cultures were

performed without hMSCs in all experiments. Error bars

represent the s.e.m. *P,0.05 and ***P,0.0001, using

one-way ANOVA and post tests.

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was also similar in the absence or presence of hMSC. However,hMSCs slightly decreased the viability of CD8+ T cells (from

81% to 75%) and had no effect on CD4+ cells (78.9% versus78.7%) (supplementary material Fig. S1A). Unlike the dramatic

decrease in CD8 expression, the relative percentage of CD8+cells was only slightly decreased (Fig. 1B), given that the majoreffect occurred within the positive CD8+ population that shifts

towards the negative peak (Fig. 1E). The percentage of CD8positive cells was evaluated by setting the cut-off at the end of

the negative peak (Fig. 1E). To determine whether the decreasein CD8 intensity was dependent on the origin of the hMSCs cell

lines, we investigated three adipose hMSC cell lines, AD1, AD4,and AD5, derived from the abdomen, breast and thigh,respectively. All hMSC derivatives dramatically downregulated

CD8 expression, with the AD5 cell line demonstrating a .70%reduction in geometric mean (GM) of CD8 fluorescence

(Fig. 1C,E). This effect was specific to CD8+ T cells, becauseno such effect was observed in the CD4+ cell subset (Fig. 1D).The downregulation of CD8 fluorescence intensity in hMSCs was

observed with several anti-CD8 antibodies generated fromdifferent clones, demonstrating that this effect was not epitope

dependent (data not shown). The same results were obtained forthe CD8b chain as well (supplementary material Fig. S1B). In

order to determine whether downregulation of CD8 was specificto hMSCs, peripheral blood mononuclear cells (PBMCs) wereco-cultured with other cell lines, such as human neonatal

fibroblasts (Fig. 1F), the HaCat cell line (Fig. 1G), or mouseembryonic fibroblasts (MEF) (data not shown). None of these

cell lines, either of juvenile or adult origin, were able todownregulate CD8 fluorescence intensity after 7 days of direct

co-culture. Altogether, these results demonstrated a strikingeffect of hMSCs on the level of CD8 expression in a culture freeof exogenous polyclonal stimulation.

Taking into account that hMSC-mediated immune suppressionis considered to be the result of both cell-cell contact and soluble

factors (Liu et al., 2012; Uccelli et al., 2008), we investigatedwhether the down-modulation of CD8 surface expressionrequired direct contact or was mediated by soluble factors. In

the absence of direct interactions (transwell supports), CD8fluorescence intensity was downregulated by 50% (P,0.001),

suggesting the involvement of soluble factors (Fig. 1H). UsinghMSC-conditioned medium collected after 7 days of culture, onlya 30% reduction in CD8 expression was observed (Fig. 1H). The

difference between the transwell and conditioned medium co-culture could be explained by the limited pool of soluble factors

in the conditioned medium, whereas the hMSCs continue to growand produce soluble factors during the entire incubation time

when seeded in transwell cultures. These results demonstrate thatthe capacity of hMSCs to downregulate CD8 is essentiallymediated by soluble factors. Previous studies have suggested that

TGF-b1 and PGE2 contribute to the immunoregulatory propertiesof hMSCs (Aggarwal and Pittenger, 2005; Groh et al., 2005). We

examined the potential role of TGF-b1 in our cell cultureconditions. Neutralization of TGF-b1 did not reverse the effect of

hMSCs on CD8 and CD44 expression (supplementary materialFig. S2A, S2B). Considering that PGE2 has been previouslyshown to mediate the immune function of hMSCs mediated by

monocytes (Chen et al., 2010; Cutler et al., 2010), and becausePGE2 is considered responsible for the inhibitory effect on DC

differentiation and function (Spaggiari et al., 2009), we evaluatedthe role of PGE2 in our experimental system. Production levels of

PGE2 in the supernatants of hMSCs or PBMCs that were cultured

alone were very low, and a significant increase (P,0.001) was

detected in co-cultures of hMSCs with PBMCs (supplementary

material Fig. S2F). However, inhibition with indomethacin did

not restore the fluorescence intensity of CD8 reduced by hMSCs

(supplementary material Fig. S2E). These data suggest that in our

experimental model, PGE2 production is not involved in the

downregulation pathway of CD8 expression.

hMSC-mediated inhibition of CD8 expression occurs at the

post-transcription level

In order to address whether the downregulation of CD8 expression

results from reduced CD8 transcription, the CD8 mRNA

expression in PBMCs cultured either alone or with hMSCs in

Fig. 2. Inhibition of CD8 expression by hMSCs occurs at the post-

transcriptional level. Unstimulated PBMCs were cultured with hMSCs at a

1:100 ratio for 7 days in transwell supports (TW) supports, to avoid the

contamination by MSCs. (A) Equivalent amounts of total RNA were used in

the real-time RT-PCR experiment to quantify the expression of CD8, CD4

and ubiquitin (UBE202) mRNA. The CD8 and CD4 mRNA values were

normalized to the ubiquitin expression (n52). (B) The surface and

cytoplasmic CD8 GM values with and without hMSCs were evaluated by

flow cytometry. For the evaluation of cytoplasmic CD8 GM values,

intracellular staining was performed (n54). (C) CD8+ cell frequency within

the lymphocyte population. To evaluate cytoplasmic CD8 cell frequency,

intracellular staining was performed (n54). The bars show s.e.m. *P,0.05

and ***P,0.001, using one-way ANOVA test followed by post tests.

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transwell conditions was quantified using real-time RT-PCR.

There was no change in the mRNA levels for CD8 (Fig. 2A),

suggesting that the reduction in CD8 expression in the CD8+ cells

when co-cultured with hMSCs was not due to reduced CD8

transcription. Consequently, it is most likely that the mechanisms

that regulate CD8 expression levels in hMSCs are post-

transcriptional. Both the surface and cytoplasmic fluorescence

intensity values of CD8 were reduced by ,60% when co-cultured

with hMSCs (Fig. 2B), whereas the relative percentage of CD8+

cells, both surface and cytoplasmic, was only reduced by ,15%

(Fig. 2C). The decrease in expression of CD8, without changes in

the mRNA levels of CD8, suggests that hMSCs regulate CD8 by

reducing its protein expression, possibly through proteolytic

cleavage (Fujimoto et al., 1984) or ubiquitylation and

degradation mechanisms (D’Agostino et al., 2011).

hMSCs downregulate the expression of activation markers

on CD8+ T cells

T cell antigen receptor (TCR)-CD3-dependent responses are regulated

by the constitutive or inducible expression of co-stimulatory receptors,

such as CD28, and the lack of CD28 expression is generally

associated with regulatory function in CD8+ T lymphocytes

(Chang et al., 2002; Cortesini et al., 2001). We tested whether

hMSCs modulate the expression of CD28 molecules on CD8+

cells. As shown in Fig. 3A, 3B, a substantial decrease in CD28

fluorescence intensity was achieved in the presence of hMSCs. We

also analyzed the expression of several markers involved in cell

adhesion and activation. The cell adhesion molecule CD31 plays a

key role in leukocyte trafficking across the endothelium (O’Brien

et al., 2003), whereas CD44 is a marker associated with cell

adhesion and migration of lymphocytes (Johnson and Ruffell,

2009). In addition, the Fas (CD95) marker is increased during

activation-induced cell death (Cohen et al., 1992; Schwartz and

Osborne, 1993). As shown in Fig. 3C, a significant decrease in

CD44 and CD95 fluorescence intensity (P,0.005 and P,0.02,

respectively) was observed in the presence of hMSCs, whereas

CD31 expression did not change. To investigate whether these

phenotypic changes were concomitant with functional alterations,

we analyzed the production level of Gzm B and IFN-c. hMSCs

induced a significant reduction (P,0.005) in the production of

Fig. 3. hMSCs downregulate the expression of activation and adhesion molecules on CD8+ T cells. (A) The kinetics of the fluorescence GM of the CD28 co-

stimulation molecule in CD8+ T cells when co-cultured with whole hMSCs, in comparison to values in the absence of hMSCs (n56, day 0 and day 3; n54, day 6).

(B) A representative CD28 expression profile on the seventh day of direct co-culture with hMSCs. The GM of CD28 expression when co-cultured is shown with a

solid line and when cultured alone, with a dotted line. (C) The fluorescence GM of CD31, CD44 and CD95 surface expression in CD8+ T cells was analyzed after

7 days of co-culture in the presence or absence of hMSCs (n54). (D) CD8+ T cells were analyzed for Gzm B production by intracellular staining, as assessed by

flow cytometry (n54). (E) The CD8+ T cells were analyzed for IFN-c secretion by intracellular staining, as assessed by flow cytometry. Results show the

individual data of four experiments, each performed in duplicate. (F) IFN-c levels were measured (in duplicate) in the supernatants of PBMCs cultured in the

absence or presence of hMSCs. (G–H) CD8+ T cells were purified from PBMCs cultured with (CD8MSC) or without hMSCs (CD8CONT) following culture with

allogeneic CD14+ monocytes for 72 hours and the fluorescence GM of CD80 (G) and ILT3 (H) in CD14+ monocytes were evaluated (n53). (I) Purified CD8MSC

or CD8CONT were co-cultured with allogeneic CFSE-labeled PBMCs for 72 hours, and the percentage of proliferating cells (low CFSE) was evaluated (n53).

Error bars represent the s.e.m. *P,0.02, **P,0.005 and ***P,0.001, using one-way ANOVA and post tests for (A) and paired t-test for C, D, F, G, H and I.

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Gzm B (Fig. 3D). A significant decrease in the level of IFN-cproduction was observed in CD8+ cells when co-cultured with

hMSCs, without external activation (P,0.01) (Fig. 3E). Because

the percentage of IFN-c-positive cells was quite low without

activation (,3%), a positive control of these experiments was

performed using external polyclonal stimulation. In this case, as

expected, the percentage of IFN-c positive cells was high

(24.962%) and was greatly reduced in the presence of MSC

(10.661.1) (supplementary material Fig. S3). IFN-c production

levels were also evaluated in tissue culture supernatants (Fig. 3F).

Interestingly, even without polyclonal stimulation, substantially

high concentrations of IFN-c (2606164 pg/ml) were produced,

and were reduced in the presence of hMSC (71623 pg/ml).

In order to address CD8+ T cell functionality further on, the

CD8+ T cell population was purified using the FACSAriaTM after

7 days of culture of PBMCs, either in the presence (CD8MSC) or

absence of hMSCs (CD8CONT). Because CD8 suppressor cells are

known to influence the expression of co-stimulatory and inhibitory

molecules on APCs (Suciu-Foca et al., 2005), we co-cultured

CD8MSC or CD8CONT cells with isolated CD14+ monocytes.

CD8MSC but not CD8CONT cells were able to downregulate the

CD80 co-stimulatory molecule (Fig. 3G), as well as to upregulate

expression of the ILT3 inhibition receptor on monocytes (Fig. 3H),

whereas CD86 was slightly decreased and ILT4 was unchanged

(data not shown). The effect of these purified cell subsets on

lymphocyte proliferation was also tested; CD8MSC or CD8CONT

cells were added to PBMCs stained with carboxyfluorescein

succinimidyl ester (CFSE). CD8MSC cells significantly decreased

the percentage of proliferating cells compared with CD8CONT cells

(Fig. 3I). Generally, such a suppression of proliferation is

attributed to CD4+ FoxP3+ Treg cells, which were not induced

by hMSCs under our experimental conditions (data not shown).

The inhibitory effect of hMSCs on CD8+ is mediated by

CD14+ monocytes

In order to investigate whether hMSCs exert their downregulatory

effect on CD8+ T cells in a direct manner, isolated CD8+ T cells

were cultured in the presence of hMSCs. The viability of isolated

CD8+ T cells was similar, whether cultured alone or in the

presence of hMSCs (77% versus 78%, respectively). Compared

with isolated CD8+ cells that were cultured alone, not only was the

level of CD8 intensity not downregulated by hMSCs, but rather the

CD8 receptor intensity was upregulated by ,30% (Fig. 4A).

These results indicate that another cell population is mediating the

decrease in CD8 expression. Because CD14+ monocytes play a

major role in modulating immune responses, we investigated

whether the down-modulation of CD8 expression was attributed to

their presence. A strong downregulation in CD8 intensity levels

was observed in the presence of monocytes, with a 70% decrease

in expression (Fig. 4A), which was similar to the effect of the

whole PBMCs. Furthermore, when monocytes were depleted from

PBMCs, the capacity of hMSCs to suppress CD8 expression was

completely abolished (data not shown).

We then explored whether the contact is crucial between these

three key players, CD8+, CD14+ and hMSCs. When the isolated

CD14+ monocytes were cultivated separately from CD8+ T cells,

the hMSCs did not exert their inhibitory effect on CD8+ cells

(Fig. 4B, third column), whereas direct contact between CD8+ and

CD14+ subsets led to a 70% reduction in CD8 expression (Fig. 4B,

fourth column). The results shown in Fig. 4B demonstrate that

CD14+ monocytes require direct contact with CD8+ T cells, rather

than with hMSCs, for the modulation of CD8 expression.

hMSCs sensitize CD14+ monocytes, which in turn mediatean inhibitory effect of hMSCs on CD8+ cells

We then investigated whether hMSCs are able to sensitize

CD14+ cells (Fig. 5A). To this end, we divided the experiment

into two steps. The first step was co-culture of isolated CD14+

with hMSCs, in an attempt to produce sensitized CD14+ cells

(sCD14+), whereas the second step was co-culture between the

‘sCD14+’ cells and isolated CD8+ cells.

When co-cultured with sCD14+ monocytes (Fig. 5A, II), the

levels of CD8 expression were inhibited by 20%, compared with

non-sensitized monocytes (Fig. 5A, I) (P,0.005). This effect

was further enhanced when CD14+ monocytes were cultured

with hMSCs continuously, where a 40% inhibition in CD8

intensity was observed compared with the non-sensitized

monocytes (P,0.001) (Fig. 5A, III). These results show that

CD14+ monocytes that have been pre-sensitized by hMSCs can

down-modulate CD8 expression, suggesting that sCD14+

monocytes acquire a suppressive phenotype in the presence of

hMSCs. Moreover, we tested some representative markers that

were significantly altered (P,0.005 for CD44 and P,0.02 for

Fig. 4. The inhibitory effect of hMSCs on CD8+ cells is mediated by

CD14+ monocytes. (A) Isolated CD8+ T cells (2.56106 per well) were

cultured in the presence or absence of hMSCs (105 per well) and in the

presence or absence of isolated CD14+ monocytes (106 per well) for 7 days.

The numbers of CD8+ and CD14+ cells that were isolated were set according

to the ratio of these cells in the general population of PBMCs (25% and 10%,

respectively). When the isolated CD8+ T cells were co-cultured with hMSCs,

an upregulation of ,30% at the CD8 receptor intensity was observed. The

addition of CD14+ monocytes to the CD8+ purified cells restored the CD8

modulation by hMSCs. **P,0.005 using the paired t-test. (B) The CD14+

monocytes require direct contact with CD8+ T cells (fourth column), rather

than with hMSCs (third column) for the CD8 expression modulation (n53).

***P,0.001 using one-way ANOVA test and post tests.

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CD95) on CD8+ cells by hMSCs (i.e. CD44 and CD28)

(Fig. 3C). The same inhibitory pattern was observed for both

activation markers (Fig. 5B). These results further support the

suppressor phenotype of sCD14+ cells.

Phenotypic characterization of the CD14+ monocytes is

presented in Fig. 5C. Both the CD80 and CD86 co-stimulatory

molecules were significantly downregulated, with a .60%reduction in fluorescence intensity. The HLA-ABC and HLA-DR

histocompatibility molecules, as well as CD16 expressionassociated with a proinflammatory status of monocytes (Ziegler-Heitbrock, 2007), were also subjected to downregulation byhMSCs (data not shown). Both the ILT3 and ILT4 inhibitory

receptors were upregulated after incubation with hMSCs.Neutralization of TGF-b1 did not reverse the effect of hMSCson ILT3 and CD80 expression in monocytes (supplementary

material Fig. S2C,D). Along with the functional sensitization ofCD14+ cells, the hMSCs also displayed a direct effect on the sizeof CD14+ monocytes compared with control cultures through

kinetics (supplementary material Fig. S4A,B). The downregulationof co-stimulatory and histocompatibility molecules by hMSCstogether with the upregulation of the inhibitory ILTs receptorssupport the fact that hMSCs modulate CD14+ monocytes towards

a suppressor phenotype.

hMSCs exert similar inhibitory effect in vivo in humanizedNSG mice

In order to address whether hMSCs could present similar featuresin vivo, we used immunodeficient NSG mice humanized with

CD34+ hematopoietic stem cells according to protocolsdescribed previously (Marodon et al., 2009). Most mice had aclear reconstitution of the human immune system in the periphery(peripheral blood, spleen) (supplementary material Fig. S5). The

level of humanization was evaluated by analyzing the CD45+human cells in the peripheral blood. We then selected the micewith the best reconstitution and injected them with hMSCs

intraperitoneally. After 7 days, the mice were killed, andexpression of CD8, CD28 and CD44 was analyzed within thehuman CD45+ (huCD45+) cells in the spleen and bone marrow

from both experimental groups. Analysis of the bone marrowshowed that expression of human CD8 (Fig. 6A), CD44(Fig. 6B) and CD28 (Fig. 6C) was downregulated in the human

CD45+ gate in the NSG mouse injected with hMSCs, comparedwith the control NSG mice. Although the fluorescence intensityof CD8 decreased, the main change observed in the NSG micewas the decreased percentage of CD8+ cells. This difference with

our in vitro data could be owing to the nature of the models. Theimmune system in NSG mice obtained by reconstitution withneonatal CD34+ cells is not completely mature (Watanabe et al.,

2009), whereas the cells in vitro originated from adult blood.Furthermore, our in vitro analysis showed that APCs wereessential for the effects on CD8 expression, whereas in the NSG

mice, the involvement of the APCs could not be evaluated.Another possibility is that the decrease in CD8 expressionpreceded the drop in CD8 cell number, and in the mouseexperiments, the first step was very fast. This hypothesis fits with

the in vitro experiments (Fig. 1B), which show that thepercentage of CD8+ cells decreased more strikingly between 6and 12 days, whereas the fluorescence intensity starts to decrease

from day 3. Similar results were observed in the spleen (data notshown). In addition, we investigated the effect of hMSCs on themobilization of human cells in the blood of NSG mice, under

non-activated or LPS-activated conditions, by calculating theblood:BM ratio of human CD45+ cells. We observed asignificant reduction in the mobilization in NSG mice treated

with MSC (Fig. 6D) under both conditions, suggesting that MSCsalter the migratory properties of human lymphoid cells in theNSG mice.

Fig. 5. hMSCs sensitize CD14+ monocytes, inducing their suppressive

function and modulating their phenotype. (A) hMSCs are capable of

inducing sensitization of CD14+ cells, named ‘sCD14+ cells’. The standard

culture duration of 7 days was divided into two steps. During the first 3 days,

isolated CD14+ monocytes were cultured either in the presence (sCD14+) or

absence (CD14+) of hMSCs in transwell conditions. The upper chambers, in

which the isolated CD8+ cells were added, were then relocated for 3 additional

days into a newly prepared lower chamber containing either fresh hMSC

medium (I and II) or freshly prepared hMSC culture (III). The monocytes that

were cultured without the hMSCs served as the negative control in this

experiment (I). Data shown are for the GM of CD8 fluorescence on the sixth

day of culture (n53). (B) The normalized GM of fluorescence for CD8, CD28

and CD44 when the standard culture duration of 7 days was divided into two

steps, as described above. White bars indicate the control fluorescence GM

expression level in CD8+cells that are cultured with control monocytes. Light

grey bars and dark grey bars represent the fluorescence GM values when the

sCD14+ monocytes were cultured either with fresh hMSC medium or with

freshly prepared hMSC culture, respectively. Results show the means of three

experiments. (C) Phenotypic characterization of CD14+ monocytes after 6 days

of transwell co-culture in the presence and absence of hMSCs (n53). The bars

show the s.e.m. *P,0.02, **P,0.005 and ***P,0.001, by one-way ANOVA

followed by post tests for A and B and paired t-test for C.

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DiscussionThe aim of this study was to investigate the immunoregulatory

mechanisms of hMSCs in a steady-state situation. The main

results of this study are as follows: (1) hMSCs exert a striking

downregulatory effect on expression of the CD8 receptor in CD8+

cells, which is associated with features of reduced cytotoxicity (i.e.

a reduction in the production of IFN-c and Gzm B), and an

increased regulatory phenotype (CD28lo, CD44lo and CD95lo);

(2) CD8+ cells that are preconditioned by MSCs decrease the

expression of costimulatory molecules on monocytes and

inhibit lymphocyte proliferation; (3) downregulation of CD8 is

essentially mediated by soluble factors and occurs at the post-

transcriptional level; (4) the immunomodulatory effect of hMSCs

on CD8+ T cells requires the presence of CD14+ monocytes,

which acquire a suppressive phenotype; and (5) hMSCs are found

to present similar features in a humanized mouse model.

hMSCs down-modulate CD8 expression

In this study, we have shown for the first time that hMSCs exert a

strong inhibitory effect on expression of the CD8 receptor

(,60% decrease), whereas the relative percentage of CD8+ cells

was only slightly reduced (,15% decrease). This inhibitory

effect was shown to be specific for hMSCs, because other cell

lines that were tested did not show the same features. Although

hMSCs have substantial effects on CD4+ cells, such as a decrease

in several activation markers (data not shown), we decided to

focus on CD8+ cells because of the striking effect of hMSCs on

CD8 detuning, which has not been previously reported. Although

most studies addressing the immunoregulatory properties of

hMSCs have used different means of external activation, we have

used none. Once T cells are activated through their T cell

receptor, they display changes in the expression of surface

markers and in the balance between proliferation and cell death.

By avoiding external activation, we assume that the functional

activity of hMSCs on immune cells mimics the steady state

condition. This difference could explain why this down-

modulation of CD8 has never been reported before.

Reduction in CD8 expression is associated with impaired cell

interaction, leading to loss of cytotoxity (Xiao et al., 2007).

Therefore, we suggest that the inhibitory effect exerted by

hMSCs when co-cultured with unstimulated PBMCs shifts the

cytotoxic CD8+ T cells towards a less cytotoxic and more

suppressive phenotype. This hypothesis is supported by our data

showing that purified CD8+ cells from co-culture with hMSCs

were able to reduce the expression of CD80 and to increase

expression of the ILT3 receptor on monocytes. Interestingly,

these cells were also able to substantially reduce the percentage

of proliferating lymphocytes. In addition, because the absence of

CD28 is one of the CD8+ T-cell hallmarks of a suppressor state

(Cortesini et al., 2001), we analyzed CD28 expression on CD8+

cells and showed its decreased expression when co-cultured with

hMSCs.

A substantial decrease in expression of CD44 was also noted in

the presence of hMSCs. The cell adhesion molecule CD44 is

responsible for mediating adhesion to the extracellular matrix

glycosaminoglycan and hyaluronan (Johnson and Ruffell, 2009).

Considering the role of CD44 in the recruitment of leukocytes in

pathological situations, its decreased expression on CD8+ cells

might lead to inhibition of CD8+ cell migration to inflammatory

sites. Our in vivo data in NSG mice showing inhibition of cell

migration after treatment with hMSCs are compatible with this

hypothesis, and confirm recently published data (Chiesa et al.,

2011). Given the correlation of reduced CD44 expression with

reduced cytotoxic activity in CD8+ CTL clones (Rodrigues et al.,

1992), our results strengthen the idea that hMSCs shift CD8+ cells

towards a non-cytotoxic and/or suppressive phenotype. This

assumption is confirmed further by the decreased secretion levels

of Gzm B and IFN-c in CD8+ cells when co-cultured with hMSCs.

CD14+ monocytes play a central role in the effect of hMSCs

on CD8+ T cells

Zhang and colleagues (Zhang et al., 2004) showed that dendritic

cells were the primary target of the immunosuppressive activity

of MSCs, which affected all major stages of the DC life cycle.

Here, we demonstrate that CD14+ monocytes, which were

sensitized by hMSCs were crucial in the inhibitory effect that

hMSCs exerted on CD8+ T cells.

DCs play a central role in the initiation and regulation of

immune response (Steinman et al., 2003), and their ability to

either initiate an immune reaction or induce tolerance relies on

the transition of co-stimulatory molecules CD80 and CD86, as

well as the upregulation of MHC class II on the cell surface

(Mellman and Steinman, 2001). Immature or regulatory DCs,

which are deficient in co-stimulatory molecules, can induce T

Fig. 6. hMSCs exert an inhibitory effect on human CD8+ cells in NSG mice. A representative histogram comparing (A) CD8; (B) CD44; and (C) CD28

expression in human CD45+ cells in the bone marrow of four three-month-old reconstituted NSG mice, injected either with PBS (dotted line) or with hMSCs

(solid line). Similar trends were observed in the spleen and the peripheral blood. (D) The blood mobilization rate of human cells, in non-activated or LPS-activated

conditions, in NSG mice treated with or without hMSCs. Results show the means of four to seven mice per group. The bars show the s.e.m. *P,0.02 using

Student’s t-test.

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cell anergy, generate regulatory T cells and promote alloantigen-

specific tolerance (Smits et al., 2005). Considering that hMSCs

downregulate the expression of MHC antigen and CD16 (data not

shown), as well as both co-stimulatory molecules CD80 and

CD86 in CD14+ monocytes, we suggest that hMSCs shift

the APC population towards a suppressive phenotype. High

expression of surface inhibitory molecules, such as ILT-3 and

ILT-4, is considered to be a biomarker for tolerogenic APCs

(Chang et al., 2002), and these molecules were also increased in

the monocytes that were co-cultured with hMSCs.

To date, several groups have reported that the inhibitory effect of

hMSCs is mediated by soluble factors (Le Blanc et al., 2004;

Rasmusson et al., 2003), whereas others claim that cell-to-cell

contact is necessary (Krampera et al., 2003). Here, we report that the

down-modulation of CD8 surface expression by hMSCs is the result

of soluble factors that are secreted by hMSCs, which target the

monocytes. However, the CD8 cells require direct contact with the

CD14+ monocytes, probably through the MHC-TCR and

interactions with co-stimulatory molecules. The nature of the

soluble factors involved in the down-modulation of CD8 is still

unclear. Neutralization of TGF-b1 and PGE2 soluble factors did not

reverse the effect of hMSCs on CD8 expression, although a

substantial increase of PGE2 production levels was noted under the

co-culture conditions. These data highlight that the mechanisms of

action of MSCs are diverse, and that downregulation of CD8 is not

dependent on PGE2, whereas inhibition of lymphocyte proliferation

appeared to be, at least in part, dependent upon PGE2 (Spaggiari

et al., 2009).

The mechanisms of action of hMSC are complex, and the

sequence of events is not easy to define. However, based on our

experiments, we can propose the following scenario (illustrated in

supplementary material Fig. S6). First, a soluble factor produced by

hMSCs modulates the functional properties of the CD14+ monocyte

towards a suppressor phenotype (downregulating MHC antigens and

co-stimulatory molecules and increasing ILT3 and ILT4 inhibitory

molecules); second, upon the interaction with MSC-conditioned

monocytes (and in the absence of hMSCs), CD8+ cells have a

decreased expression of CD8 and CD28, and shift towards a

suppressor CD28neg-low phenotype; third, the CD8lo cells (purified

by FACS) were able to reduce the proliferation of fresh lymphocytes

as well as to reduce co-stimulatory molecules on monocytes,

probably maintaining their regulatory phenotype. Although we

cannot exclude a direct effect of conditioned monocytes on the

proliferation of lymphocytes, our data clearly describe one potential

sequential mechanism. The mandatory presence of CD14+ cells

might explain why the hMSCs inhibitory effect is more pronounced

at high lymphocyte concentrations. Indeed, under these conditions, a

higher number of CD14+ cells could be sensitized and exert their

effects on the CD8+ cells.

Moreover, hMSCs were found to induce memory over CD14+

cells, as pre-sensitized monocytes succeeded to reduce surface

expression of CD8, even in the absence of hMSCs. Considering that

hMSCs are capable of inducing pre-sensitized CD14+ cells, we

suggest that the ability of hMSCs to prolong allograft survival is the

consequence of this tolerogenic-inducing effect. Indeed, clinical

studies have revealed that a single intravenous administration of

MSC might be sufficient for achieving long-term tolerance. One

possible explanation is that hMSCs sensitize monocytes and

dendritic cells towards a suppressor phenotype. The duration of

this effect needs to be investigated further.

Physiological relevance of the inhibitory effect of hMSCson CD8+ T cells

In vivo, hMSCs have been shown to be capable of preventing

graft rejection in several transplantation models (Bartholomewet al., 2002; Casiraghi et al., 2008). The ability of hMSCs to protect

allografts from rejection could be mediated by targeting the

effector function of alloreactive T cells. Because the CD8 receptor

plays a crucial role during activation of CD8 T cells (Couedel et al.,1999; Gao and Jakobsen, 2000; Holler and Kranz, 2003), and as

the CD8+CD282 cells could be protective for the graft (Coley

et al., 2009; Colovai et al., 2003; Sindhi et al., 2005), we propose

that the beneficial effects of hMSCs observed in the therapeuticcontext of transplantation occur through the inhibition of

CD8 expression and costimulatory molecules. Conversely, this

mechanism could explain why MSCs could promote cancer

growth (Muehlberg et al., 2009; Prantl et al., 2010). The functionalconsequence of CD8 inhibition by hMSCs is associated with

substantially reduced levels of IFN-c production, as it has been

reported that IFN-c cytokine secretion enhances the rejection of

skin grafts (Mattarollo et al., 2010). Our data obtained from thehumanized mouse model, support that this mechanism of action

does occur also in vivo. A recent paper by Perico and colleagues

(Perico et al., 2011) strengthens our results, because these authors

report that infusion of autologous hMSCs in recipients of kidneyfrom related living donors induces a profound reduction of CD8+

cell activity in the transplanted patients.

The appearance of regulatory CD8+ CD282 T suppressor

cells is associated with a reduced need for maintenance

of immunosuppression in pediatric liver-intestine transplant

recipients (Sindhi et al., 2005), as well as in adult-to-adult livingdonor liver transplantation (Lin et al., 2009). Studies in heart

transplant patients indicate that CD8+ CD282 T suppressor cells

upregulate the inhibitory receptors ILT-3 and ILT-4 on APCs

(Chang et al., 2002). Therefore, it is possible that, in our system,there is a cross-talk between the CD8+ cells and the monocytes.

Not only do the monocytes influence the CD8+ cell phenotype, but

the newly generated CD8+CD28lo cells might also increase the

immunosuppressive properties of the monocytes, as assessed bythe upregulation of ILT-3 and ILT-4 inhibitory molecules. Our

experiments using purified CD8+ cells after co-culture with

hMSCs confirm this hypothesis, because ILT3 was significantlyincreased in monocytes co-cultured in presence of CD8MSC.

In conclusion, our study demonstrates a new immunomodulation

mechanism of action of hMSCs through the modulation of CD8+cells towards a non-cytotoxic and/or suppressive phenotype. This

mechanism of action must be taken into account in clinical trials,

where it should be beneficial in grafts and autoimmune diseases, butpotentially detrimental in malignant diseases.

Materials and MethodsGeneration of human adipose-derived MSCs

The hMSCs were isolated from healthy adult females undergoing a routinelipoaspiration procedure. Informed consent was obtained in accordance with theDeclaration of Helsinki. Adipose tissue cells were washed with phosphate-bufferedsaline (PBS) (Gibco, USA), digested by using 0.1% collagenase I (Worthington,USA) for 1 hour at 37 C, and then centrifuged for 10 minutes at 2000 rpm. Thecell pellet was then re-suspended and passed through a 100 mm filter for removalof debris. The resultant cellular fraction was plated in hMSC complete medium:Dulbecco’s modified Eagle’s medium: Ham’s F12 expansion media (DMEM/F12)(Beit Haemek, Israel), containing 10% fetal calf serum (FCS) (Hyclone, USA),5 ng/ml basic fibroblast growth factor (bFGF) (Biological Industries, Israel),2 mM L-glutamine (Gibco, USA); 1% streptomycin and penicillin solution(Biological Industries, Israel). The cells were incubated in a humid atmospherecontaining 5% CO2 at 37 C and allowed to adhere for 72 hours, after which the

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non-adherent cells were removed by washing with PBS. The culture medium wasrefreshed twice per week thereafter.

When the cells had reached 70–80% confluence, the adherent cells weretrypsinized with Trypsin-EDTA solution B at 37 C for 5 minutes (BiologicalIndustries, Israel), harvested and re-plated in 75 cm2 flasks. After the thirdpassage, a homogenous cell population was obtained, and characterization of theadherent cells was performed by flow cytometry analysis. The adipose-derivedadherent cells were phenotyped as hMSCs, using mouse anti-human antibodiesagainst CD31, CD34, CD45, CD29, CD73, CD44, CD105, HLA-AB and HLA-DR(eBioscience). The typical phenotype of hMSCs is: CD312, CD342, CD452,CD29+, CD73+, CD44+, CD105+, HLA-ABC+ and HLA-DR2. Human isotypeantibodies served as respective controls (eBioscience). hMSCs were maintained inculture for no more than six passages. MSCs derived from different tissue sources,such as abdominal (AD1), breast (AD4) and thigh adipose tissue (AD5), werestudied. Human fibroblasts from foreskin and the HaCat cell line were used ascontrols for hMSCs.

Separation of PBMCs and blood-cell subtypes from whole blood

Blood donations from healthy donors were purchased from the Magen DavidAdom National blood bank service (MADA, Tel Hashomer Hospital, Tel Aviv,Israel). Approximately 50 ml of peripheral blood received per donation wasdiluted with PBS 1:3 and layered on Ficoll-Histopaque density gradient solution(Histopaque 1077; Sigma-Aldrich, Munich, Germany). The mononuclear cellsobtained were enumerated using Trypan blue coloration before fluorescencestaining. The PBMCs were cryo-preserved in freezing media containing 90% fetalbovine serum (FBS) and 10% dimethyl sulfoxide (DMSO), with a cell numberadjusted to 107 cells/ml in cryotube vials.

Subsets of mononuclear cells, including CD4+ T cells, CD8+ T cells and CD14+monocytes, were enriched using the RosetteSep negative-selection method, accordingto manufacturer’s instructions (StemCell Technologies, BC, Canada). The purity ofthe cell populations was 9065% for CD4+ T cells, 8865% for CD8+ T cells and7966% for CD14+ monocytes (6 s.e.m.), according to the flow cytometry data.

For depletion of CD14+ monocytes, PBMCs were labeled with mouse anti-human CD14-PE antibody (ImmunoTools, Friesoythe, Germany), followed byincubation with mouse anti-PE microbeads. The magnetically labeled CD14+ cellswere bound on an MS column using the MiniMACS separator (Miltenyi Biotech,Bergisch Gladbach, Germany), and non-CD14+ cells were recovered.

Direct and transwell co-cultures

The hMSCs (105/well) were seeded in a complete growth medium in six-wellculture plates and were allowed to attach overnight. Allogeneic PBMCs wereadded on the following day, and cells were co-cultured for 3 to 11 days. PBMCsfrom several donors were tested at different ratios, and the inhibitory effect wasfound to be optimal for the hMSC:PBMC ratio of 1:100. Unless otherwisespecified, the experiments were performed with the time frame of 7 days.

For the investigation of CD8+ T cell functionality, CD8+ cells were purified usingFACSAriaTM cell sorter (BD Biosciences, Immunocytometry Systems, MountainView, CA, USA) from PBMCs cultured with (CD8MSC) or without hMSCs(CD8CONT). The purity of CD8+ T cells was .95%. The CD8MSC or CD8CONT cellswere then cultured for 72 hours at 56105 per well, in fresh medium in the presenceof PBMCs to assess their effect on the proliferation of lymphocytes, or in thepresence of purified CD14+ cells to assess their effect on co-stimulation markers.

For co-culture experiments using transwell supports, preventing direct cellinteraction, the hMSCs were seeded in six-well culture plates and were allowed toadhere overnight. Allogeneic PBMCs were added to the upper transwell chamber(30 mm diameter) with a 0.4 mm pore membrane (transwell chamber, Costar).

In several experiments, the hMSCs were replaced by conditioned medium fromhMSC cultures. One day before passaging, the hMSC culture supernatant washarvested, centrifuged and filtered through a 0.2-mm Millipore filter. Allexperiments were performed in duplicates.

Inhibition assay and ELISA

In some experiments, anti-TGF-b1 antibodies (R&D Systems) or indomethacin(Sigma-Aldrich) were added to the co-cultures at concentrations of 2 mg/ml and 5–50 mM, respectively. Controls were treated with an equivalent volume of therelevant carrier solution. IFN-c and PGE2 levels were measured in tissue culturesupernatants by ELISA following the manufacturer’s instructions (R&D Systems,MN, USA).

Mice

NOD SCID RAG22/2cc2/2 (NSG) immunodeficient mice strain were purchased

from the Jackson Laboratories (Bar Harbor, ME, USA). All animal experimentswere approved and properly conducted according to the local Institutionalguidelines IACUC (IL-035-04-2008).

For humanization, 24–48-hour-old NSG mice were irradiated at 100 cGy, and105 purified human CD34+ cells were directly injected into the liver using a 1 mlinsulin syringe (Terumo, Tokyo, Japan), as described previously (Marodon et al.,

2009). For LPS-activated conditions, mice were intraperitoneally injected withLPS (Escherichia coli, Sigma-Aldrich, St Louis, MO) dissolved in saline solutionat a dosage of 50 mg/kg body weight, or given an equal volume of PBS, serving ascontrol PBS group.

CD34+ hematopoietic stem-cell purification

Human umbilical cord blood (hUCB) was obtained from healthy womenundergoing full-term caesarian deliveries at the Rambam Health Care Campus(Haifa, Israel). Informed consent of the mother was obtained in accordance withthe Declaration of Helsinki. Approximately 30 ml of hUCB received per donationwere diluted with PBS 1:3 and layered on Lymphoprep solution (Axis-Shield PoCAS, Oslo, Norway) for density gradient separation.

The mononuclear cells were then labeled with mouse anti-human CD34-PEantibody (ImmunoTools, Friesoythe, Germany), followed by incubation withmouse anti-PE microbeads. The magnetically labeled CD34+ cells were purifiedon an MS column by using the MiniMACS separator (Miltenyi Biotech, BergischGladbach, Germany).

Transfer of hMSCs in humanized NSG mice

16106 hMSCs (AD5) at passage three were re-suspended in 1 ml PBS and injectedintraperitoneally into each humanized mouse at the age of 7–9 months. Seven daysafter the transfer of hMSCs, the mice were killed and samples of peripheral blood,bone marrow and spleen were harvested from the recipient mice. Levels of CD8,CD28 and CD44 expression were determined in the human CD45+ cell gate byflow cytometry. The blood mobilization rate was determined according to theblood:bone marrow ratio of human CD45+ cell number in the NSG mice, treatedor not with MSC. Non-activated and LPS-activated conditions were tested.

Flow cytometry analysis

Cell phenotype was evaluated using conjugated antibodies. To determine thephenotype of the cell surface antigen, single-cell suspensions were incubated withthe following antibodies: anti-CD3-PE-Cy5, anti-CD4-PE-Cy5, anti-CD8a-FITC,anti-CD8a-APC, anti-CD8b-PE, anti-CD25-PE, anti-CD14-PE and anti-CD19-FITC antibodies (ImmunoTools, Friesoythe, Germany); anti-CD105-PE and anti-CD95-PE antibodies (eBioSciences, San Diego, CA); anti-CD8b-FITC antibodies(Immunotech, Marseille, France); anti-CD16 PE, CD80-Alexa 647 and anti-CD86-APC antibodies (BioLegend, San Diego, CA); and anti-HLA-ABC-PE antibodiesand anti-HLA-DR-PE mAb (Dako, Glostrup, Denmark). Isotype-matched antibodieswere used as control. The cells were incubated with the respective fluorescentantibodies at a concentration of 1 mg per 106 cells per 100 ml for 30 minutes at 4 C inthe dark. The cells were then washed twice with ice-cold PBS (pH 7.2), containing1% FCS and 1% bovine serum albumin (BSA), and re-suspended in 300 mL PBS. Inorder to distinguish between surface and intra-cytoplasmic expression of CD8,surface CD8 was stained (using anti-CD8a-APC antibodies) before the inner CD8(using anti-CD8a-FITC antibodies), saturating CD8 surface expression.

Viability of T cell subsets

Dying lymphocytes are characterized by morphological changes, and have a lowerforward scatter (FSC) and a higher side scatter (SSC) compared with the livingcells, which in conjunction with Annexin-PI staining, allow the definition of livingand dead cell gates (Moulian et al., 2001). Accordingly, cell viability wasdetermined in CD4+ and CD8+ cell subsets, cultured either alone or with hMSCs.

Cell proliferation

PBMCs (105/ml in PBS) were incubated with 10 mM CFSE (Invitrogen) at 37 Cfor 15 minutes. Cells were then re-pelleted and re-suspended with fresh pre-warmed medium for another 30 minutes at 37 C. CFSE-labeled PBMCs werewashed twice in PBS and stimulated with beads coated with anti-CD2, anti-CD3and anti-CD28 antibodies, at a 1:2 bead-to-cell ratio (Milteny Biotec) to induce asignificant proliferation. After 24 hours of culture, CFSE-labeled mononuclearcells were incubated with allogeneic highly purified CD8+ T cells (1:5 ratio) fromconditioning cultures, in the presence (CD8MSC) or in the absence of hMSCs(CD8CONT). After 3 days, the cells were washed and resuspended in PBS, and theCFSE level was measured in the CFSE-labeled cells.

For intra-cytoplasmic cytokine expression, a fixation and permeabilization kitwas used according to manufacturer’s instructions (eBioscience, San Diego, CA).The cells were then stained with anti-IFN-c-PE mAb (eBioscience, San Diego,CA) or anti-Granzyme B-FITC antibodies (BioLegend, San Diego, CA).Intracellular FoxP3 was stained using the same kit that included the anti-FoxP3antibody (eBioscience, San Diego, CA).

Data for three- and four-color analysis were collected on a FACSCaliburTM(BD Biosciences, Immunocytometry Systems, Mountain View, CA, USA) andanalyzed using the Flow Cytometry Analysis Software ‘Flowjo’ (Tree Star, Inc.).GraphPad Prism software (San Diego, CA, USA) was used for presentation of theresults and statistical analysis.

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Real-time RT-PCR

For real-time RT-PCR analysis, the PBMCs were cultured in transwell conditionsto avoid any potential contamination with detaching MSCs. Isolation of RNA fromPBMCs and conversion to cDNA were performed according to manufacturer’sinstructions, using the Aurum total RNA kit (Bio-Rad, CA, USA) and theiScriptTM cDNA synthesis kit (Bio-Rad, CA, USA). PCR amplification wasperformed using the SensiMix Plus SYBR (Quantance, CA, USA) according tomanufacturer’s instructions, in the Stratagene Mx3005P real-time PCR platform(Stratagene, La Jolla, CA). The following primers were used: CD8 59 left primer,59-CCCTGAGCAACTCCATCATGT-39; CD8 39 right primer, 59-GTGGGCTT-CGCTGGCA-39; CD4 59 left primer, 59-GTCCCTTTTAGGCACTTGCTTCT-39;CD4 39 right primer, 59-TCTTTCCCTGAGTGGCTGCT-39; UBE202 59 leftprimer, 59-AATGGCAGCATTTGTCTT-39; UBE202 39 right primer, 59-CACA-CAACAGAGAACAGATGGAC-39. UBE202 was used as a housekeeping gene, asdescribed previously (Aquea et al., 2008).

Statistical analysis

Most experiments were performed independently at least three times, and technicalduplicates were systematically performed. Results are given as mean 6 s.e.m. ofindependent experiments throughout the manuscript. The statistics were analyzedusing GraphPad Prism software (San Diego, CA). Throughout the manuscript,either the paired or unpaired two-tailed Student’s t-test (for paired and unpaireddata, respectively) was used for comparison of the two groups. We chose to use theStudent’s t-test and not a nonparametric test because of the low statistical power ofthe nonparametric test for small samples. However, we checked the equality ofvariances. In addition, because in the t-test the assumption is that the data follow aGaussian distribution, we tested the normality of the decreased CD8 expressioninduced by hMSCs, by combining the results from all the experiments (n542) thatwere performed in the study by Kolmogorov-Smirnov, D’Agostino-Pearson andShapiro-Wilk tests, and we found that the distribution was normal. Therefore, weassumed that the distribution was also normal for other markers. However, whenn.3, the nonparametric test was also used and similar results were obtained. Tocompare three or four conditions, we used one-way variance (ANOVA) and posttests: Dunnet’s multiple comparison test was used to compare the differentexperimental conditions with the control, and the Bonferroni test was used tocompare all pairs of columns. Throughout the manuscript, the P values are asfollows: ***P,0.001; **P values between 0.001 and 0.01; *P values between0.01 and 0.05.

AcknowledgementsWe thank I. Petit for helpful discussions and critical review of themanuscript, V. Morad for her contribution in the establishment andcharacterization of the human MSC lines, and O. Shenker and Y.Sakoury for their help in flow cytometry experiments.

FundingThis study was supported in part by the French Association AgainstMyopathies; MYASTAID [grant number LSHM-CT-2006-037833 toS.B.-A]; and FIGHT-MG [grant number HEALTH-2009-242-210 toS.B.-A.] from the European Community.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108860/-/DC1

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