Experimental Hematology 2010;38:922–932

Modulated expression of adhesion molecules and galectin-1:Role during mesenchymal stromal cell immunoregulatory functions

Mehdi Najar, Gordana Raicevic, Hicham Id Boufker, Basile Stamatopoulos, Cecile De Bruyn,Nathalie Meuleman, Dominique Bron, Michel Toungouz, and Laurence Lagneaux

Laboratory of Experimental Hematology, Institut Jules Bordet, Universite Libre de Bruxelles, Brussels, Belgium

(Received 22 February 2010; revised 18 May 2010; accepted 19 May 2010)

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doi: 10.1016/j.exph

Objective. As mesenchymal stromal cells (MSCs) have been proposed as a tool for manage-ment or prevention of graft-vs-host disease, we investigated their immunoregulatory proper-ties, their expression of adhesion molecules and galectin-1, and the impact of environmentcontext on these functions.

Materials and Methods. The effects of MSCs on T-cell proliferation were analyzed using car-boxyfluorescein diacetate N-succinimidyl ester labeling. We evaluated the expression of adhe-sion molecules and galectin-1 by MSCs and the impact of an inflammatory or infectiousenvironment on these expressions. Using neutralizing antibodies against adhesion moleculesand a galectin-1 inhibitor, we assessed the role of these molecules in MSC functions.

Results. MSCs inhibition of T-cell proliferation depended on MSC concentrations, cellcontact, and culture environment. Expression of adhesion molecules and secretion ofgalectin-1 by MSCs are tightly regulated. Coculture with activated T cells upregulated expres-sion of CD54 (intercellular adhesion molecule 1) and CD58 (lymphocyte functionLassociatedantigen 3) and secretion of galectin-1 by MSCs. Interestingly, in an inflammatory or infectiousenvironment, expression of adhesion molecules and galectin-1 by MSCs was differentiallymodulated. Furthermore, blocking galectin-1 activity prevented the suppressive potential ofMSCs. Neutralization of adhesion molecule activity had no effect on MSC inhibition.

Conclusion. Galectin-1 plays an important role in MSC immunoregulatory functions, whichare depending on cell environment. The present study provides new insights concerning MSCphysiology and will increase the safety and efficiency of MSCs in clinical settings. � 2010ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Mesenchymal stromal cells (MSCs) are multipotent progen-itors that can be derived from many sites, including both adultand fetal tissues [1–3]. MSCs are characterized by theirability to differentiate into multiple mesenchymal andnonmesenchymal lineages [4,5] and to exert potent immuno-modulatory effects. Because of these properties, MSCs haveemerged as promising tools for tissue repair, regenerativemedicine, management of graft-vs-host disease (GVHD),and modulation of autoimmune disorders [6].

MSCs exert immunosuppressive effects in vitro throughregulation of different immune cells by several mecha-nisms. MSCs are able to suppress T-lymphocyte activationand proliferation induced by mitogens, polyclonal activa-

: Mehdi Najar, Ph.D., Universite Libre de Bruxelles,

Laboratoire d’Hematologie Experimentale, Blvd de

00 Bruxelles, Belgium; E-mail: mnajar@ulb.ac.be

nt matter. Copyright � 2010 ISEH - Society for Hematol

em.2010.05.007

tors, and cognate antigens [7]. Nevertheless, conflictingdata still exist in the literature regarding the mechanismsby which MSCs modulate immune cells. The potentialmechanisms include both direct cell-to-cell contact andproduction of immunoregulatory factors. Specific effectsinclude the induction and expansion of regulatory T cells;production of soluble immunoregulatory factors, includingprostaglandin E2, hepatic growth factor, transforminggrowth factor�b, interferon (IFN)-g, interleukin (IL)-10,leukemia inhibitory factor, and human leukocyte antigen(HLA) G; and the creation of a tryptophan-depleted envi-ronment via expression of indoleamine 2,3-dioxygenase[8]. In addition to T lymphocytes, MSCs also target otherimmune cells, including B lymphocytes, natural killer cells,and dendritic cells. Regulation of these cells by MSCs ispromoted by several other mechanisms [9]. In cocultures,interaction between MSCs and T lymphocytes seems to

ogy and Stem Cells. Published by Elsevier Inc.

923M. Najar et al./ Experimental Hematology 2010;38:922–932

be crucial for immunomodulation. The galectins area family of soluble lectins characterized by their affinityfor b-galactoside residues [10]. These proteins haverecently attracted increasing attention because of theirinvolvement in various physiological and pathologicalprocesses. Galectin-1 is one of the best characterizedmembers of this family; it possesses diverse functionsand is expressed in many tissues.

Given the importance of adhesion molecules andgalectin-1, we investigated their expression by MSCs indifferent contexts, as well as their roles during MSC immu-noregulatory functions. These careful characterizations ofMSC physiology and the effects of environmental contexton their function will increase the safety and efficiency ofMSCs in clinical settings.

Materials and methods

Human MSC culture and expansionBone marrow (BM) was harvested from the sternum or iliac crestof 10 healthy donors after informed consent was obtained. Meanage of donors was 33 6 2 years (range, 16�41 years). MSCswere isolated using the classical adhesion method. Briefly, mono-nuclear cells were isolated by density gradient centrifugation(LinfoSep; Biomedics, Madrid, Spain), washed in Hank’s bufferedsalt solution (Lonza Europe, Verviers, Belgium), and seeded at 2� 104 cells/cm2 in Dulbecco’s modified Eagle’s medium withlow glucose (Lonza) supplemented with 15% fetal bovine serum(Sigma-Aldrich, Bornem, Belgium), 2 mM L-glutamine, and 50U/mL penicillin (both from Lonza). Cell cultures were incubatedat 37�C in a 5% CO2 humidified atmosphere. After 48 hours, non-adherent cells were removed by washing and the medium waschanged twice a week. When subconfluency (80�90%) wasachieved, adherent cells were trypsinized (Lonza) and expandedby replating at a lower density (1,000 cells/cm2).

MSCs were immunophenotypically characterized by flow cy-tometry using the following monoclonal antibodies: anti�CD166-fluorescein isothiocyanate (FITC) (DakoCytomation, Glostrup,Denmark); anti�CD45-FITC and anti�HLA-DR-phycoerythrin(PE) (Exalpha Biologicals, Maynard, MA, USA); anti�CD34-PE and anti�CD73-PE (BD Biosciences Pharmingen, San Diego,CA, USA); and anti�CD14-PE, anti�CD105-FITC and anti�CD90-PE (R&D Systems, Minneapolis, MN, USA). The colony-forming unit�fibroblast (CFU-F) assay was performed aftereach passage to estimate the number of mesenchymal progenitorsin the culture. To study their multilineage potential, MSCswere cultured in the appropriate induction media to assess adipo-genic, osteogenic, and chondrogenic differentiation as describedpreviously [11].

MSC culture in an inflammatory or infectious environmentAn inflammatory environment was mimicked using a combinationof the following cytokines: 25 ng/mL IL-1b (Peprotech, RockyHill, NJ, USA), 103 U/mL IFN-g, 50 ng/mL tumor necrosisfactor�a, and 3 � 103 U/mL IFN-a (all from Prospec Inc, Reho-vot, Israel). An infectious environment was mimicked bytriggering toll-like receptors (TLRs) expressed on MSCs. Thefollowing TLR agonists were used: 30 mg/mL polyriboinosinic

polyribocytidylic acid [poly(I:C)] (viral infections) for TLR3and 10 mg/mL lipopolysaccharide (LPS) (bacterial infections)for TLR4. Both agonists were purchased from Sigma-Aldrich.These protocols were designed according to studies describedpreviously [12,13].

MSC immunomodulatory potentialPurification of T lymphocytes. Peripheral blood samples were ob-tained from healthy donors after informed consent was obtained.Mononuclear cells were isolated as described for BM mononu-clear cells. CD3þ T lymphocytes were purified by positive selec-tion using the MACS system (Miltenyi Biotec GmbH, Bergisch,Germany) according to the manufacturer’s instructions. The purityof the selected cells was always O95%, as determined by flowcytometry.

T-cell stimulation. Direct T-cell receptor activation by T-cellreceptor agonist anti�CD3/anti�CD28-coated Dynabeads (Dynal,Biotech, Oslo, Norway) was used to stimulate lymphocyte prolif-eration. This method provides full activation and expansion ofT lymphocytes in a purified system. Lymphocyte proliferationwas also induced by mitogenic lectin or allogeneic peripheralblood mononuclear cells (PBMC), as described previously [14].

Stimulation of T lymphocytes by T-cell receptor agonist and bymitogenic lectin or allogeneic PBMC was performed in theabsence of MSCs and then added to cocultures.

Cocultures of MSCs and T cells. Activated T lymphocytes (1� 105)were added to plated MSCs (1 day ahead cocultures) and incubatedfor 5 days of culture in RPMI-1640 medium supplemented with10% fetal bovine serum. MSCs and T-cell cocultures were run at1:1 and 1:80 cell ratios corresponding to high and low MSC concen-trations, respectively. In order to assess the importance of cellularinteractions in MSC-mediated immunomodulation, we used a Trans-well system (Transwell Permeable Supports, Life Sciences, Acton,MA, USA). In some experiments, MSC-conditioned medium(50% final volume) was added to T-cell cultures to evaluate therole of soluble factors.

T-cell proliferation assay. Lymphocyte proliferation was assessedusing the CellTrace CFSE Cell proliferation kit (Invitrogen,Molecular Probes, Eugene, OR, USA). Briefly, 10 mM carboxy-fluorescein diacetate N-succinimidyl ester dye was used to stain107 T cells before coincubation with MSCs. After 5 days of cocul-ture, carboxyfluorescein succinimidyl ester fluorescence wasanalyzed by flow cytometry, and results were expressed as thepercent inhibition of T-cell proliferation. Samples were run ona FACSCalibur (BD Biosciences, San Jose, CA, USA) andanalyzed using CellQuest software (BD Biosciences).

Microarray analysisTotal MSC RNA was extracted in a single step using TriPure Isola-tion Reagent (Roche Applied Science, Vilvoorde, Belgium).Microarray analysis was performed using 1.5 mg RNA with an Af-fymetrix GeneChip Human Genome U133 Plus 2.0 Array, whichcontains more than 54,000 probe sets for the analysis of about47,000 transcripts (Affymetrix, High Wycombe, UK). Briefly,double-stranded complementary DNA was synthesized using theOne-Cycle cDNA synthesis Kit (Affymetrix). The complementaryRNA was synthesized and biotinylated using the IVT Labeling kit

924 M. Najar et al./ Experimental Hematology 2010;38:922–932

according to manufacturer’s recommendations, and then hybrid-ized. Image analysis and probe quantification were done with Af-fymetrix software, which produced raw probe intensity data in theform of Affymetrix .CEL files. Normalization was done using therobust multi-array method [15] to process a group of .CEL filessimultaneously. Genes defined as ‘‘absent’’ by the Affymetrixalgorithm in five or six of the six arrays were not considered forfurther analysis.

Reverse transcriptase polymerase chain reaction (RT-PCR)Total RNA from each cell culture was extracted using the Tripuremethod (Roche Applied Science). The samples (1 mg RNA) weretreated with DNase (Invitrogen Life Technologies, Merelbeek,Belgium) at 37�C for 30 minutes in a final volume of 10 mL con-taining 1 mL DNase, 10� buffer, and 1 U DNase RQI. The reversetranscription was performed with 1 mg DNase-treated RNA usingMoloney murine leukemia virus reverse transcriptase (InvitrogenLife Technologies) in a final volume of 20 mL containing 4 mLfirst-strand buffer, 10�2 M dithiothreitol, 1 mM of each deoxynu-cleoside triphosphate, 50 U RNase inhibitor, and 100 U Moloneymurine leukemia virus reverse transcriptase. The reverse transcrip-tion produced 5 ng complementary DNA, which was then used fora PCR reaction in a final volume of 50 mL containing 25 mL multi-plex PCR mix (QIAGEN Venlo, The Netherlands) and 200 nmolupstream sense and downstream antisense primers (Sigma-Genosys, Pampisford Cambs, UK). After activation at 95�C for15 minutes, PCR was performed for 35 cycles for each primerset with the following conditions: denaturation at 94�C for 30seconds, annealing at 60�C for 90 seconds, and extension at72�C for 90 seconds. After a 10-minute elongation step at 72�C,PCR products were separated by electrophoresis on 2% (w/v)agarose gels and visualized by ethidium bromide staining. Gelswere scanned with an FLA-5000 imaging system (Fujifilm,Raytest, Straubenhardt, Germany) and Image Reader software(Raytest). Band intensities were quantified using AIDA Image An-alyser 3.45 software (Raytest) and normalized against band inten-sities of b-actin as an endogenous loading control. A negativecontrol of the RT-PCR was performed without RT to verifycomplete DNase treatment and exclude possibility of genomicDNA amplification.

The following primers were used:

Galectin-1 (forward) 50- AAC CTG GAG AGT GCC TTCGA-30

(reverse) 50- GTA GTT GAT GGC CTC CAG GT-30

b-actin (forward) 50- CTG GCA CCC AGC ACA ATG -30

(reverse) 50- CCG ATC CAC ACG GAG TAC TTG -30

Human recombinant galectin-1 assayThe influence of human recombinant galectin-1 (R&D Systems)on lymphocyte proliferation was also evaluated. We added 30mg/mL recombinant galectin-1 to 5-day lymphocyte cultures.

Analyses of MSC surface molecule expressionAnalyses of cell surface molecule expression on MSCs were per-formed by flow cytometry. MSCs were labeled with the followingconjugated monoclonal antibodies (Abcam, Inc., Cambridge, MA,USA): CD49e-FITC, CD106-PE, CD54-FITC, CD102-FITC, andCD58-PE.

Human galectin-1 quantification assayHuman galectin-1 levels were measured in cell culture superna-tants using an enzyme-linked immunosorbent assay technique ac-cording to manufacturer’s instructions (Antigenix America Inc,Huntington Station, NY, USA). The detection sensitivity of thisenzyme-linked immunosorbent assay is approximately 0.3 ng/mL.

Blocking and neutralization experimentsThe neutralizing human antibodies against CD49e, CD106, CD54,CD102, and CD58 were used to block interactions between MSCsand T cells. All of these antibodies were purchased from R&DSystems and used at a concentration of 10 mg/mL. The sugarbinding activity of galectin-1 is inhibited by D-galactopyranosyl-b-D-thiogalactopyranoside (thiodigalactoside). This galectin-1 inhib-itor, was purchased from Sigma and used at a concentration of20 mM. All compounds were applied to the cocultures for 5 days.

Statistical analysisData are expressed as mean 6 standard error of the mean. Statis-tical comparisons were performed using the Wilcoxon test forpaired samples; p ! 0.05 was considered to be statisticallysignificant.

Results

Obtaining and characterizing human MSCsMSC cultures were obtained from 20 mL BM aspiratesfrom 10 healthy donors. Mean time for the primary cultureto reach subconfluence was 13 6 2 days. After one passage(P1), adherent cells displayed a fibroblast-like morphologyin culture plate. MSCs were immunophenotypically charac-terized by flow cytometry. This analysis (Fig. 1) revealedthat MSCs were uniformly positive for CD73 (87% 6

1%), CD90 (97% 6 1%), CD105 (96% 6 1%), andCD166 (98 6 1%), but negative for CD14 (0.7% 6

0.2%), CD34 (0.05% 6 0.03%), CD45 (0.8% 6 0.12%),and HLA-DR (0.3% 6 0.1%). The mean number ofCFU-F obtained from the bone marrow samples was 45 6

8/106 cells, confirming the low level of MSCs present inBM. After P1, the mean number of CFU-F was 90 6 16� 103, demonstrating the expansion of MSCs. The CFU-F efficiency remained stable throughout the duration ofthe culture. To confirm their differentiation potential,MSCs were plated in specific induction media for genera-tion of adipocytes, osteoblasts, or chondrocytes. After 2to 3 weeks, lipid vacuoles, calcium deposits, or chondro-genic matrix were observed, respectively, demonstratingthe multipotent nature of the MSCs (data not shown).

Immunosuppressive effects of MSCs on activated T cellsLymphocyte proliferation induced by CD3/CD28 agonists,mitogenic lectin, and allogeneic PBMC, were evaluatedin the presence or absence of MSCs. After 5 days of cocul-ture, MSCs inhibited T-cell proliferation regardless of thestimulation used to activate lymphocyte. We also observedthat this inhibition was MSC dose- and contact-dependent.

Figure 1. Characterization of human mesenchymal stromal cells (MSCs) by flow cytometry. Bone marrow MSCs at passage 1 were stained with specific

monoclonal antibodies (black line) against CD105, CD73, CD90, human leukocyte antigen�DR, CD14, CD34, CD166, and CD45. White lines indicate

isotype-matched mouse immunoglobulin G antibody control staining. A representative histogram from 1 of 10 independent experiments is presented.

925M. Najar et al./ Experimental Hematology 2010;38:922–932

Independently of T-cell activation, at low ratio (1:80) MSCsdid not inhibit T cells (0% inhibition) and proliferation re-mained unchanged. We reported an optimal inhibition athigh ratio (1:1), where induced lymphocyte proliferationwas suppressed by more 60% 6 5% in all case. For thenext experiments, we decided to conduct MSC/T-cell cocul-ture at 1:1 ratio corresponding to an optimal lymphocytesuppression. A representative example of MSC-mediatedinhibition is shown in Figure 2. After 5 days of coculture,MSCs suppressed lymphocyte proliferation in a contact-dependent manner. In the presence of direct contactbetween MSCs and T cells, lymphocyte proliferation wasefficiently and significantly (p ! 0.02) reduced by O60%.In the absence of direct contact (using the Transwellsystem), the inhibition was less effective. Interestingly,poly(I:C) or LPS treatment of MSCs significantly decreasedtheir ability to suppress T-cell proliferation. Indeed, thepercentage of T-cell inhibition was strongly reduced andreached only 17% 6 3%.

Conditioned medium (CM) from MSCs cultured alone,even at high concentrations, failed to inhibit proliferationof T cells. However, CM from MSC/T-cell cocultures(Fig. 3) was able to suppress the lymphocyte response(p ! 0.03). This inhibition was also dependent on thecell-to-cell contact between MSCs and T cells. CM ob-tained from direct contact coculture, however, was more

immunosuppressive than CM obtained from cultureswithout contact.

Microarray analysisIn order to evaluate MSC expression of molecules involvedin cellular interactions, we performed microarray analysis.Different families of cell adhesion molecules, such asthe immunoglobulin superfamily, integrins and galectins,have been found to be involved in cell interactions. Forthis study, we investigated three different samples of BM-MSCs at passage 1. Our microarray analysis revealed thatthe MSCs expressed the genes for intercellular adhesionmolecule 1 (ICAM-1) (CD54), ICAM-2 (CD102), vascularcell adhesion molecule (VCAM) (CD106), and lymphocytefunction�associated antigen 3 (LFA-3) (CD58), whichbelong to the immunoglobulin superfamily, and the integrinvery late activation antigen (VLA)-a5 (CD49e). Amongmembers of the galectin family, we only detected geneexpression of galectin-1.

Expression of cell surface molecules on MSCsFlow cytometry analysis revealed that MSCs constitutivelyexpressed ICAM-1 (CD54), ICAM-2 (CD102), VCAM(CD106), LFA-3 (CD58), and VLA-a5 (CD49e) at differentlevels. VCAM and VLA-a5 showed the highest expression,while the other proteins were only weakly positive. The

Figure 2. Mesenchymal stromal cells (MSCs) inhibit activated T-cell proliferation. In each experiment, carboxyfluorescein succinimidyl ester (CFSE)�labeled

T cells were activated by CD3/CD28 agonists, and MSCs (1� 105) were added to the lymphocyte coculture (1:1 ratio) in the presence or absence of direct contact.

(A) Representative CFSE histogram plot. CFSE fluorescence was obtained by flow cytometry analysis. (B) Percentage of T-cell inhibition. Data are expressed as

mean percentage of T-cell inhibition 6 standard error of mean from seven independent experiments. p Values are denoted as: **p ! 0.02.

926 M. Najar et al./ Experimental Hematology 2010;38:922–932

MSCs were able to modulate the expression of somemolecules, depending on the environment (Table 1). Amongthese molecules, only CD54 and CD58 were considerablyupregulated (increase of the percentage of positive cells)when the MSCs were cultured in the presence of activatedT cells or in an inflammatory/infectious environment.

Figure 4 displays a representative example of the shiftobserved in CD54 expression. As shown in Table 1, themedian fluorescence intensity of both CD54 and CD58were significantly increased and the variation was morerelevant under indicated conditions. As the expressionlevels of CD106 and CD49e were constitutively high, we

Figure 3. Mesenchymal stromal cell (MSC) conditioned medium (CM)

inhibits T-cell proliferation. CM obtained from 5 days of culture under

the indicated conditions was tested for immunomodulatory effects on

lymphocyte proliferation. Data are expressed as mean percentage of T-

cell inhibition 6 standard error of mean from seven independent

experiments. p Values are denoted as: *p ! 0.03.

927M. Najar et al./ Experimental Hematology 2010;38:922–932

could not observe any modulation of these molecules, evenafter coculture with T cells. These observations werefurther confirmed by intracellular staining confirming theupregulation of CD54 and CD58.

However, exposure of MSCs to inflammatory/infectiousenvironments resulted in increased T-cell migration. Thepercentage of T cells transmigrating into the MSC layerwas significantly enhanced (data not shown).

Galectin-1 expression in MSCsWe evaluated the messenger RNA expression of galectin-1 inMSCs obtained from five donors, and all samples showed

Table 1. Expression of adhesion molecules by mesenchymal stromal cells

Surface antigen MSC Control MSC/T cells

CD102

% 3.75 6 0.7 3.8 6 0.2

MFI 42 6 2 38 6 1.5

CD54

% 18.4 6 0.6 77 6 3.3*

MFI 15 6 1.6 322 6 20**

CD106

% 58 6 0.7 57 6 1.2

MFI 14.5 6 1 12.7 6 1.6

CD58

% 17.5 6 1,6 51 6 2.3*

MFI 12 6 1.5 96 6 9*

CD49e

% 95 6 1 84 6 2.7

MFI 24 6 2 27 6 1.6

Flow cytometry analysis was carried out using specific monoclonal antibodies.

lymphocytes or in the presence of an inflammatory or infectious environment. M

antibody (Miltenyi) to exclude T cells. Data obtained from seven independent

of cells expressing each surface antigen and corresponding median fluorescence

ribocytidylic acid.

p Values are denoted as: *p ! 0.031; **p ! 0.01.

positive expression (Fig. 5A). We then used semi-quantitative RT-PCR to measure this expression using b-actinas an internal control. As shown in Figure 5B, we confirmedthe constitutive expression of galectin-1 messenger RNA byMSCs. In addition, no significant difference in galectin-1messenger RNA expression was detected between thedifferent donors.

Soluble recombinant galectin-1 suppresses T-cellproliferationAs galectin-1 was expressed by MSCs, we assessed theinfluence of human recombinant galectin-1 on activatedT cells. The addition of recombinant galectin-1 (30 mg/mL) to cultured lymphocytes inhibited their response, asshown by a significant (p ! 0.02) decrease in T-cellproliferation (Fig. 5C).

Production of soluble galectin-1 by MSCsUsing an enzyme-linked immunosorbent assay technique,we evaluated the level of galectin-1 in culture supernatants(n 5 5). The secretion of galectin-1 by MSCs depended onculture conditions. Under normal conditions, the MSCsconstitutively produced 8 6 1 ng/mL galectin-1 (Fig. 6A).Cocultures of MSCs with activated T cells significantlyincreased galectin-1 production by more than fourfold ina cell contact-dependent manner. In the absence of contact,galectin-1 levels remained approximately equivalent to thecontrol level (10 6 2.3 ng/mL). Interestingly, the increasedgalectin-1 secretion by MSCs during lymphocyte coculturewas independent of the stimulus (allogeneic PBMC,phytohemagglutinin/IL-2 cocktail) used to activate the

MSC/inflammation MSC/PIC MSC/LPS

4 6 0.75 3.7 6 0.65 3.5 6 0.6

48 6 3 46 6 2.2 44 6 2.5

93 6 2* 69 6 3* 72 6 3*

240 6 12** 132 6 10** 105 6 9*

60 6 1 61 6 1.4 57 6 0.85

19 6 2 16.5 6 0.8 17 6 1.2

43 6 2.1* 32 6 0.7* 37 6 0.9*

60 6 5* 51 6 7* 44 6 4*

97.5 6 0.5 97 6 1 96.5 6 0.7

31 6 2.2 25 6 1.7 30 6 2.4

Mesenchymal stromal cells (MSCs) were cultured either with activated T

SCs were also stained with an anti�CD45-VioBlue-labeled monoclonal

experiments are presented as mean 6 standard error of mean percentage

intensity (MFI). LPS 5 lipopolysaccharide; PIC 5 polyriboinosinic poly-

Figure 4. Representative flow cytometry histogram of CD54 expression.

Expression and modulation of CD54 adhesion molecule on mesenchymal

stromal cells (MSCs) were assessed by flow cytometry using specific

monoclonal antibody. MSCs were cultured either with activated T lympho-

cytes or in the presence of an inflammatory or an infectious environment.

Ctrl 5 control; Infla 5 inflammation.

928 M. Najar et al./ Experimental Hematology 2010;38:922–932

T cells. Indeed, galectin-1 levels in these cocultures reached40 6 3 ng/mL and 35 6 1.9 ng/mL, respectively.

Depending on the inflammatory or infectious nature ofthe environment, the MSCs modulated their expression ofgalectin-1 differently, as shown in Figure 6B. In an inflam-matory environment, the MSCs showed increased secretionof galectin-1. In contrast, galectin-1 levels decreased underinfectious conditions (polyriboinosinic polyribocytidylicacid and LPS). Both the increase and decrease ofgalectin-1 secretion under the indicated conditions weresignificant (p ! 0.02). In the presence of simultaneousinflammation and infection, however, the MSCs did notshow modulation of galectin-1 secretion; the level remainedunchanged compared to the control.

Blockade of cell interactions and inhibitionof galectin-1 activityAdhesion and integrin molecules are not involved in thesuppressive effects of MSCs We first used human mono-clonal antibodies against CD49e, CD106, CD54, CD102,and CD58 to block the interaction between MSCs andT cells. The effects of MSCs on lymphocyte proliferationwere not altered by this approach, even when the antibodieswere used in combination (Fig. 7A).

Prevention of the immunosuppressive effect of MSCsby blockade of galectin-1 activity Treatment with thiodi-galactoside, an inhibitor of galectin-1 binding to itscarbohydrate recognition domain, confirmed the role ofgalectin-1 in the inhibition of T-cell proliferation. The

blockade of galectin-1 by thiodigalactoside preventedthe suppression of lymphocyte proliferation by theMSCs (Fig. 7B), and T-cell proliferation was restored, asshown by the significant decrease in the percentage oflymphocyte inhibition (25%).

DiscussionMSC immunomodulatory functions have generated clinicalinterest for the improvement of the efficiency of hematopoi-etic stem cell transplantation, management of GVHD, andmodulation of autoimmune disorders [6]. In this study,MSCs were characterized and defined according to theInternational Society for Cellular Therapy minimal criteria[16].

MSCs act as pleiotropic immune regulators to modu-late immune responses through the production of multiplesoluble factors and by direct cell-to-cell contact. We con-firmed the ability of MSCs to suppress lymphocyteproliferation regardless of T-cell stimulation used. Wealso observed a dose- and contact-dependent inhibition ofT-cell proliferation by MSCs. Indeed, only MSCs at highconcentrations potently inhibited lymphocyte proliferationand this effect was only optimal when a direct contactbetween the two cell populations was allowed. Theseobservations are in agreement with the literature [14,17],demonstrating that the suppressive effects of MSCs aredose-dependent but not stimulus-dependent. Exposure ofMSCs to poly(I:C) and LPS clearly antagonized theirimmunosuppressive properties and T-cell proliferationwas restored. These observations suggest that factorsinvolved in immunomodulation are released by MSCsonly after coculture with T lymphocytes and that thisproduction is sensitive to culture environment. Inflamma-tion and infection are known to be major events triggeringGVHD after allogeneic stem cell transplantation [18].MSCs are particularly sensitive to environmental signals[19] that modulate the functions and responses of MSCs[20–22]. An inflammatory and infectious environmentwere mimicked using, respectively, a cytokine cocktail(IL-1b, IFN-g, TNF-a, and IFN-a) or by triggering TLRwith their agonists (LPS and poly(I:C)) [12,13]. UsingIFN-a was of importance because this cytokine wasdescribed to confer proinflammatory function and topossess immunoregulatory functions [23,24].

The influence of such signals on expression of adhesionmolecules by MSCs is poorly understood. MSCs expressa large number of surface molecules [25], including thoseof the integrin families and adhesion molecules responsiblefor cellular interactions via binding to receptors on T cells.Our microarray analysis revealed that MSCs expressed theadhesion molecules ICAM-1 (CD54), ICAM-2 (CD102),VCAM (CD106), LFA-3 (CD58), and integrin VLA-a5(CD49e), which might be involved in MSC/T-cell interac-tions [4,26,27]. Flow cytometry analysis revealed that

Figure 5. Assessment of galectin-1 gene expression in mesenchymal stromal cells (MSCs). After obtaining and expanding the MSCs, we assessed their

messenger RNA expression of galectin-1. RNA was isolated from MSCs and subjected to semi-quantitative reverse transcriptase polymerase chain reaction

(RT-PCR) using specific primers for b-actin and galectin-1. (A) Amplified products were separated by electrophoresis. A representative agarose gel electro-

phoresis analysis of the amplified products and the negative control of the RT-PCR are presented. (B) Amplicon band densities were then quantified by

densitometry. Results were normalized by comparison with the housekeeping gene b-actin and expressed as the ratio of target gene expression to b-actin

expression. Data are presented as mean relative expression 6 standard error of mean (SEM) from 10 independent experiments. (C) The effect of human

recombinant galectin-1 on T-cell proliferation. Human recombinant galectin-1 (30 mg/mL) was added to activated lymphocyte cultures for 5 days. Data

are expressed as mean percentage of T-cell proliferation 6 SEM from seven independent experiments. p Values are denoted as: **p ! 0.02.

929M. Najar et al./ Experimental Hematology 2010;38:922–932

MSCs constitutively expressed these molecules at differentlevels. Importantly, we found that MSCs cultured with acti-vated T cells or in an inflammatory/infectious environmentupregulated CD54 and CD58 expression. Our observationsare in agreement with previous reports showing that theexpression of adhesion molecules is dependent on cultureconditions [28] and can be modulated under certain circum-stances [29–31]. Despite their enhanced expression byMSCs during coculture with activated lymphocytes, CD54and CD58 did not appear to participate directly in the inhi-bition of T-cell proliferation by MSCs as demonstrated byneutralization/blocking assays. Importantly, migration oflymphocytes into MSC surroundings is pivotal for theestablishment of immunomodulation [32]. Such migrationimplies cell interactions mainly promoted by CD54 andCD58 [33], the expressions of which were upregulated byMSCs in the presence of T cells. Moreover, the exposureof MSCs to such environments increased T-cell migrationtoward the MSCs. By increasing both lymphocyte migra-tion and CD54/CD58 expression, MSCs seem to favorand promote their own interaction with T cells.

Several soluble factors involved in the immunosuppres-sive effects of MSCs have been described in the literature[8], including transforming growth factor�b1 and HLA-G,with contradictory results. In our system, we did not observeany significant role of these molecules in the inhibition ofT-cell proliferation by MSCs. Galectins are a family of animallectins involved in modulation of immune responses and arecharacterized by their affinity for b-galactosides [34,35].Previous studies reported therapeutic functions for galectin[36], as well as potential roles during fetomaternal tolerance[37,38]. Interestingly, Baum et al. observed that galectin-1could modulate and ameliorate GVHD occurring duringallohematopoietic stem cell transplantation [39].

Given that galectin-1 has both immunosuppressive andanti-inflammatory effects [40,41] and that MSCs mediateimmunomodulation via the release of soluble inhibitoryfactors, we decided to evaluate the expression and role ofsoluble galectin-1 produced by MSCs during coculturewith T lymphocytes.

Microarray analysis revealed that the galectin-1 gene isexpressed by MSCs and this observation was confirmed

Figure 6. Detection of soluble galectin-1 by enzyme-linked immunosorbent assay (ELISA). Supernatants from different mesenchymal stromal cell (MSC)

culture conditions were collected, and soluble galectin-1 was quantified by ELISA. (A) Galectin-1 level during MSC and T-cell coculture (5 days). (B)

Galectin-1 level after exposure (1 day) of MSCs to an inflammatory or infectious condition. Data are expressed as mean 6 standard error of mean from

seven independent experiments. p Values are denoted as: **p ! 0.02. Infla 5 inflammation; LPS 5 lipopolysaccharide; PIC 5 polyriboinosinic polyribo-

cytidylic acid.

930 M. Najar et al./ Experimental Hematology 2010;38:922–932

by RT-PCR. This finding is in agreement with previousreports [42,43] that showed gene expression of galectin-1without exploring the protein level. In this study, wedemonstrated that galectin-1 protein is constitutivelysecreted by MSCs and that this secretion was markedlyincreased during direct coculture with activated T cells,but remained unchanged in the absence of cell contact.The blockade of galectin-1 activity prevented the suppres-sion of T-cell proliferation by MSCs. These resultshighlight another mechanism involving galectin-1 in the

Figure 7. (A) Adhesion and integrin molecules are not involved in the immunos

were cultured for 5 days with MSCs in the presence or absence of neutralizing ant

(CD102), vascular cell adhesion molecule (VCAM) (CD106), lymphocyte funct

(VLA)-a5 (CD49e). Seven independent experiments were performed and data are

(SEM). (B) Prevention of the MSC immunosuppressive effect by blocking galec

presence or absence of thiodigalactoside (TDG). Seven independent experimen

inhibition 6 SEM. p Values are denoted as: **p ! 0.02.

regulation of immune responses by MSCs. Recently, Lepel-letier et al. reported that galectin-1 and semaphorin-3A,both expressed by MSCs, may be involved in the immuno-suppressive effects of MSCs [44]. We demonstrated,however, that the production of galectin-1 is greatlyincreased after contact with activated T cells, leading tothe efficient suppression of lymphocyte proliferation. Littleis known about the in vitro influence of inflammatory andinfectious signals on galectin-1 secretion by MSCs. Weobserved that galectin-1 secretion by MSCs was modulated

uppressive effects of mesenchymal stromal cells (MSCs). Activated T cells

ibodies against intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2

ion�associated antigen 3(LFA-3) (CD58), and very late activation antigen

expressed as mean percentage of T-cell inhibition 6 standard error of mean

tin-1 activity. Activated T cells were cultured for 5 days with MSCs in the

ts were performed and data are expressed as mean percentage of T-cell

931M. Najar et al./ Experimental Hematology 2010;38:922–932

differently depending on the environment. In the presenceof a proinflammatory cytokine cocktail, galectin-1 secretionby MSCs was considerably increased. Similarly, humanendothelial cell expression of galectin-1 was upregulatedafter cells treatment with a mixture of inflammatory cyto-kines [45]. It is, therefore, likely that increased expressionof galectin-1 contributes to the inhibitory effect of MSCson T-cell responses, thereby reducing inflammation [46,47].

Activation of TLRs with their respective agonists resultedin decreased galectin-1 expression by MSCs and couldexplain the prevention of their immunomodulatory activityin such context. Recently, we reported that poly(I:C) andLPS treatment of MSCs result in decreased expression ofother immunomodulatory factors, such as hepatic growthfactor and prostaglandin E2 [13].

These data suggest that in the presence of a dangerousenvironment, such as infections, MSC immunosuppressivefunctions could be blockade to maintain the responsivenessof the immune system and its reactivity to pathogens. Theimmunosuppressive potential of MSCs is not constitutive,but rather induced under specific circumstances. A cross-talk between MSCs and immune cells seem to be crucial.Inflammation, infection, and immune response induce stim-ulation of lymphocytes. During coculture with theseactivated lymphocytes and probably through T-cell derivedcytokines, MSCs is activated in order to become suppres-sive. Once MSCs is triggered they acquire an inhibitoryprofile and increase the release of immunoregulatoryfactors, such as galectin-1, responsible for T-cell inhibition.

Expression of galectin-1 is modulated by particularcircumstances, such as immune response, inflammation,and infections, providing an important contribution toimmune homeostasis [48]. According to our observationsand other reports [49,50], depending on the challenge,MSC immunomodulation is tightly regulated to maintaina balanced immune response.

In conclusion, the present study provides new insightsconcerning environmental dependence of MSCs immunoreg-ulatory functions as well as their expression of adhesion mole-cules and galectin-1. We identified a pivotal role of galectin-1in MSC-mediated immunomodulation during coculture withT lymphocytes. Triggering MSCs by T-cell�derived cyto-kines could increase their secretion of galectin-1 and thusensure efficiency of the suppression. Due to their immuno-modulatory and anti-inflammatory properties, MSCs havebecome increasingly attractive as a therapeutic approach forthe prevention and treatment of GVHD and autoimmunediseases. Careful characterization of MSC physiology andthe effects of environmental context on their functions willincrease the safety and efficiency of MSCs in clinicalsettings.

AcknowledgmentsM. Najar and G. Raicevic are Televie research fellows of ‘‘LeFonds National de la Recherche Scientifique’’ (Brussels, Belgium)

(FRS-FNRS 3.4.532.07F–7.4.524.08F). This study was also sup-ported by BRUSTEM an impulse program of ‘‘Institut d’encour-agement de la Recherche Scientifique et de l’Innovation deBruxelles’’ (IRSIB; Brussels, Belgium).

Conflict of Interest DisclosureNo financial interest/relationships with financial interest relatingto the topic of this article have been declared.

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