Human Mesenchymal Stem Cells Promote ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS Human Mesenchymal Stem Cells Promote Survival of T Cells in a Quiescent State FEDERICA BENVENUTO,a,b STEFANIA FERRARI,a,b EZIO GERDONI,a FRANCESCA GUALANDI,c FRANCESCO FRASSONI,c VITO PISTOIA,d GIANLUIGI MANCARDI,a,b ANTONIO UCCELLIa,b a

Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Genoa, Italy; bCentre of Excellence for Biomedical Research, University of Genoa, Genoa, Italy; cDepartment of Haematology, San Martino Hospital, Genoa, Italy; dLaboratory of Oncology, Istituto di Ricovero e Cura a Carattere Scientifico, G. Gaslini, Genova, Italy Key Words. Mesenchymal stem cells • Apoptosis • T cells • Fas

ABSTRACT Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC damp-

ened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. STEM CELLS 2007;25:1753–1760

Disclosure of potential conflicts of interest is found at the end of the article.

INTRODUCTION Mesenchymal stem cells (MSC) are nonhematopoietic cells first identified in the late sixties [1] as a component of the bone marrow stromal scaffold supporting hematopoiesis. As a consequence, MSC have been proposed to promote marrow engraftment in vivo and favor immune reconstitution following allogeneic transplantation [2]. MSC have been identified also in other tissues, both fetal and adult, including umbilical cord blood, fetal liver, and amniotic fluid, as well as lung, adipose tissue, cartilage, synovium, dental tissues, and peripheral blood [3]. MSC are also considered a promising strategy for tissue repair, as they have been shown to differentiate into almost any cell type derived from all lineages [4]. Besides their transdifferentiating capacity, MSC mediate immunomodulatory effects. Several groups demonstrated that MSC inhibit different effector functions of immune cell populations including T cells, B cells, dendritic cells (DC), and natural killer cells [5]. In particular, MSC have been proposed to inhibit T-cell proliferation through the induction of cell division arrest but not apoptosis [6]. Similar results have been observed also on B cells [7] and DC [8]. This immunomodulatory effect of MSC is at the basis of their in vivo capacity of prolonging skin graft survival [9], ameliorating

autoimmune experimental encephalomyelitis (EAE) [10, 11], and treating human graft-versus-host disease [12]. Recent data suggest that the MSC therapeutic effect may be poorly linked to cell transdifferentiation but is mainly due to protective effects on the surrounding tissue [13, 14]. These results are in line with an in vitro [15] and an in vivo antiapoptotic effect exerted by MSC on neurons [11]. Recently, Ramasamy and colleagues demonstrated that MSC can inhibit leukemia cell proliferation and also their apoptosis, suggesting that MSC may “freeze” cells in a resting state [16]. In this study, we addressed the capacity of MSC to inhibit, under different experimental conditions, T-cell proliferation, but also T-cell apoptosis induced through T-cell receptor (TCR) engagement. In addition, we provide compelling evidence that inhibition of apoptosis is achieved through a direct effect on the Fas-FasL pathway. These results support the idea that MSC support T-cell survival in a quiescent condition.

MATERIALS

AND

METHODS

MSC In Vitro Culture and Expansion Human bone marrow samples were obtained from healthy donors undergoing bone marrow explant for allogeneic transplantation procedures as described elsewhere [7]. Briefly, bone marrow mononu-

Correspondence: Antonio Uccelli, M.D., Neuroimmunology Unit, Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132 Genoa, Italy. Telephone: ⫹39 010 3537028; Fax: ⫹39 010 3538639; e-mail: [email protected] Received January 29, 2007; accepted for publication March 20, 2007; first published online in STEM CELLS EXPRESS March 29, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0068

STEM CELLS 2007;25:1753–1760 www.StemCells.com

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clear cells were isolated by density gradient centrifugation (1,077 g/ml; Lympholyte Cell Separation Media; Cedar Lane, Hornby, ON, Canada, http://www.cedarlanelabs.com) and seeded at the density of 25–30 ⫻ 106 cells per 75-cm2 flask (Sarstedt, Nu¨mbrecht, Germany, http://www.sarstedt.com/php/main.php) in Human MesenCult Basal Medium additioned with its specific supplement (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell. com) and incubated at 37°C and 5% CO [2]. Bone marrow nonadherent cells were removed after 4 days, and culture medium was refreshed twice per week thereafter. At 80% confluence, cells were harvested with 0.05% trypsin and 0.02% EDTA (Euroclone, Milan, Italy, http://www.euroclone.net) and plated in 75-cm2 flasks at the density of 7 ⫻ 105 cells. Characterization of MSC in culture was achieved by flow cytometry. Typical CD34⫺ CD45⫺ CD14⫺, CD73⫹ CD44⫹ CD105⫹ cells were usually obtained after three passages in culture. The MSC immunophenotype was defined utilizing the following monoclonal antibodies: anti-CD34 fluorescein isothiocyanate (FITC), CD14 phycoerythrin (PE), CD73 PE, CD44 FITC (Becton, Dickinson and Company, Franklin Lakes, NJ, http:// www.bd.com), CD45 PE-CY5 (Serotec Ltd., Oxford, U.K., http: //www.serotec.com), and CD105 PE (Ancell, Bayport, MN, http:// www.ancell.com).

Media, Stimuli, and Reagents Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood of healthy donors and separated by density gradient centrifugation as previously described. PBMC were suspended in RPMI 1640 (Euroclone) enriched with 2 mM Lglutamine, penicillin, and streptomycin (Gibco, Grand Island, NY, http://www.invitrogen.com) and 10% fetal calf serum (FCS; Gibco). Jurkat leukemic T cells were provided by American Type Culture Collection (ATCC, Manassas, VA, http://www.atcc.org) and cultured in RPMI 1640 plus 10% FCS at the density of 0.5 ⫻ 106 cells per milliliter and grown at 37°C in a humidified 5% CO2 atmosphere. Cells were routinely subcultured every 2–3 days and medium refreshed 24 hours before each experiment. Anti-Fas IgM monoclonal antibody (mAb) (CH11) and anti-Fas IgG1 blocking mAb (ZB4) were both purchased by Immunotech (Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp). Human recombinant IL-2 (Proleukin) was obtained from Chiron Corporation (Siena, Italy, http://www.chiron.com) and used at 100 IU/ml for both T-cell proliferation and activation induced cell death (AICD) protocols. A monoclonal antibody directed against human CD3 was purified from supernatants of the hybridoma cell line OKT3 (obtained from ATCC). Etoposide (VP16) was purchased from Bristol-Myers Squibb (New York, http:// www.bms.com).

Isolation of Human Thymocytes Human thymocytes were isolated from thymic lobes obtained from pediatric patients undergoing thoracic surgery upon parents’ informed consent. Briefly, thymic lobes, dissected out aseptically, were transferred into ice-cold Dulbecco’s phosphate-buffered saline (DPBS; Euroclone) containing 2% FCS, washed thoroughly, and passed, with a syringe plunger, through a 70 ␮M pore fine nylon mesh to achieve single cell suspension. Thymocytes were then suspended in RPMI 1640 supplemented with 10% FCS, stained by flow cytometry with anti-CD38 PE and anti-CD1a PE monoclonal antibodies (both from Becton, Dickinson), and plated.

Proliferation and AICD Assays PBMC were dispensed at 2 ⫻ 106 cells per milliliter in flat bottom microtiter 200 ␮l/well plates (Sarstedt) at the concentration of 1 ⫻ 105 cells per well, and 20 ␮l of OKT3 was added to each well. Cells were cultured alone or in the presence of two different MSC concentrations (1:1 and 1:4 MSC/T cells, respectively) for 4 days, pulsed with 0.5 ␮Ci of [3H]thymidine for 8 hours (5 Ci/mmole specific activity; GE Healthcare Europe GmbH, Milan, Italy, http:// www.gehealthcare.com), and then harvested on MultiScreen Harvest Plates (Millipore, Billerica, MA, http://www.millipore.com) using a cell Harvester 96 (Tomtec, Hamden, CT, http://www. tomtec.com). MultiScreen Harvest Plates were scintillated by Max-

ilight scintillation liquid (Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com) and counted in a beta counter (Chameleon 425-104 Multilabel Counter; Bioscan, Washington, http://www.bioscan.com). Results are given as mean value of triplicate well and expressed as Kcpm (cpm ⫻ 1,000). For the induction of AICD, PBMC were plated at the density of 1 ⫻ 106 cells per milliliter in complete medium with 100 U/ml IL-2 for 3 days at 5% CO2 and 37°C. Then, cells were harvested, washed once with complete medium, counted, and then stimulated with 20 ␮l of OKT3 supernatant as described above for proliferation assay. On day 4, cells were harvested, washed twice with DPBS, and then analyzed by flow cytometry as described below.

Induction of Apoptosis Jurkat leukemic T cells were treated with an anti-Fas mAb capable of triggering apoptosis (clone CH11) at the concentration of 0.5 ␮g/ml for 6 hours, at 5% CO2 and 37°C, alone or in presence of MSC at two concentrations (1:1 and 1:4 MSC/Jurkat). We also treated MSC, for 1 hour at 37°C, with clone ZB4 (1 ␮g/ml), an anti-Fas mAb that renders CD95 unavailable on the MSC surface, thus preventing apoptosis. Upon death receptor blockade, MSC were washed once with complete medium to remove the excess of blocking antibody. Next, Jurkat cells were added and treated with anti-Fas stimulating mAb as previously described. Apoptosis of Jurkat cells was assessed by flow cytometric detection of Annexin V positive cells as described below. In order to investigate the effect of MSC on stress mediated apoptosis affecting the mitochondrial pathway, we utilized etoposide (50 ␮M), a chemotherapeutic agent inhibiting selectively the topoisomerase II, and valinomycin, a potent potassium ionophore capable of perturbing the mitochondrial membrane potential and included as positive control in the MitoShift Kit (Trevigen Inc., Gaithersburg, MD, http://www.trevigen.com; see below). Detection of apoptotic cells was achieved by flow cytometry as described below.

Flow Cytometry Flow cytometric evaluation of Annexin V positive cells was performed with the rH Annexin V FITC Kit (Bender MedSystems GmbH, Vienna, Austria, http://bendermedsystems.com) according to manufacturer instructions. Briefly, upon incubation with an antiCD3 PE-conjugated mAb (Becton, Dickinson), cells were stained with Annexin V FITC, counterstained with propidium iodide, and immediately analyzed. Expression of Fas ligand and Fas receptor (CD95) was detected by flow cytometry utilizing a mouse antihuman CD95 PE mAb (Serotec) and a mouse anti-human Fas ligand FITC mAb (Bender MedSystems). Intracellular expression of Bcl-2 was tested by flow cytometry using clone 124, mouse IgG1, and FITC-conjugated mAb from DakoCytomation (Glostrup, Denmark, http://www.dakocytomation.com). Cells were fixed by using IntraStain Reagent A and then permeabilized by IntraStain Reagent B (DakoCytomation). Evaluation of total caspases by flow cytometry was carried out utilizing the Vybrant FAM Poly Caspases Assay Kit (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) according to manufacturer instruction. Granzyme B expression was evaluated by flow cytometry utilizing the anti-granzyme B FITC conjugated mAb (Becton, Dickinson) upon permeabilization with FACS Permeabilizing Solution according to the recommended procedure. The loss of mitochondrial potential was evaluated by MitoShift Kit according to manufacturer instructions. All the abovedescribed stainings were performed by gating on the CD3 positive population detected by a mouse anti-human CD3 PE-Cy5 or CD3 PE from Serotec. Flow cytometry analysis was performed by FACSCalibur and FACSCanto flow cytometers and data were analyzed with Cell Quest and Diva software, respectively (Becton, Dickinson).

Statistical Analysis Statistical analysis was performed on independent samples, namely cells cultured in the presence of MSC versus cells cultured alone (controls), using unpaired two-tailed Student’s t test, which takes into account mean values and standard deviation. Analysis for

Benvenuto, Ferrari, Gerdoni et al.

Figure 1. MSC arrest T-cell division but do not induce apoptosis. (A): Anti-CD3 stimulated peripheral blood mononuclear cells were cultured with MSC at 1:4 (gray bar) and 1:1 (white bar) ratios and in the absence (left) or presence (right) of IL-2. (B): Downregulation of intracellular Ki67 by MSC at 1:4 (gray bar) and 1:1 (white bar) ratios following anti-CD3 stimulation without (left) or with (right) IL-2. (C): Percentage of CD3⫹ Annexin⫹ cells upon anti-CD3 stimulation in the presence of MSC at 1:4 (gray bar) and 1:1 (white bar) with (right) or without (left) IL-2. Results represent the mean of five independent experiments. Abbreviation: Kcpm, cpm ⫻ 1,000.

repeated measures was performed by repeated measures analysis of variance (ANOVA), which tests the equality of means (SPSS 13.0; SPSS Inc., Chicago, http://www.spss.com).

RESULTS MSC Arrest T-Cell Division but Do Not Induce Apoptosis We investigated the effect of MSC on T cell proliferation induced upon TCR engagement. Almost complete inhibition of T-cell proliferation was achieved in the presence of MSC at the ratio of 1:1 and 1:4 (MSC/T cells) and was not reverted by the addition of IL-2 (Fig. 1A). To investigate the effect of MSC on T-cell division, we measured the expression of Ki67, a proliferation antigen linked to the active phases of the cell cycle. As shown in Figure 1B, the expression of Ki67 on CD3 positive cells was strikingly downmodulated by MSC in a dose-dependent manner (p ⬍ .05 and p ⬍ .01 at 1:4 and 1:1, respectively), suggesting that the inhibition of proliferation was due to an arrest of the cell cycle in the G0-G1 phase. Culture supplemenwww.StemCells.com

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Figure 2. MSC support survival of unstimulated T cells. (A): Unstimulated peripheral blood mononuclear cells (PBMC) from healthy donors were cultured in serum-free medium for 4 days either alone (MSCFCS⫺) or with MSC at 1:4 (MSC1:4 FCS⫺, gray bar) and 1:1 (MSC1:1 FCS⫺, white bar) ratios and analyzed by flow cytometry to evaluate the frequency of CD3⫹ Annexin⫹ cells compared with PBMC cultured in complete medium (MSC-FCS⫹, light gray bar). Results are expressed as mean of five independent experiments. (B): Unstimulated PBMC were cultured according to the same experimental condition as above for 8 days, and the frequency of CD3⫹ Annexin⫹ cells was assessed at four different time points. Results represent the mean of three independent experiments. Abbreviations: ANOVA, analysis of variance; FCS, fetal calf serum.

tation with IL-2 did not significantly revert the MSC-induced cell division arrest, as demonstrated by the marginal increase of Ki67⫹ cells. Then, we investigated whether TCR-triggered nondividing T cells undergo apoptosis. As presented in Figure 1C, the percentage of Annexin V positive T cells upon TCR stimulation in the presence or absence of MSC was similar and was not affected by exposure to IL-2. These findings confirm that MSC do not increase the number of T cells undergoing physiological cell death upon TCR triggering.

MSC Support Survival of Unstimulated T Cells Next, we asked whether MSC could support the survival of T cells cultured in vitro under starving conditions. To this end, we cultured PBMC in serum-free conditions for 8 days with or without MSC and determined the proportions of CD3⫹, Annexin V⫹ apoptotic cells by flow cytometry. At day 4, the percentages of the latter cells in cultures with no FCS but containing MSC at both the 1:1 and the 1:4 ratios were (a) significantly lower than in control cultures without MSC (p ⬍ .05 for both MSC/T-cell ratios) and (b) similar to those detected in PBMC incubated for the same time with 10% FCS (Fig. 2A). The protective effect of MSC was prolonged over time as shown

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Figure 3. MSC rescue human thymocytes from spontaneous apoptosis. Human thymocytes were cultured with MSC at 1:4 (gray bar) and 1:1 (white bar) ratios for 2 days. Percentage of Annexin V⫹ cells was assessed on CD1a⫹ CD38⫹ cells. Results are expressed as mean of five independent experiments.

by a significant time per treatment interaction following analysis with repeated measures ANOVA (p ⬍ .01 for time effect and p ⬍ .05 for treatment effect) (Fig. 2B).

MSC Rescue Human Thymocytes from Spontaneous Apoptosis In order to verify whether MSC support the survival of proliferating cells prone to spontaneous apoptosis, thymocytes were isolated from the thymic stroma and cultured. The frequency of Annexin V positive cells was investigated after 48-hour culture with or without MSC by gating on CD1a⫹, CD38⫹ thymocytes. Following their removal from the thymic stroma, these cells proliferated vigorously irrespective of the lack of TCR engagement, as assessed by the high expression of Ki67 (data not shown). The number of apoptotic thymocytes decreased upon coculture with MSC, and this was statistically significant at the 1:1 ratio (Fig. 3). These findings indicate that MSC can protect from death not only resting T cells, but also dividing thymocytes cultured in the absence of trophic factors.

MSC Inhibit AICD by Downregulating the Death Receptor Pathway Next, we investigated whether the antiapoptotic effect of MSC could counteract activation induced cell death. In the following experiments, T cells were exposed to IL-2 for 3 days before TCR triggering with OKT3. Under these experimental conditions, the proportion of CD3⫹ Annexin V⫹ T cells was much higher than that observed in a standard proliferation assay lacking IL-2 pretreatment (mean value ⫽ 29.76 vs. 61.69, respectively; p ⬍ .01). When AICD was induced in the presence of MSC, we observed a dose-dependent decrease of CD3⫹ Annexin V⫹ cells compared with controls (Fig. 4A). Programmed cell death of activated cells is triggered mainly through the Fas receptor, and both Fas and its ligand (FasL) are normally induced on T cells upon activation [17]. Therefore, we analyzed the expression of both Fas and FasL on activated CD3⫹ T cells in the presence or absence of MSC. Indeed, MSC coculture strikingly decreased the expression of both Fas and FasL on T cells undergoing AICD, as shown in Figure 4B and 4C, respectively. These findings suggest that MSC mediated rescue of T cells from AICD involves downregulation of Fas receptor and Fas ligand on the T-cell surface.

Figure 4. MSC inhibit activation induced cell death (AICD) by downregulating the death receptor pathway. AICD was induced in the presence of MSC at 1:4 (gray bar) and 1:1 (white bar) ratios, and the percentage of Annexin V⫹ CD3⫹ cells was measured (A). Under the same experimental condition, we measured the expression of Fas receptor (CD95) (B) and Fas ligand (C) on CD3⫹ cells. Results are the mean of five independent experiments.

MSC Affect the Activity of Proteases Involved in Cell Death Fas engagement triggers a proteolytic cascade whereby activated caspases cleave and activate in succession downstream caspases, leading finally to apoptosis. Thus, we studied intracellular total active caspases by flow cytometry. As shown in Figure 5A and 5B, we detected a decrease of active total caspases in CD3⫹ cells cultured in the presence of MSC compared with controls. Granzyme B can induce cell death through caspase-dependent and -independent mechanisms involving also the inactivation of pro-cell growth factors [18]. Thus, we investigated whether MSC could affect the intracytoplasmic release of granzyme B by activated CD8⫹ T cells. Upon TCR engagement in the presence of MSC, we observed a striking decrease of intracellular granzyme B compared with CD8⫹ T cells alone (Fig. 5 C) (p ⬍ .01 at both MSC/T-cell ratios). These results suggest that MSC can protect T cells from cell death induced via TCR triggering through the inhibition of pro-apoptotic proteases.

Benvenuto, Ferrari, Gerdoni et al.

Figure 5. MSC affect the activity of proteases involved in cell death. (A): Flow cytometric experiment depicting the decrease of total active caspases on CD3⫹ cells following activation induced cell death (AICD) induction in the presence of MSC. (B): Expression of total caspases on CD3⫹ cells in the presence of MSC at 1:4 (gray bar) and 1:1 (white bar) ratios. Similarly, the expression of granzyme B on CD3⫹ CD8⫹ cells undergoing AICD in the presence of MSC at two concentrations (1:4 ratio, gray bar; 1:1 ratio, white bar) is depicted (C). Results are expressed as mean of five independent experiments. Abbreviations: FAM, carboxyl-fluorescein; FITC, fluorescein isothiocyanate; FMK, floromethyl-ketone; PE, phycoerythrin; VAD, Val-Ala-Asp.

MSC Inhibit Anti-Fas Induced Apoptosis of Jurkat Cells In order to confirm the protective role of MSC on Fas induced T-cell apoptosis, we utilized Jurkat leukemic T cells, constitutively expressing CD95 on their surfaces, to provoke cell death upon triggering with the anti-Fas mAb CH11. As expected, MSC markedly inhibited proliferation of Jurkat cells inducing cell division arrest (not shown). Interestingly, MSC at both ratios significantly decreased the proportion of Annexin V positive Jurkat cells following incubation with the anti-Fas CH11 mAb compared with controls (Fig. 6A, left histogram). As human MSC express variable levels (mean value ⫽ 53, SD ⫽ 32) of CD95 on their surfaces (data not shown), we investigated whether MSC underwent apoptosis upon Fas ligation with the CH11 mAb. Up to 45% of MSC treated with the anti-Fas antibody were annexin V positive (mean ⫽ 39.16%, SD ⫽ 10.57) compared with only 16.05% (mean; SD ⫽ 1.76) of MSC treated with an isotype-matched irrelevant antibody (p ⬍ .01). These findings demonstrate that CD95 expressing MSC are susceptible to Fas induced apoptosis. www.StemCells.com

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Figure 6. MSC inhibit anti-Fas induced apoptosis of Jurkat cells. (A): The percentage of Annexin V⫹ CD3⫹ Jurkat leukemic T cells treated with CH11 (anti-Fas monoclonal antibody [mAb]) to induce apoptosis in absence or presence of MSC at two concentrations (1:4, gray bar and 1:1, ruled bar, respectively) (left histograms) and upon pretreatment of MSC with the blocking anti-Fas mAb ZB4 to make CD95 unavailable for induction of apoptosis of MSC (right histograms). Results are expressed as mean of three independent experiments. (B): A representative experiment is shown. Dot plot A: anti-Fas treated Jurkat cells; dot plot B: ⫹MSC 1:1; dot plot C: ⫹MSC 1:1 pretreated with blocking anti-Fas mAb (ZB4); dot plot D: ⫹MSC 1:4; dot plot E: ⫹MSC 1:4 pretreated with blocking anti-Fas mAb ZB4. Annexin V⫹ cells are displayed on the upper right and lower right quadrants of each dot plot, whereas viable cells are displayed on lower left quadrants. Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.

Thus, we pretreated MSC with the blocking anti-Fas mAb ZB4 in order to make the CD95 expressed by MSC unavailable for ligation with the CH11 antibody utilized to induce apoptosis of Jurkat cells. Under these circumstances, the capacity of MSC, at both 1:1 and 1:4 ratios, to rescue Jurkat cells from apoptosis was similar (p ⬍ .01 for both MSC/Jurkat cell ratios), demonstrating a remarkable inhibitory effect of MSC on apoptosis induced by triggering of Fas receptor on target cells (Fig. 6A, right histogram).

MSC Does Not Act Through Bcl-2 The cytoplasmic membrane-associated protein Bcl-2 is a potent inhibitor of apoptosis induced under various conditions of cell stress, but it is known to provide little protection against cell death induced through the Fas pathway [19]. Thus, we analyzed the intracellular expression of Bcl-2 in PBMC undergoing AICD, as previously described, in the presence or absence of MSC. Upon TCR engagement, the percentage of T cells expressing Bcl-2 activated in the presence of MSC at 1:1 and 1:4 ratios was 97.44% (SD ⫽ 3.32) and 98.05% (SD ⫽ 3.69),

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Figure 7. MSC do not affect chemical induced apoptosis. Jurkat cells were treated with etoposide (50 ␮M) and valinomycin (500 nm) as described in Material and Methods in the absence or in the presence of MSC at a 1:1 concentration (white bar). The percentage of Annexin V⫹ CD3⫹ cells (histograms [A] and [C]) as well as mitochondrial membrane depolarization (histograms [B] and [D]) were evaluated on CD3⫹ cells by flow cytometry at the end of chemical treatments. Histograms (A) and (B) show the results when Jurkat cells were exposed to chemicals in the presence of MSC at a 1:1 ratio. Histograms (C) and (D) show the results of experiments when Jurkat cells were exposed to MSC (1:1) for 24 hours before adding toxic compounds. Results are expressed as the mean of five independent experiments. Abbreviation: MM, mitochondrial membrane.

respectively, compared with 94.8% (SD ⫽ 10.20) in the control cells. These results suggest that the MSC effect on T cells is not due to the upregulation of the antiapoptotic molecule Bcl-2.

MSC Do Not Affect Chemical Induced Apoptosis As Bcl-2 protects mainly from cytotoxic insults, we asked whether MSC could protect T cells from chemically induced apoptosis, a process affecting mitochondria integrity. Changes in mitochondrial membrane integrity involve both the inner and the outer mitochondrial membranes and lead to disruption of the inner transmembrane potential (⌬⌿m). Thus, we treated Jurkat cells with either etoposide (50 ␮M) or valinomycin (500 nM), two chemotherapeutic drugs inducing apoptosis through the breakdown of mitochondrial membrane potential. Following 20-hour treatment with etoposide, 78.95% (mean) of Jurkat cells were Annexin V positive compared with 68.27% when the treatment was carried out in the presence of MSC (p ⬎ .05). Similarly, 73.27% (mean) of Jurkat cells showed depolarization of the mitochondrial membrane potential compared with 66.42% in the presence of MSC, which was not significant (Fig. 7A). Upon 4 hours treatment of Jurkat cells with valinomycin, we observed a decrease of Annexin V positive Jurkat cells when cells were exposed to MSC compared with controls (36.21% vs. 26.96%, respectively) (Fig. 7B). Likewise, only a minimal difference was observed in the percentage of depolarization of the mitochondrial membrane when Jurkat cells tested alone were compared with those cocultured with MSC (71.02% vs. 68.07%, respectively) (Fig. 7B). These findings suggested that, in the presence of MSC, there is a moderate but not statistically significant decrease of T-cell apoptosis induced by etoposide and valinomycin. Then we hypothesized that the marginal protective effect of MSC detected in these experiments depended on a direct cytotoxic on MSC effect of the chemicals utilized. Indeed, when we exposed MSC to etoposide treatment, the number of Annexin V positive cells was 35.1% (mean; SD ⫽ 7.31) and was 23.05% (SD ⫽ 4.67) when MSC were treated with valinomycin; 20.95%

(SD ⫽ 3.22) of untreated MSC resulted Annexin V positive. When we looked at ⌬⌿m, mitochondrial membrane depolarization of MSC exposed to valinomycin was 29.3% (SD ⫽ 5.47), as compared with 12.35% (SD ⫽ 3.29) of etoposide treated and 4.01% (SD ⫽ 1.98) of untreated MSC. Based on these results, we decided to culture Jurkat cells with MSC (at a 1:1 ratio) for 24 hours before adding the toxic drug at the same concentration as that one utilized before in order to verify whether pretreatment with MSCs in the absence of the chemicals could enhance survival of Jurkat cells. Again, the presence of MSC, before the treatment with the toxic drug, did not significantly decrease the frequency of Annexin positive Jurkat cells, either upon etoposide or valinomycin treatment, compared with controls (Fig. 7C). Similar results were obtained as to the assessment of mitochondrial membrane potential, which was modestly affected by pretreatment of Jurkat cells with MSC (at a 1:1 ratio) (Fig. 7D). Accordingly, Jurkat cells treated with valinomycin did not significantly increase Bcl-2 expression in the presence of MSC compared with controls (data not shown). These findings suggest that MSC have a moderate but not significant antiapoptotic effect when cell death is induced by chemical stress, a process regulated by the Bcl-2 protein family, which is involved in maintaining the integrity of the mitochondrial membrane.

DISCUSSION Mesenchymal multipotent progenitor cells, more commonly defined as mesenchymal stem cells, are part of the stromal nonhematopoietic component of the bone marrow tightly connected with osteoblasts and endothelial cells. Thus, MSC contribute to the formation of the “hematopoietic stem cell (HSC) niche,” the unique microenvironment supporting survival of HSC and controlling proliferation, maturation, and differentiation of blood born cells [20 –22]. As a consequence, MSC have been proposed as tool to hasten marrow engraftment after allogeneic transplantation and help immune reconstitution [23, 24].

Benvenuto, Ferrari, Gerdoni et al. MSC are physiologically committed to differentiate into tissues of the mesenchymal lineage but, under some circumstances, they have been shown to transdifferentiate into almost all tissues [4]. More recently, MSC have been demonstrated to have a number of effects on immune cells [5]. In particular, MSC have been reported to inhibit proliferation of T cells through a major histocompatibility complex independent mechanism [25], leading to cell division arrest [6]. Although the mechanisms involved in such inhibition of T-cell proliferation are still poorly understood, a veto-like activity has been reported [26], and a role for soluble molecules including prostaglandin E2 [27], transforming growth factor-␤ [28], and indoleamine 2,3-dioxygenase [29] has been proposed. In this manuscript, we provide, for the first time, compelling evidence that MSC can support cell survival when T cells are subjected to stress conditions leading to cell apoptosis. First, we observed that T cells cultured under starving conditions and thymocytes, a cell population physiologically prone to spontaneous cell death [30], are significantly rescued from apoptosis by the presence of MSC. More importantly, we demonstrate that MSC play a dual role on activated T cells. Although, on one side, MSC can hamper T-cell proliferation through the inhibition of cell division and subsequent accumulation of cells in the G0 phase of the cell cycle as previously reported [6], on the other hand, they can prevent cell death when T cells are overstimulated through TCR engagement to achieve AICD. It is of note that the proliferation assay and the AICD assay utilized in this study are carried out via identical TCR triggering by antiCD3 and differ exclusively for T-cell pretreatment with IL-2 in the AICD assay, leading to Fas upregulation and subsequent induction of apoptosis. The protective effect of MSC targets mainly the “death receptor” pathway of apoptosis, as suggested by the downregulation of Fas receptor and Fas ligand on TCR activated T cells. The role of the Fas/FasL pathway was confirmed by the inhibition of apoptosis of Jurkat cells upon Fas triggering in the presence of MSC. Accordingly, induction of apoptosis in the presence of MSC resulted in a decreased activation of total caspases involved in cell death and in the inhibition of the intracellular release of granzyme B, a serine protease that initiate apoptosis by cleaving and activating caspases and inactivating cellular factors involved in cell survival [18]. We also showed for the first time that MSC, variably expressing Fas on their surfaces, undergo Fas mediated apoptosis. These findings add to others suggesting that MSC can become, under certain circumstances, targets of immune responses [31– 33], and therefore their putative immune-privilege should be considered cautiously. Finally, we demonstrated that the antiapoptotic effect of MSC on T cells is virtually independent of Bcl-2, a cytoplasmic molecule highly involved in the mitochondrial pathway of apoptosis [34], which is known to be distinct from the Fas receptor pathway [19, 35]. Accordingly, preservation of the integrity of mitochondrial membrane potential upon chemical stress, such as that induced by etoposide and valinomycin, is marginally influenced by MSC. Interestingly, Spees and colleagues recently reported on the capability of MSCs to rescue aerobic respiration through mitochondrial transfer [36]. We propose that chemical opening of the mitochondrial permeability transition pore and

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subsequent collapse of the inner transmembrane potential, a central irreversible checkpoint of the apoptotic program [37], cannot be reverted by a limited exposure to MSCs. These findings are of remarkable relevance, as they support a model of interaction between MSC, key cells involved in the HSC niche, and T cells and, most likely, any blood born cells, including HSC. Based on this model, MSC are capable, by means of yet poorly defined mechanisms involving “cell to cell” contact and soluble molecules, to keep T cells quiescent in the G0 phase of the cell cycle while supporting their survival. Similarly, MSC can arrest division of other immunocompetent cells including B cells [7], dendritic cells [8], and leukemic cells [16]. Interestingly, an MSC-mediated protective effect has been reported also on apoptotic neurons [15, 38] and tumor cells [16]. Moreover, MSC have been suggested to play an in vivo protective role from tissue damage and cell death in EAE [11], in a model of acute renal failure [13], experimental diabetes [14], and lung fibrosis [39]. Interestingly, we detected, within a set of genes highly expressed by immunomodulatory MSCs from C57B6/J mice, several molecules involved in the hematopoietic stem cells niche [40]. Some of these genes, including, for example, thrombospondin (Thbs)-1 and Thbs-2 [41], osteopontin [42], angiopoietin-1 [43], and galectin-1 [44], have been reported to have an antiapoptotic and antiproliferative effect and support HSC survival and maintenance in a quiescent state inside the niche.

CONCLUSION These results suggest that MSC are endowed with the intrinsic capacity of promoting survival of T cells in a resting state, possibly through the recapitulation of their physiological activity on HSC within the niche.

ACKNOWLEDGMENTS This study was supported by Grants from the Italian Foundation for Multiple Sclerosis (2004/R/20, A.U.), the Fondazione Cariplo (2004.1372/10.4898, A.U.) and the Fondazione CARIGE (2003.0904-1, G.L.M. and A.U.), the Ministry of Health (Ricerca Finalizzata Ministeriale 2005-57, A.U.), and the Ministry of Research and University (PRIN 2005-2005063024_004, G.L.M. and A.U.). Authors are grateful to Lucio Zanini, who kindly supplied human thymuses, and Maria Pia Sormani and Irene Malaspina for help in statistical analysis, artwork, and editing of the manuscript. Authors declare that they do not have competing financial interest and that this work has never been published, completely or in part, elsewhere and is not under submission to another journal.

DISCLOSURE

Friedenstein AJ, Petrakova KV, Kurolesova AI et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968;6:230 –247.

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CONFLICTS

The authors indicate no potential conflicts of interest.

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