Role for Interferon- in the Immunomodulatory Activity of Human Bone Marrow Mesen - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Mauro Kramperaa, Lorenzo Cosmib, Roberta Angelib, Annalisa Pasinia, Francesco Liottab, Angelo Andreinia, Veronica Santarlascib, Benedetta Mazzinghib, Giovanni Pizzoloa, Fabrizio Vinantea, Paola Romagnanib, Enrico Maggib, Sergio Romagnanib, Francesco Annunziatob作者单位：a Department of Clinical and Experimental Medicine, Section of Haematology, University of Verona, Verona;b Excellence Center of University of Florence, DENOthe, Florence, Italy % j6 K8 m# ~) i  o
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      【摘要】
   Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA-unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4  and CD8  T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell-activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact-dependent and required the presence of interferon (IFN)- produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN-. The suppressive effect of IFN- was related to its ability to stimulate the production by MSCs of indoleamine 2,3-dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft-versus-host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell¨C derived IFN-.
      【关键词】 Mesenchymal stem cells Immune suppression Indoleamine -dioxygenase: A& S8 S0 c* I
   INTRODUCTION% t5 ^0 Z2 j1 u) w: j7 E
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Bone marrow (BM) mesenchymal stem cells (MSCs) are multipotent nonhematopoietic progenitor cells that can differentiate into BM stromal cells, osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, neurons, and cells of visceral mesoderm .

In the last few years, it has become clear that MSCs also possess immunoregulatory properties. The BM microenvironment has been found to provide appropriate support for T-cell development in the absence of thymus, because in this condition most T cells adhering to BM stroma exhibit an immature phenotype . However, the mechanisms involved in the immunoregulatory activity of MSCs on T lymphocytes are still partially obscure.6 Y3 ]' P6 r. O' J2 R. T. s8 t

In this study, we demonstrate that human BM-derived MSCs are able to inhibit the proliferation not only of CD4  and CD8  T lymphocytes but also of NK cells, whereas they have no effect on the proliferation of B lymphocytes. The inhibitory effect of MSCs on the proliferation of T lymphocytes was neither related to the lack of their activation nor to the direct induction of apoptosis. The suppressive activity of MSCs was completely abrogated by the addition of an anti¨Cinterferon- receptor (IFN-R) monoclonal antibody (mAb). Taken together, these findings indicate that IFN- produced by T lymphocytes or NK cells may promote the immunomodulatory activity of MSCs, which in turn suppress T- or NK-cell proliferation. Accordingly, in the presence of exogenous IFN-, B lymphocytes also became susceptible to the inhibitory activity of MSCs. The ability of IFN- to induce the suppressive effect of MSCs on cell proliferation appeared to be, at least in part, related to the enhancement of the indoleamine 2,3-dioxygenase (IDO) activity. These findings provide an explanation of why coinfusion of MSCs may be beneficial in the treatment of GvHD, a disorder that is primarily mediated by IFN-¨Cproducing type 1 T helper (Th1) cells .9 y/ l' \/ v! H, V( h+ {7 d
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MATERIALS AND METHODS; J8 y- V2 \4 H5 v9 A/ @- c# _% }
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Generation of MSCs

MSCs were generated from BM aspirates of healthy donors, recruited after informed consent. BM cells were obtained with density-gradient centrifugation (Lymphoprep, Nycomed Pharm, Oslo, Norway) and cultured in 25-cm2 flasks (BD Falcon, Becton Dickinson, Milan, Italy) at a concentration of 30 x 106 nucleated cells in 5 ml of Dulbecco¡¯s modified Eagle¡¯s medium, with high glucose concentration, GLUTAMAX I, 15% heat-inactivated adult bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Gibco, Milan, Italy, http://www.invitrogen.com). Cultures were incubated at 37¡ãC in a 5% CO2 atmosphere. After 72 hours, nonadherent cells were removed. When 70%¨C80% adherent cells were confluent, they were trypsinized (0.05% trypsin at 37¡ãC for 5 minutes, Gibco), harvested, and expanded in larger flasks. A homogenous cell population is normally obtained after 2 to 3 weeks of culture.; g* ?; P6 N/ y3 k9 ]7 `: P

Reagents' X6 n# P+ d* o4 Y. ^

Anti-CD105 (endoglin), -CD73, -CD106 (VCAM-1), -CD44, -CD90, -CD29, -CD45, -CD14, -CD34, -CD80, -CD86, -IFN--R (CD119), -CD8, -CD4, -CD25, -CD69, -CD152, -HLA (class I and II), -perforin, and -granzyme A mAbs, as well as Annexin V and BrdU detection kits, were purchased from BD Biosciences (San Diego, CA, http://www.bdbiosciences.com). Recombinant human IFN-, interleukin (IL)-4, IL-7, and IL-15, as well as anti¨Ctransforming growth factor (TGF)-ß1 mAb, were purchased from R&D System (Minneapolis, http://www.rndsystems.com). Human rIL-2 was a kind gift from Eurocetus (Milan, Italy).- ?9 s0 B8 J9 _$ ]

Flow Cytometric Analysis

Flow cytometric analysis was performed as detailed elsewhere . Briefly, 105 cells were incubated with the specific or the isotype control mAb at  4¡ãC for 30 minutes; cells were then washed and analyzed on a BDLSRII cytofluorimeter using the Diva software (BD Biosciences). A total of 104 events for each sample were acquired.
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Intracellular synthesis of IL-4 and IFN- at single-cell level was performed on polyclonally stimulated T lymphocytes, as described . To assess the expression of perforin and granzyme A, cells were fixed, washed, permeabilized, and then incubated for 15 minutes at room temperature with the specific or the isotype control mAb. Cells were then washed and analyzed on a BDLSRII cytofluorimeter.

MSC Differentiation Assay: [: G7 v5 t4 B9 i, H- E
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Cell stemness was assessed by testing the ability of MSCs to differentiate into adipocytes, osteoblasts, and chondrocytes, as previously described . Oil red O, von Kossa, and toluidine blue dyes were used to identify adipocytes, osteoblasts, and chondrocytes, respectively. More than 90% of the cells differentiated depending on the time left in culture with the differentiating agent.
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Isolation from Peripheral Blood of CD4 , CD8 , CD4 CRTH2 , CD4 CD25¨C T Cells, NK Cells, and B Cells

Negative selection from peripheral blood (PB) of CD4 , CD8  T cells, and NK cells was performed by high-gradient magnetic-activated cell sorting, as described elsewhere .+ x! [" G$ Q5 |

Proliferation Assays& D2 o6 F0 c2 E+ m, d, K. C

A total of 105 purified CD4  or CD8  human T cells were stimulated with 105 irradiated (9,000 rad) allogeneic T-cell¨Cdepleted PBMCs and 1 µg/ml of anti-CD3 mAb (clone HIT3a) (BD Biosciences) in the absence or presence of different numbers of MSCs (104, 103, 102, 10/well). On day 5, cultures were pulsed for 8 hours with 0.5 µCi (0.0185 MBq) of 3H-TdR (Amersham, Little Chalfont, UK, http://www.amersham.com) and harvested, and radionuclide uptake was measured by scintillation counting. In some experiments, neutralizing anti¨CIFN-R (5 µg/ml), neutralizing anti-CD152 (10 µg/ml), neutralizing anti¨CTGF-ß1 (10 µg/ml), or the same amount of isotype-matched mAbs were added to the cultures. In another series of experiments, human rIL-2 (10 IU/ml), rIL-7 (1 ng/ml), rIL-4 (2 ng/ml), or rIL-15 (7 ng/ml) was added to the cultures. In another set of experiments, two IDO inhibitors, 1-methyl tryptophan (250¨C1,000 µM; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and Norharmane (125¨C500 µM; Sigma-Aldrich) were added to the cultures.' H2 Q4 Q: q+ k1 @7 `! J
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A total of 105 human NK cells were stimulated with 105 irradiated (9,000 rad) allogeneic T cell¨Cdepleted PBMCs and IL-2 (100 U/ml) in the absence or presence of different numbers of MSCs (104, 103,102, 10/well). On day 5, after 8 hours of 0.5 µCi 3H-TdR pulsing, cultures were harvested and radionuclide uptake was measured by scintillation counting. In some experiments, the proliferation assay was performed using different numbers of NK cells (50 x 103, 25 x 103, 12.5 x 103, 6.25 x 103/well). In other experiments, an anti-CD119 (5 µg/ml) or the same amount of an isotype-matched mAb was added to the cultures.
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A total of 105 human B cells were stimulated with 105 irradiated (9,000 rad) allogeneic T cell¨Cdepleted PBMCs and 2 µg/ml of the CpG-containing DSP30F oligodeoxynucleotide (ODN) (MWG Biotech, Ebersberg, Germany, http://www.mwg-biotech.com) in the absence or presence of different concentrations of MSCs (104, 103, 102, 10/well). On day 5, after 8-hour pulse with 0.5 µCi 3H-TdR, cultures were harvested and radio-nuclide uptake was measured by scintillation counting. In some experiment, 2 ng/ml of human rIFN- was added at the beginning of cultures.% M  h8 Y+ `9 U# L
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CarboxyFluorescein Diacetate, Succinimidyl Ester (CFSE) Labeling and BrdU Uptake

Labeling of lymphocytes with CFSE was performed as described previously . Briefly, cells were extensively washed and resuspended at a final concentration of 107 cells/ml in phosphate-buffered saline. CFSE was added at a final concentration of 5 µM and incubated for 4 minutes at room temperature. The reaction was stopped by cell washing with 10% heat-inactivated fetal calf serum¨C containing RPMI 1640. Cells then received the allogeneic stimulus in the presence or absence of MSCs for 5 days. BrdU was added to the medium during the last 6 hours of culture at a final concentration of 10 µM. Cells were then harvested and treated according to manufacturer¡¯s instructions (BrdU Flow Kits; BD Biosciences). Cells were then analyzed on a BDLSR cytofluorimeter (BD Biosciences) using both the FACSDiva and the ModFit LT3.0 softwares. Cell division was characterized by sequential halving of CFSE fluorescence, generating equally spaced peaks on a logarithmic scale. Seven individual peaks and corresponding regions were identified (G0 ¨CG6). The symbols related to each peak (G0 ¨CG6) were indicated, the undivided T cells (G0) residing in the rightmost peak and T cells that have divided six times (G6) residing in the leftmost peak.
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Transwell Experiments6 \- x; p0 B, J# Y' C
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Transwell experiments were performed in 24-well transwell plates (0.22-µm pore size, Costar, Corning, NY). A total of 5 x 105 CFSE-labeled CD4  T lymphocytes were stimulated with 5 x 105 irradiated T cell¨Cdepleted allogeneic PBMCs and 1 µg/ml of anti-CD3 mAb in the absence or presence of 5 x 104 MSCs, placed in the same or in another chamber. On day 4, after 6 hours of pulsing with BrdU, cells were harvested and evaluated by flow cytometry for CFSE and BrdU contents. In additional experiments, an anti-CD119 or an isotype-matched mAb (5 µg/ml) was added to the cultures., p: j3 f8 z2 T

Evaluation of Apoptosis and NK Cytotoxicity* E& o, v1 L1 q) I( ~* K7 n) H
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The evaluation of apoptosis in CD4  and CD8  T cell populations was performed using the annexin V kit (BD Biosciences) following the manufacturer¡¯s instructions. Cells were then analyzed on a BDLSRII cytofluorimeter (BD Biosciences) using the FACSDiva software.* D7 E/ Y2 h* J! F9 L6 s

To evaluate the effects of MSCs on the cytotoxic activity of NK cells, these cells were purified and cultured in the absence or presence of MSCs (ratio, 10:1) for 5 days. On day 5, NK cells were collected and tested for their ability to kill K562 target cells by using flow cytometry. Briefly, K562 target cells were cultured for 5 hours in the absence or presence of different numbers of untreated or MSC-precultured NK cells (ratio, 1:1, 1:2, and 1:4). Cells were then collected, stained with anti-CD56 mAb and annexin V, as detailed elsewhere .7 [- q1 O. A/ p

Real-Time Quantitative Reverse Transcription¨CPolymerase Chain Reaction (TaqMan)
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Foxp3 quantitation was performed using Assay on Demand (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) as described elsewhere ./ b7 b5 P8 ]! k$ C9 j6 v

Quantitation of IFN- Concentration in Supernatants* o, B5 Z! R" y3 [* |8 Y4 }( `% T2 h, ~

For the quantitation of IFN- in cell supernatants, a commercially available ELISA kit was used (R&D System), according to the manufacturer¡¯s instructions.& Q6 o3 B& g8 b1 u8 H" u! W
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Statistics( S! P8 }, ^  t# ?  a5 ?/ N' Y3 g4 G

Statistical comparison of the proliferation assay arms was carried out according to the Student¡¯s t-test. Differences were considered statistically significant with p
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RESULTS
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MSCs Inhibit the Proliferative Response of T and NK but Not B Cells" p: Q6 ~- w7 f9 ]2 a8 s& S

Human MSCs were obtained from BM aspirates of healthy volunteers, appropriately cultured, and purified to homogeneity. Figure 1 shows the immunophenotype and the differentiative potential of MSCs used in our experimental system. Constitutive expression of CD105, CD73, CD29, CD44, CD90, CD106, HLA class I, and CD119, but not of CD80, CD86, CD45, and HLA-DR, was observed (Fig. 1A). The same cells exhibited multilineage differentiation potential, as assessed by culturing in adipogenic, osteogenic, or chondrogenic medium (Fig. 1B).& G& ^# F- l+ `' Y. a5 o

Figure 1. Immunophenotype and differentiative potential of human mesenchymal stem cells (MSCs). (A): Proliferating MSCs were analyzed by flow cytometry for the expression of CD105 (endoglin), CD73, CD29, CD44, CD90, CD106 (vascular cellular adhesion molecule-1), CD80, CD86, CD45, HLA-class I and II (HLA-DR), and CD119 (interferon-R). (B): Multilineage differentiation potential was assessed by culturing MSCs for 2 weeks with adipogenic (AM), osteogenic (OM), and chondrogenic medium (CM) and then staining the cells with Oil red O, von Kossa, and toluidine blue dyes, respectively.
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The activity of increasing numbers of MSCs on the proliferative response to allogeneic stimulation of total T lymphocytes purified from PB of healthy donors was then evaluated. As shown in Figure 2A, the addition of MSCs in cultures of activated T lymphocytes significantly inhibited their proliferation only at 1:10 ratio (104 MSCs vs. 105 T lymphocytes), whereas at lower ratios it was ineffective. To evaluate the kinetics of T cell proliferation in the absence or presence of MSCs, we performed parallel experiments in which the proliferation tracer CFDA-SE and the uptake of the thymidine analogue BrdU at different time points of cell cultures were contemporaneously evaluated. As shown in Figures 2B and 2C, during the first 3 days of culture, T-cell proliferation was not affected by the presence of MSCs. The suppressive effects of MSCs became significant on day 4 and even more on day 5 (Figs. 2B, 2C). As shown in Figure 2D, after 5 days of culture in the presence of MSCs, there was a significant accumulation of T cells in generation (G) 1, G2, and G3, with a significant decrease in G4, G5, and G6.( R! |" K/ G3 K; Q. P7 L
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Figure 2. Inhibitory effect of mesenchymal stem cells (MSCs) on T-cell proliferation induced by allogeneic stimulation. (A): Proliferation of purified T lymphocytes induced by allogeneic stimulation in the absence or presence of different numbers of MSCs was evaluated on day 5 by 3H-TdR uptake. Results are expressed as mean values (¡À standard deviation ) of cpm obtained in 10 separate experiments. (B): Proliferation of purified T lymphocytes induced by allogeneic stimulation in the absence (black columns) or presence (white columns) of 104 MSCs was evaluated at different time points as percentage of BrdU  cells. Columns represent mean values (¡ÀSD) of the percentages of BrdU  cells obtained in six separate experiments at different times of culture. (C): Histograms represent the CFSE cell content in the indicated culture condition, as evaluated at different times by flow cytometry. One representative of six separate experiments is shown. (D): T cell generations (G0-G6) induced by allogeneic stimulation in the absence (black columns) or presence (white columns) of 104 MSCs were evaluated on day 5 by CFDA-SE contents, as described in Materials and Methods. Columns represent mean values (¡ÀSD) of cell percentages in the indicated generation obtained in nine separate experiments. *p

To establish whether MSCs can exert their inhibitory effect on both CD4  and CD8  T lymphocytes, the proliferation in response to allogeneic stimulation of CD4  or CD8  T cells, purified from PB of the same donors and cultured in absence or presence of increasing numbers of MSCs, was assessed. Again, the proliferation of both CD4  and CD8  T lymphocytes was inhibited only when 104 MSCs were added to 105 CD4  or CD8  T cells. Of note, CD8  T cells seemed to be more susceptible than CD4  lymphocytes to the MSC suppression (p
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Figure 3. Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4  and CD8  T cells, as well as both proliferation and cytolytic activity of natural killer (NK) cells. CD4  (A) and CD8  (B) T cell generations (G0¨CG6) induced by allogeneic stimulation in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) were evaluated on day 5 by CFDA-SE contents, as described in Materials and Methods. Columns represent mean values (¡À standard deviation ) of cell percentages in the indicated generation from six separate experiments. (C): NK cells were cultured with allogeneic T cell-depleted peripheral blood mononuclear cells (PBMCs) and rIL-2 in the absence or presence of different proportions of MSCs, as described in Material and Methods. Results are expressed as mean values (¡ÀSD) of cpm obtained in five separate experiments. (D): Supernatants of NK cells cultured with allogeneic T-cell¨Cdepleted PBMCs and rIL-2 in the absence (black column) or presence (white column) of MSCs (ratio, 10:1) were collected on day 5 and assessed for interferon (IFN)- content by an appropriate ELISA. Gray columns represent the level of IFN- produced by MSCs cultured with allogeneic T cell¨Cdepleted PBMCs. Results are expressed as mean values (¡ÀSD) of ng/ml of IFN- found in six separate experiments. (E): Cytolytic activity of NK cells evaluated by annexin V staining of target cells (K562) at different NK/K562 ratios. Columns represent percentages (¡ÀSD) of annexin V¨Cstained K562 cells after culturing in the absence (grey) or presence of MSC-untreated NK cells (black) or in the presence of MSC-pretreated NK cells (white). Data from six separate experiments are reported. (F): B lymphocytes were cultured in the presence of allogeneic cells and the CpG-containing ODN DSP30F in the absence or presence of different proportions of MSCs, as described in Material and Methods. Results are expressed as mean values (¡ÀSD) of cpm obtained in five separate experiments. *p ' i: |& k; ]1 Q( J
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We then asked whether MSCs were also able to suppress the proliferation and the functional activities of NK and B cells. To this end, purified CD56  cells (purity > 98%) were stimulated with allogeneic cells in the presence of rIL-2. As shown in Figure 3C, MSCs significantly suppressed the NK cell proliferation. In addition, measurement of IFN- levels in culture supernatants revealed a significant decrease of this cytokine when NK cells were cultured in the presence of MSCs (Fig. 3D). More importantly, the ability of NK cells to lyse K562 target cells seemed to be significantly impaired, at least at the 4:1 (NK:K562) cell ratio, when NK cells were precultured for 5 days in the presence of MSCs (Fig. 3E). Finally, to test the effect of MSCs on B cell proliferation, CD19  cells purified from PB (purity > 98%) were stimulated with the DSP30F CpG-containing ODN plus allogeneic T-cell¨Cdepleted PBMCs. As shown in Figure 3F, MSCs did not exhibit any suppressive effect on B cell proliferation.$ S' y6 G6 y4 n" g

Inhibitory Effect of MSCs Is Not Contact-Dependent and Is Not Due to Their Interference on Cell Activation, Induction of Apoptosis, or Involvement of T Regulatory Cells& P0 i8 F% \7 j
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The mechanisms possibly responsible for the inhibitory effects of MCSs on human T-cell proliferation were then investigated. To evaluate whether the suppressive activity of MSCs was mediated by cell contact or via the release of soluble factors, CFDA-SE¨Clabeled CD4  cells were stimulated with allogeneic cells in the absence or presence of MSCs (10:1 cell ratio), which were added in the same well of a Transwell plate, separated or not by a porous septum. The inhibitory effect of MSCs on CD4  T-cell proliferation was not affected by the physical separation of the two populations, as shown by both CFDA-SE and BrdU stainings, suggesting that the inhibitory effect of MSCs was not mediated by cell contact but was dependent on the release of soluble factors (Figs. 4A, 4B).

Figure 4. The inhibitory effect of mesenchymal stem cells (MSCs) on T-cell proliferation does not require cell-to-cell contact. (A): CD4  T cells were stimulated with T cell¨Cdepleted allogeneic peripheral blood mononuclear cells (PBMCs) in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) placed in the same or in the other chamber (grey columns) of a Transwell plate. Cell generations (G0-G6) were evaluated on day 4 by CFDA-SE contents, as described in Materials and Methods. Columns represent mean values (¡À standard deviation) of percentage of cells in the indicated generation obtained in six separate experiments. (B): CD4  T lymphocytes were cultured with allogeneic T cell¨Cdepleted PBMCs in the absence or presence of 5 x 104 MSCs. The scheme of the Transwell experiment is depicted at the top of the figure. Plots represent the proliferation of CD4  T lymphocytes, evaluated by the contemporaneous assessment of CFSE versus BrdU uptake in each culture condition. One representative of three separate experiments is shown.

In previous reports . However, a mixture of the same anti¨CCTLA-4 and anti¨CTGF-ß1 mAbs that had been found able to revert the suppressive effect of Treg cells showed no effect on the suppressive activity of MSCs (Fig. 5A). Moreover, none of the above-mentioned cytokines, at least at the concentration tested, was able to rescue T cells from the suppressive activity exerted by MSCs (Fig. 5B).
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Figure 5. The inhibitory effect of mesenchymal stem cells (MSCs) on T-cell proliferation does not involve T cell activation or the activity of Treg cells. (A): CD4  T cells were stimulated with T cell-depleted allogeneic peripheral blood mononuclear cells (PBMCs) in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) in the absence (medium) or presence of indicated monoclonal antibodies, and 3H-TdR uptake on day 5 was evaluated. Results are expressed as mean values (¡À standard deviation ) of cpm obtained in four separate experiments. (B): CD4  T cells were stimulated with T cell-depleted allogeneic PBMCs in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) in the absence (medium) or presence of indicated recombinant cytokines, and 3H-TdR uptake on day 5 was evaluated. Results are expressed as mean values (¡ÀSD) of cpm obtained in four separate experiments. (C): Expression of activation markers CD69, CD25, and CTLA-4 by CD4  and CD8  T cells after allogeneic stimulation. Mean values of percentages of cells showing the indicated marker (¡ÀSD) from three separate experiments are shown. (D): Annexin V staining of CD4  or CD8  T cells stimulated with T cell-depleted allogeneic PBMCs in the absence (black columns) or presence (white columns) of MSCs (ratio, 10:1). Columns represent percentages (¡ÀSD) of annexin V-stained cells obtained in four separate experiments. (E): Total CD4  or CD4 CD25¨C T cells were stimulated with T cell-depleted allogeneic PBMCs in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1), and BrdU uptake was evaluated on day 5 of culture. Columns represent mean values (¡ÀSD) of percentage of BrdU  cells obtained in three separate experiments. (F): CD4  T cells were stimulated with T cell-depleted allogeneic PBMCs in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1), and Foxp3 mRNA expression was evaluated on day 5 of culture by quantitative reverse transcription¨Cpolymerase chain reaction. Three separate experiments are reported. Columns represent mean values (¡ÀSD) of Foxp3 mRNA obtained in three replicates. *p . d" I# u4 q6 k8 a
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We also tried to establish whether the inhibitory effect of MSCs was due to their interference with cell activation processes. To this end, the expression of activation markers, such as CD69, CD25, and CTLA-4, by both CD4  and CD8  T-cell subsets stimulated with allogeneic cells in the absence or presence of MSCs was assessed. No significant difference in CD69, CD25, or CTLA-4 expression by CD4  and CD8  T cells was detected at any time (6, 24, 48 hours) (Fig. 5C). To exclude the possibility that the inhibition of T-cell proliferation induced by MSCs was due to cell death, the presence of apoptotic cells on day 5 was evaluated by flow cytometry in cocultures of MSCs/ CD4  or MSCs/CD8  cells, cultured at a 1:10 cell ratio. No significant differences in the proportion of annexin V¨Cpositive cells was observed in both CD4  and CD8  T cell populations (Fig. 5D).9 G& L% I2 L8 e8 l2 l$ Z+ D; @* s

Finally, to evaluate a possible involvement of CD4 CD25  Treg cells in the inhibitory activity of MSCs, we tested the ability of MSCs to suppress the proliferation induced by allogeneic stimulation of CD4 CD25¨C T cells. As shown in Figure 5E, MSCs exerted the same suppressive effects on the proliferation of either total CD4  or CD4 CD25¨C T cells. In addition, the evaluation on day 5 of mRNA for Foxp3, a marker highly expressed by CD4 CD25  Treg cells (13,083 ¡À 3,727 fgs of Foxp3 cDNA/50,000 cells, n = 3), showed low levels of expression on total CD4  T cells cultured in the absence or presence of MSCs. More importantly, no significant differences in Foxp3 mRNA levels between CD4  T cells cultured under the two conditions were detected (Fig. 5F).; A, W8 ~, z, B. A
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Inhibitory Effect of MSCs on Cell Proliferation Requires the Presence of IFN-, Which, at Least in Part, Acts by Enhancing Their IDO Activity2 E* ?0 ?% z- z) j9 m

In the course of this study, we were intrigued by the observation that MSCs could inhibit the proliferation of either T or NK cells, whereas they did not exhibit any effect on B cell proliferation. Therefore, the possibility that some factors produced by both T and NK cells but not by B cells may allow, or even trigger, the suppressive activity exerted by MSCs was hypothesized. We focused our attention on IFN- because this cytokine represents one of the factors produced by several CD4  and CD8  T lymphocytes, as well as by NK cells, but not by B lymphocytes. In addition, IFN- can potentially modify the MSC immunological phenotype, as it is able to upregulate the expression of HLA class I molecules and to induce de novo expression of HLA class II molecules. To provide evidence on the possible role of IFN-, the suppressive activity of MSCs toward CD4  and CD8  T cells in the absence or presence of a neutralizing anti¨C IFN-R mAb was assessed. The addition in culture of the anti¨C IFN-R mAb completely reverted the suppressive effect exerted by MSCs on CD4  and at least partially even on CD8  T cells, as assessed by both thymidine uptake (Fig. 6A) and CFSE-BrdU assays (data not shown). In addition, the capacity of the anti¨C IFN-R mAb to revert the suppressive activity exerted by MSCs on NK cell proliferation was achieved only when the number of NK cells in culture was reduced, in agreement with the notion that NK cells are able to produce high IFN- concentrations (Fig. 6B). More importantly, the addition of the anti¨C IFN-R mAb in the condition in which MSCs and CD4  T cells were separated by the porous septum in the Transwell system completely restored the proliferation of the CD4  T cells (Fig. 6C).
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Figure 6. Role of interferon (IFN)- in the suppressive activity of mesenchymal stem cells (MSCs). (A): CD4  or CD8  T lymphocytes obtained from three different donors were stimulated with T cell-depleted allogeneic peripheral blood mononuclear cells (PBMCs) in the absence or presence of MSCs (cell ratio, 10:1) in the absence (black columns) or presence (white columns) of a neutralizing anti- IFN-R or an isotype-matched (grey columns) monoclonal antibody (mAb). Results are expressed as mean values (¡À standard deviation ) of cpm obtained in three separate experiments. (B): Different numbers of natural killer (NK) cells were cultured in the presence of allogeneic stimulation and rIL-2, as described in Materials and Methods, in the absence (squares) or presence (circles) of a neutralizing anti-IFNR or an isotype-matched (triangles) mAb. The suppressive activity is expressed as percent inhibition of thymidine uptake compared with that obtained in the absence of MSCs (considered as 100%). Results are expressed as mean values (¡ÀSD) of cpm obtained in three separate experiments. (C): CD4  T cells were stimulated with T cell-depleted allogeneic PBMCs in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) placed in the other chamber of a Transwell plate without any mAb in the presence of anti- IFN- R mAb (grey columns) or an isotype-matched mAb (hutched columns). Columns represent mean values (¡ÀSD) of percentage of BrdU  cells obtained in three separate experiments. (D): B lymphocytes obtained from three different donors were stimulated with T cell-depleted allogeneic PBMCs and CpG-containing ODNs in the absence or presence of MSCs (cell ratio, 10:1), without (black columns) or with (grey columns) rIFN-. Results are expressed as mean values (¡ÀSD) of cpm obtained in three separate experiments. (E): Proliferative response of total CD4  (triangles), purified CD4 CRTH2  (circles), and CD4 CRTH2¨C (squares) lymphocytes after allogeneic stimulation in the absence or presence of different numbers of MSCs. Results are expressed as mean values (¡ÀSD) of cpm obtained in three separate experiments. *p & R: f! D0 f" G* W( r
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To further support the role of IFN- on the suppressive activity of MSCs, two additional experiments were performed. First, the suppressive activity of MSCs on the proliferation of B lymphocytes (which are unable to produce this cytokine) in the absence or presence of exogenously added IFN- was assessed. As shown in Figure 6D, the addition in culture of IFN- allowed MSCs to suppress even the proliferation of B lymphocytes. Second, purified PB CD4  T lymphocytes were separated into CRTH2  and CRTH2¨C fractions, a cell marker that has been previously associated with Th2 cells , and then assessed for their responsiveness to allogeneic stimulation in the absence or presence of MSCs. As expected, purified CRTH2 CD4  T cells produced IL-4, but not IFN-, whereas CRTH2¨CCD4  T cells produced mainly IFN- (supplemental online Fig. 2). As shown in Figure 6E, only the proliferation of total CD4 , as well as of CD4 CRTH2¨C, T cells was inhibited in a dose-dependent fashion by MSCs, whereas the proliferation of CD4 CRTH2  cells was not. Of note, even if the proliferation of CD4 CRTH2  cells was lower than the proliferation of total CD4  or CD4 CRTH2¨C T cells, the ability of all T cell populations to respond to allogeneic stimulation was similar, the stimulation index (cpm stimulated ¨C cpm unstimulated/cpm unstimulated cells) of CD4 CRTH2 , total CD4 , and CD4 CRTH2¨C T cells being equal to 90.5 ¡À 19.6, 106.0 ¡À 26.3, and 129.7 ¡À 29.8, respectively.

Finally, the mechanisms by which IFN- can trigger, or contribute to, the immunosuppressive activity of MSCs were investigated. It has recently been demonstrated that human MSCs express IDO, which catalyzes the conversion from tryptophan to kynurenine, and whose expression is upregulated by IFN- . As shown in Figure 7, both these molecules exerted a partial, but consistent, inhibition of the suppressive activity of MSCs on T-cell proliferation.3 S3 X0 f# l* E! e  a. ?$ Z
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Figure 7. Competitive inhibitors of indoleamine 2,3-dioxygenase (IDO) activity reduce the suppressive effect exerted by mesenchymal stem cells (MSCs) on T-cell proliferation. CD4  T cells were stimulated with T cell-depleted allogeneic peripheral blood mononuclear cells in the absence (black columns) or presence (white columns) of MSCs (cell ratio, 10:1) without or with different concentrations of the IDO inhibitors 1-methyl tryptophan or norharmane, and 3H-TdR uptake was evaluated on day 5 of culture. Columns represent mean values (¡À standard deviation) of cpm obtained in three separate experiments. *p
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DISCUSSION, _8 L6 o! Z) U+ y4 m" o: d6 t
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BM transplantation is largely used for the treatment of several pathological conditions, despite the lack of suitable BM donors, representing a limit to further expand this kind of therapeutic approach. The donor-recipient histoincompatibility is associated with a high risk of both graft rejection and GvHD, situations in which strategies to diminish immune responses after transplantation are mandatory. Recently, a great interest in MSCs contained in BM has been developed, inasmuch as several reports suggest that these cells not only have multipotential differentiation ability . Thus, the infusion of MSCs in conjunction with the donor organ or BM might provide a useful tool for favoring the engraftment and to reduce the incidence and/or the intensity of GvHD. However, the development of this novel therapeutic strategy requires that the mechanisms involved in the immunosuppressive activity of MSCs are better clarified.% h% w+ B  N; ]* i
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In the present study, we evaluated the activity of human MSCs on the proliferation of different lymphocyte populations and also tried to provide further information on the mechanisms possibly involved in their suppressive effect. To address these points, we tested the activity exerted by MSCs obtained from human BM on the proliferation of T lymphocytes, both CD4  and CD8 , NK cells, and B lymphocytes. MSCs were able to inhibit in a dose-dependent fashion the proliferative response of CD4  and CD8  T lymphocytes, as well as of NK cells, whereas they did not exert any inhibitory effect on B-cell proliferation. The evaluation of the proliferation tracer CFDA-SE and the uptake of the thymidine analogue BrdU on allogeneic-stimulated T cells at different time points of cell cultures revealed that the proliferation of T cells began to be suppressed by MSCs only after 3 days. These data indicate that to trigger the suppressive activity of MSCs, a crosstalk between MSCs and target cells is needed during the first phase of culture, an event that cannot occur with B lymphocytes. Moreover, we clearly showed that NK cells precultured for 5 days with MSCs were partially inhibited in their ability to lyse the K562 target cells. Why these data are apparently at variance with those reported by Rasmusson et al.  is unclear. One possible explanation may be that in this study the ability of MSCs to suppress NK cell¨Cmediated lysis of K562 cells was directly assessed during the 4 hours of MSC/NK cell coculture of the cytolytic assay, which may be a time not sufficient to reveal the suppressive effect.
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Because the suppressive activity of MSCs seems to be limited to T lymphocytes and NK cells, as that exerted by natural CD4 CD25  Treg cells .
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To provide further information on the mechanisms involved in the suppressive activity of MSCs, we took advantage of two orders of observations. First, the suppressive activity of MSCs did not require cell-to-cell contact, suggesting the possibility that it was mediated through some soluble factors. Second, the suppressive effect of MSCs seemed to be stronger on CD8  rather than on CD4  T lymphocytes, and even more on NK cells, whereas it did not affect the proliferation of B lymphocytes. The latter finding allowed us to hypothesize that some mediators actively produced upon stimulation by both NK and T, but not by B, lymphocytes may trigger, or at least contribute to, the suppressive activity of MSCs. An obvious candidate for this activity was IFN-, which is produced at high concentrations by NK and CD8  T cells and at lower concentrations by CD4  T cells but is not produced by B cells. In agreement with this possibility, the addition in culture of a neutralizing anti¨C IFN-R mAb consistently inhibited the suppressive activity of MSCs on both CD4  and CD8  T cells and even on NK cells, provided that lower numbers of these latter cells were tested in culture. This finding is consistent with the observation that NK cells are able to produce extraordinarily high amounts of IFN-, which can, at least partially, bypass the mAb-mediated blocking of the IFN-R. The crucial role of IFN- in MSC-driven suppression was further supported by another series of experiments. First, MSCs were able to suppress the proliferation of PB CD4 CRTH2¨C T cells, which contain a mixture of cells, including those able to produce IFN-, whereas they had no inhibitory effect on the proliferation of purified CD4 CRTH2  T cells, which represent a pure population of Th2 cells able to produce IL-4 but not IFN- . Finally, and most importantly, the addition in culture of exogenous IFN- also made the proliferative response of B lymphocytes susceptible to the inhibitory activity of MSCs.
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The mechanisms by which IFN- can trigger, or contribute to, the immunosuppressive activity of MSCs were finally investigated. It has recently been demonstrated that human MSCs express IDO, which catalyzes the conversion from tryptophan to kynurenine and whose expression is upregulated by IFN- , results in a complete abrogation of the MSC suppressive activity.
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The demonstration that human MSCs can interact with HLA-unrelated immune cells modulating their proliferative response and that IFN- is crucial to evoke this capacity may have important implications in transplantation biology. Acute GvHD is a Th1-mediated condition  in which NK and CD8  T cells also play an important role. However, the results of this study provide evidence that the most important cytokine produced by these cells, i.e., IFN-, plays an important role in activating the immunomodulatory effects of MSCs, thus favoring, in turn, the suppression of GvHD itself. Based on these findings, it is likely that the coadministration of MSCs with the transplant could be useful in controlling both graft rejection and GvHD. However, additional studies are needed to clarify the cell contact signals by which MSCs exert their immunosuppressive activity on the proliferation of several cell types, including Th1 cells. Moreover, the finding that MSCs can even suppress the proliferation of B cells in the presence of exogenously added IFN- supports the possibility that lymphoproliferative disorders or autoimmune diseases, which are characterized by strong B cell activation, also could benefit from therapeutic approaches based on the use of allogeneic or autologous MSCs.1 r  M8 t8 E8 Q8 L' w( @

ACKNOWLEDGMENTS

M.K. and L.C. contributed equally to this work. This work was supported by Italian Ministry of University and Scientific Research, Italian Association for Cancer Research (AIRC), Italian National Research Council (CNR), Fondazione Cariverona, and the Ministry of Health of Tuscany Region.

DISCLOSURES

The authors indicate no potential conflicts of interest.. G( Y( G1 `) b" Z8 S
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