Inhibition of T-Cell Proliferation by Murine Multipotent Mesenchymal Stromal Cells is Mediated by CD39 Expression and Adenosine Generation

Abstract

Multipotent mesenchymal stromal cells (MSCs) are bone marrow-derived cells of nonhematopoietic origin with immunoregulatory properties. Although some functions of MSCs have been identified, there are still features that are not explained thus far. The aim of the present study was to identify novel factors involved in MSC-mediated inhibition of T-cell proliferation. We here demonstrate that the surface molecule CD39 is coexpressed in concert with CD73 on murine MSCs catalyzing the generation of adenosine, which can directly act on activated T cells via the adenosine A2A receptor. Blocking of the adenosine pathway either by the A2A receptor antagonist SCH58261 or the specific CD39 inhibitor polyoxotungstate 1 (POM-1) blocked MSC-mediated suppression of T-cell proliferation almost completely. We conclude that CD39/CD73 coexpression is a novel important component of the immunoregulatory functions of murine MSCs. Our findings may both be important to improve our understanding of MSC function and for the development of immunomodulatory cellular therapies.

Keywords

Mesenchymal stromal cells (MSCs)Immunoregulation CD39/CD73 T-cell inhibition Adenosine

Introduction

Multipotent mesenchymal stromal cells (MSCs) are adult stem cells of nonhematopoietic origin in the bone marrow. MSCs, first described by Friedenstein et al. as “colony-forming unit fibroblasts,” are undifferentiated cells with high self-renewal capacity that have the potential to give rise to cells of different lineages (21). They are typically isolated from bone marrow but can be purified from other adult tissues such as adipose tissue, umbilical cord blood, spleen, lung, and skeletal muscle as well (8,21,55). MSCs are relatively rare in bone marrow (0.001–0.01%) but offer the advantage that they can be easily cultured and expanded in vitro (21,43). One of the major functions of MSCs in vivo is to provide an environment for the maturation of hematopoietic cells in the bone marrow and to repair damaged tissues (19,43).

A number of studies showed that MSCs possess immunomodulatory properties in vitro and in vivo, which makes them a promising alternative to standard immunosuppression after allogenic transplantation. MSC treatment prolongs skin allograft survival in baboons (2), prevents rejection of allogenic melanoma cells in mice (16), and attenuates graft-versus-host disease in mice and in humans (35,51). Therefore, MSCs seem to affect only alloreactive responses without compromising other reactions of the recipient immune system (4). Several features found in MSCs contribute to their immunoregulatory effects. Suppression of allogenic immune responses appears to be mediated both by cell contact and by soluble factors depending on the experimental conditions (31,59). In human MSCs, it has been shown that immunosuppression is at least partly mediated by the expression of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and the use of an IDO inhibitor reduces but does not completely abrogate immunosuppression (30,38). Other factors such as transforming growth factor-β1 (TGF-β1), hepatocyte growth factor (HGF), prostaglandin E₂ (PGE₂), or matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) were described to suppress T-cell proliferation (13,14,40,41). Apparently different mechanisms mediate the regulatory capacity of MSCs depending on the type of stimulating agent. While TGF-β1 and HGF seem to be most important after allogenic stimulation, PGE₂ dominates the suppressive function after mitogenic stimulation (1,15,46). Additionally, the mechanisms of immunomodulation by MSCs may vary depending on the method of isolation and culture conditions.

In the present study we tested the effects of murine MSCs on suppression of T-cell proliferation. We show for the first time that the generation of adenosine from extracellular ATP/ADP is an important mechanism involved in suppression of T-cell proliferation. This effect is mediated by the novel MSC cell surface marker CD39, which catalyses the production of adenosine in concert with CD73 (17). Our data indicate that murine MSCs share features of immunoregulatory T cells, which has intriguing implications for the development of cell-based immunosuppressive therapies in autoimmune diseases or after organ transplantations.

Materials and Methods

Animals

Five- to 16-week-old male BALB/c and C57BL/6 mice (Harlan-Winkelmann, Borchen, Germany) were used for the experiments. Animal procedures were conducted in accordance with the German Law on the use of Laboratory Animals and approved by the regional authorities.

Isolation and Culture of Bone-Marrow (BM)-Derived Multipotent MSCs

Murine MSCs were isolated and expanded using a modification of the methods of da Silva Meirelles et al. (8) and Tropel et al. (52). Bone marrow from male BALB/c mice was extracted by flushing out femurs and tibias using a 27-gauge needle. Red blood cells were removed by hypotonic lysis and the remaining cells were cultured at a density of 1.4 × 10⁶/cm² in isolation medium consisting of low glucose DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin (P/S), and 3.7 g/L HEPES (Invitrogen, Karlsruhe, Germany). Nonadherent cells were discarded 4 days later. At day 7 CD45-positive cells were removed by magnetic sorting with CD45 magnetic beads (Miltenyi, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. CD45-negative cells were seeded at a density of 8 × 10³/cm² in isolation medium supplemented with recombinant murine EGF (10 ng/ml; Tebu-Bio, Offenbach, Germany) and recombinant human PDGF-BB (1 ng/ml; R&D Systems, Wiesbaden, Germany). In some experiments formalin-fixed MSCs (4% PFA for 20 min) were used.

Characterization of Multipotent MSCs

For flow cytometric characterization cells were detached by incubation with EDTA. Unspecific binding sites were blocked by preincubation with Fc-Block (eBioscience, Frankfurt, Germany). The following PE-or FITC-conjugated monoclonal antibodies were used for staining: CD117-FITC, CD44-FITC, CD45-FITC, CD62L-FITC, CD31-PE (Immunotools, Friesoythe, Germany), VCAM-FITC, CD140b-PE, CD105-PE, CD34-PE, CD73-PE, CD39-PE, CD90.2-PE, Flk1-PE, ICAM-PE, CD133-FITC (eBioscience), Sca1-PE (BD Biosciences, Heidelberg, Germany), CD29-FITC (BioLegend, Eching, Germany). Antibody specificity was controlled using matched isotype antibodies. Flow cytometry was performed using FACS Calibur instrumentation (BD Biosciences). Data was analyzed with WinMDI 2.8 software (© Joseph Trotter).

Differentiation Assays

To demonstrate that the isolated MSCs possess multipotent abilities, MCSs were differentiated into osteoblasts, adipocytes, and chondrocytes using specific stimuli. For osteoblastic differentiation the cells were cultured for 18 days in DMEM (1.0 g/L glucose) supplemented with dexamethasone (0.1 μM), β-glycerophosphate (10 mM), and ascorbic acid (0.3 mM) and assayed by Von Kossa staining. To induce adipogenic differentiation cells were treated over 10 days with DMEM (4.5 g/L glucose) supplemented with 10% FCS, isobutyl-methylxanthine (0.1 mg/ml), indomethacin (200 μM), dexamethasone (1 μM), and insulin (10 μg/ml). Differentiation was detected by Oil Red-O staining. For chondrocytic differentiation, cells were incubated according to the previously described micromass culture technique (11,49). Briefly, 10 μl of a concentrated cell suspension (8 × 10⁶ cells/ml) were plated into the center of each well of a 12-well culture plate. The cells were allowed to attach at 37°C for 2 h, overlaid with differentiation medium [α-MEM with 1% FCS, 6.25 μg/ml insulin, 50 nM ascorbate-2-phosphate, 10 ng/ml human TGF-β1 (Peprotech, Hamburg, Germany), and 1% P/S] and incubated for 2 weeks, followed by staining with Alcian blue (all chemicals from Sigma, Munich, Germany).

RT-PCR

MSCs were plated in six-well plates at a density of 3 × 10⁴/well, cultured overnight, and then stimulated with interferon-γ (IFN-γ) (100 U/ml) ± tumor necrosis factor-α (TNF-α; 10 ng/ml; Peprotech) for 48 h. Messenger RNA was isolated after stimulation using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) according to manufacturer's instructions and reversely transcribed with the Omniscript RT kit (Qiagen, Hilden, Germany). cDNA was analyzed for the expression of IDO and β-actin. The following PCR primer sets were used: 5′-ACCCACACTGTGCCCATCTA-3′ and 5′-GCCACAGGATTCCATACCCA-3′ (for β-actin); 5′-GTACATCACCATGGCGATTG-3′ and 5′-GCTTTCG TCAAGTCTTCATTG-3′ (for IDO).

Western Blot Analysis

Expression of IDO by murine MSCs on the protein level was analyzed by Western Blot. MSCs were plated and stimulated for 48 h with IFN-γ (100 U/ml) ± TNF-α (10 ng/ml). Cells were lysed in RIPA buffer containing protease inhibitors, applied to 10% SDS gel electrophoresis according to the method of Laemmli (33), and transferred to Hybond-P PVDF membrane (GE Healthcare, Munich, Germany). Recombinant mouse IDO was used as positive control (0.25 μg; Alexis/Axxora, Lörrach, Germany). The membrane was blocked in Tris buffered saline (TBS) with 1% Tween-20 and 5% skim milk and incubated with an anti-mouse-IDO monoclonal antibody (1:5000, clone: mIDO-48; BioLegend) at 4°C overnight. The membrane was washed, incubated with alkaline-phosphatase-conjugated goat-anti-rat-Ig (1:2000; BioLegend) and stained with alkaline phosphatase substrate NBT/BCIP (Roche).

Kynurenine Measurement in Cell Culture Supernatants

Analysis of the tryptophan catabolite kynurenine in cell culture supernatants was used to determine IDO activity in murine MSCs according to a method of Däubener et al. (9).

T-Cell Proliferation Assay

Proliferation assays were performed in 96-well round-bottom plates in a total volume of 160 μl MLR medium consisting of Iscove basal medium (Biochrom, Berlin, Germany), 10% FCS, 1% P/S, 50 μM β-mercaptoethanol (Gibco, Paisley, UK) in triplicate. MSCs were treated with 50 μg/ml mitomycin C (Sigma) for 2 h to prevent cell proliferation, washed three times, and then plated at a density of 0.5 × 10⁵ cells/well. T cells were purified from C57BL/6 splenocytes by magnetic activated cell sorting (MACS) using CD90 MicroBeads (Miltenyi) according to the manufacturer's instructions. Sorted cells were >95% positive for CD45 excluding contamination with splenic MSCs. C57BL/6 T cells (responder cells) were plated at 0.5 × 10⁵. T cells were stimulated after 24 h with 5 μg/ml concanavalin A (ConA; Sigma). After an additional 72 h, 1 μCi [³H]thymidine (Perkin-Elmer, Rodgau, Germany) was added to each well and incubated for another 18 h. Cells were harvested onto filter plates (Perkin-Elmer) and [³H]thymidine uptake was measured on a TopCount beta counter (Perkin-Elmer).

The following inhibitors or antibodies were used for functional analysis of MSC-mediated effects: the adenosine A2A receptor antagonist SCH58261 (25 μM), the CD39 inhibitor POM-1 (50 and 100 μM; all chemicals from Sigma), an anti-HGF receptor antibody (10 μg/ml; GeneTex/Biozol, Eching, Germany), and a monoclonal anti-TGF-β1 antibody (1 μg/ml; Abcam, Cambridge, UK). Specificity of the used antibodies was controlled by using matched isotype controls.

Transwell Experiments

C57BL/6 T cells (2 × 10⁵) were plated in the lower chamber of a 24-well transwell plate (0.4 μm pore size; Corning, Schiphol-Rijk, The Netherlands) in a volume of 600 μl MLR medium. Mitomycin C-treated MSCs (2 × 10⁵) were plated in the upper chamber. T-cell proliferation was induced by the addition of ConA (5 μg/ml) to each well 24 h after plating. After 4 days cells from the lower chamber were transferred to a 96-well round-bottom plate in triplicate (100 μl/well). Medium (60 μl) from the transwell cultures was added to a final volume of 160 μl/well. The cells were pulsed with 1 μCi [³H]thymidine (GE Healthcare) and harvested 18 h later.

Malachite Green Assay for the Detection of Ectonucleotidase Activity

Ectonucleotidase activity was measured in MSCs without addition of T cells using phosphate generation as the readout. The assay was performed as described previously by Baykov et al. (3) and Wu et al. (58). MSCs were plated in 24-well plates and used for measuring ectonucleotidase activity at 80% confluency. Phosphate levels were determined from standard curves using increasing concentrations of KH₂PO₄.

Measurement of Adenosine Production by High Performance Liquid Chromatography (HPLC)

For analysis of adenosine in cell culture supernatants various numbers of MSCs were incubated for 45 min at 37°C with 50 μM ATP in phosphate-free incubation buffer (20 mM HEPES, pH 7.5, 10 mM glucose, 5 mM KCl, 120 mM NaCl, 2 mM CaCl₂, 5 mM tetramisole; all chemicals from Sigma). Supernatants were taken, evaporated under N₂, resuspended in 200 μl H₂O bidest., and analyzed by HPLC. Chromatographic analysis was performed on a Shimadzu LC-10 AD instrument. A SPD 20AV detector was used with a LiChrospher RP 18e 5 μm guard and a LiChrospher 250-4, 5 μm RP 18e column (Merck, Darmstadt, Germany). Adenosine was measured isocratically with 7% (v/v) acetonitril in 17 mmol/L phosphate buffer as mobile phase, with a flow rate of 1.0 ml/min. Absorbance was measured at 260 nm. Retention time was 7.5 min. Class VP software was used for system control and data analysis.

Statistical Analysis

Statistical analysis was performed by t-test using Prism software (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant at p < 0.05. In cases where multiple comparisons were made, a Bonferroni correction was performed. A value of p < 0.05 was considered significant. Data are presented as the mean value ± SD of at least three independent experiments.

Results

Phenotypic Characterization of Multipotent MSCs

Murine MSCs were generated from bone marrow of BALB/c mice. They constitute a rapidly proliferating adherent cell population with fibroblast-like morphology that can be cultured for many passages in vitro (>60) in contrast to human MSCs, which often stop proliferating after 4–15 passages (14,54). Multipotency of MSCs was confirmed by their ability to differentiate into chondrocytes, adipocytes, and osteoblasts using specific stimuli (Fig. 1A–D). FACS analysis showed that MSCs were positive for the MSC-associated markers CD105, CD73, CD140b (PDGF receptor), CD29, Sca-1, and CD44, but negative for CD45 and CD31 excluding contamination by hematopoietic or endothelial cells (Fig. 1E). CD90 and VCAM were weakly expressed. The cells did not express CD117, CD62L, CD133, CD34, CD25, ICAM, or Flk1.

Figure 1.

Characterization of murine MSCs. (A) Undifferentiated MSCs (H&E staining); (B) osteoblastic differentiation (Von Kossa staining); (C) chondrogenic differentiation (Alcian-Blue staining); (D) adipogenic differentiation (Oil Red-O staining). Scale bars: 100 μm. (E) FACS staining: BALB/c MSCs expressed MSC-associated markers such as CD105, CD44, CD73, CD29, Sca-1, and CD140b; CD90 and VCAM were weakly expressed; CD45, CD34, CD117, ICAM, CD62L, CD133, CD31, and Flk1 could not be detected. Staining with specific monoclonal antibody (filled histogram) and matched isotype control (open histogram).

MSCs Strongly Inhibit T-Cell Proliferation

Purified T cells from C57BL/6 mice were stimulated with ConA and cultured with or without BALB/c MSCs. MSCs were used as unstimulated cells or prestimulated with IFN-γ or IFN-γ + TNF-α. As shown in Figure 2A, MSCs suppressed T-cell proliferation significantly by up to 75%, reducing proliferation from 30289 ± 9304 to 7695 ± 4638 cpm. No significant differences in their suppressive potential were detected between unstimulated or the two groups of prestimulated MSCs. MSCs alone did not proliferate. Formalin-fixed MSCs had no suppressive effect on T cells, indicating that metabolically active MSCs are needed for the suppressive activity (Fig. 2B). This effect was not dependent on direct contact between T cells and MSCs because suppression of T-cell proliferation was still present after cell separation using transwell chambers (Fig. 2C).

Figure 2.

MSCs suppress T-cell proliferation. (A) BALB/c MSCs significantly suppress proliferation of purified C57BL/6 T cells after ConA stimulation. No difference in suppressive potential was detected between prestimulated or unstimulated MSCs (^***p < 0.001; n = 15). (B) Formalin-fixed MSCs lose their ability to suppress T-cell proliferation (n = 5). (C) Suppression of T-cell proliferation by MSCs is unaffected by separation of MSCs and T cells using transwell chambers, which prevent direct cell contact (^***p < 0.001; n = 3).

Suppression of Mitogen-Activated T Cells by MSCs Is Not Mediated by HGF, TGF-β, or IDO

Previous studies have shown that the immunoregulatory abilities of MSCs can be partly explained by the expression of the tryptophan-degrading enzyme IDO, PGE₂, and the secretion of TGF-β and HGF, respectively (9,22,23,48). PGE₂ as well as HGF and TGF-β could be detected in cell culture supernatants of BALB/c MSCs (data not shown). In blocking experiments using monoclonal antibodies against TGF-β and the HGF receptor no significant influence on T-cell proliferation was observed (Fig. 3A). Only minimal expression of IDO was detected by RT-PCR in unstimulated cells but mRNA levels increased after stimulation of MSCs with IFN-γ or IFN-γ + TNF-α. However, no expression of IDO protein was detected by Western blot (Fig. 3B). Consistent with this finding, kynurenine, the end product of tryptophan degradation (9), was not detected in supernatants of our MSCs. This was in sharp contrast to human MSCs, which produced high levels of kynurenine after IFN-γ stimulation (data not shown).

Figure 3.

MSC-mediated suppression of mitogen-induced T-cell proliferation is not mediated by TGF-β1, HGF, or IDO. (A) Addition of an anti-HGF receptor antibody or an anti-TGF-β1 antibody does not influence MSC-mediated suppression (p > 0.05; n = 4). (B) MSCs do express IDO mRNA after stimulation with IFN-γ or IFN-γ + TNF-α but do not express IDO on the protein level (n = 3–5).

MSCs Express CD39 and Generate Immunosuppressive Adenosine

Adenosine generation stimulated by hypoxia, organ injury, traumatic shock, or at sites of inflammation is in part mediated by the action of the two sequential enzymes: CD39 and CD73. The effect of adenosine is based on the engagement of the corresponding adenosine receptor A2A expressed by T cells. FACS analysis showed that isolated MSCs expressed not only the well-known surface marker CD73 but also CD39, which represents the rate-limiting enzyme in the sequential degradation of ATP to adenosine (Fig. 4A) (10). MSCs actively catabolize ATP to adenosine and phosphate as shown by measurement of adenosine by HPLC and measurement of phosphate by malachite green assay (Fig. 4B, C). The formation of phosphate could be blocked in a dose-dependent manner by addition of the specific CD39 inhibitor POM-1 (Fig. 4C). POM-1 as a polyoxometalate belongs to a new class of NTPDase inhibitors that have recently been described to be highly specific and more potent than conventional inhibitors such as ARL67156 (39,56). We further investigated the role of CD39/CD73-dependent adenosine production on suppression of T-cell proliferation in a mixed lymphocyte reaction by using POM-1 and the adenosine A2A receptor antagonist SCH58261. Both POM-1 and SCH58261 were able to significantly decrease MSC-mediated inhibition of T-cell proliferation (Fig. 5A, B).

Figure 4.

MSCs express the ectoenzymes CD39 and CD73 and are capable of generating adenosine from ATP. (A) Coexpression of the ectoenzymes CD39 and CD73 on the cell surface. Staining with specific monoclonal antibody (filled histogram) and matched isotype control (open histogram). (B) MSCs produce adenosine as detected by HPLC. The amount of generated adenosine is dependent on cell number. (C) MSCs catalyze degradation of ATP to phosphate and adenosine. Generation of phosphate is detectable after addition of ATP and can be blocked by addition of the specific CD39 inhibitor POM-1 in a dose-dependent manner (^*p < 0.05; ^**p < 0.005; n = 4).

Figure 5.

MSC suppress T-cell proliferation by generation of adenosine. (A) Suppression of T-cell proliferation in a mixed lymphocyte reaction can be reversed by addition of 100 μM POM-1 while addition of 50 μM POM-1 does not alter immunosuppression by MSCs significantly (^***p < 0.001; n = 4). (B) Suppression of T-cell proliferation by MSCs is blocked significantly by addition of the adenosine A2A receptor antagonist SCH58261 (^***p < 0.001; n = 8).

Discussion

MSCs are multipotent cells with a high immunomodulatory capacity that is mediated by soluble factors and cell surface molecules. Here, we identified that MSCs isolated from bone marrow of BALB/c mice express the ectonucleotidases CD39 and CD73, supporting the generation of adenosine and thereby promoting strong immunosuppression of effector T cells. This mechanism has been recently identified to play a major role in the immunomodulatory activity of regulatory T cells (Tregs) (5,10,28). We here demonstrate for the first time that BALB/c MSCs share these important functions for the modulation of inflammatory immune responses with Treg cells and that the expression of enzymatic active CD39/CD73 is a major player by which MSCs can down-regulate mitogen-induced T-cell proliferation.

In the present study we investigated immunosuppressive activity of bone marrow-derived MSCs on allogenic mitogen stimulated T cells. Previous studies reported that several pathways are involved in MSC immunomodulation, including secretion of anti-inflammatory cytokines TGF-β1 and HGF, the formation of PGE₂, and the expression of IDO. In our studies we observed that HGF and TGF-β1 play only a minor role in our experimental setting. The discrepancies to some previous studies may be explained by differences in culture conditions for the isolation of MSCs and the factors used to stimulate T-cell proliferation. While TGF-β1 and HGF seem to be most important after allogenic stimulation, they do not play a role after mitogenic stimulation (1,14,45,46).

Expression of the tryptophan-degrading enzyme IDO has been described in some studies as mediator of MSC-associated immunosuppression (18,38). Other experiments could not confirm these results. In particular, expression of IDO by murine MSCs is quite controversial (30,47,53). In the present study we demonstrate that BALB/c MSCs only express IDO at the mRNA but not on the protein level, and therefore do not possess tryptophan degrading properties as assessed by the lack of kynurenine production. Using similar culture conditions we were able to isolate human MSCs with high expression of functional active IDO (data not shown). These findings support the hypothesis that there are major species differences in MSC properties.

To further characterize MSC functions, we studied expression of cell surface molecules involved in the modulation of cellular immune responses. Recently, several groups independently reported on regulatory CD4/CD25, Foxp3-positive Tregs expressing CD73 and CD39 (5,10,28). Coexpression of the phosphohydrolytic enzymes CD73/CD39 on the cell surface of Treg cells was shown to catalyze the sequential generation of adenosine by degradation of extracellular ATP/ADP to 5′-AMP (CD39) and conversion of 5′-AMP to adenosine (CD73) (17,60) leading to strong downregulation of T-cell proliferation and a decreased secretion of proinflammatory cytokines (7,20,42). Since CD73 is a characteristic molecule expressed on MSCs, we here analyzed the presence of CD39 and the generation of bioactive adenosine. We provide evidence that both molecules, CD39 and CD73, are constitutively expressed by BALB/c MSCs leading to the generation of adenosine. Our blocking experiments with a specific CD39 inhibitor and a specific A2A receptor blocker revealed that adenosine exerts its effect directly on T cells even in the absence of antigen-presenting cells. This coincides with the findings of Erdmann et al., who showed that the activation of the adenosine A2A receptor on T cells is independent of the presence of dendritic cells (20).

The appearance of extracellular nucleoside triphosphates (NTP) is a well-known indicator of tissue and cell damage that activates immune and endothelial cells and induces inflammatory responses. This reaction is controlled and counteracted by the degradation of NTP by specific ectonucleotidases on the surface of various cells. CD39 is a member of this enzyme family and is mainly expressed on immune cells such as B cells, dendritic cells, and subsets of T cells (10). Our findings on CD39 and CD73 expression on MSCs suggest that murine MSCs may be involved in downregulation of tissue inflammation by the generation of adenosine. There is general agreement that adenosine inhibits T-lymphocyte activation and effector functions and thereby has strong anti-inflammatory effects, can limit exaggerated immune reactions, and can protect tissue from cell damage (6,25). Sensitivity of T cells to adenosine-mediated inhibition depends on the expression of the A2A receptor regulated by their state of activation. Expression is highest in effector cells, decreases in memory cells, and is lowest in naive cells (50). Activated T cells with high inflammatory potential may possess the highest susceptibility to adenosine mediated suppression. Our data may provide a novel explanation for the beneficial effects of MSC treatment on tissue destruction described in animal models of myocardial infarction, organ transplantation, or cell regeneration (26,27,32,36,37,44). At present, it is not known whether or not CD39/CD73 is also coexpressed on human MSC populations and whether some immunoregulatory MSC functions described in human studies can be explained by adenosine generation (24, 29,34,57). Therefore, the relevance of this novel mechanism to promote immunosuppression in vivo and the importance for clinical trials need to be shown in future studies.

In conclusion, we demonstrate that CD39/CD73 coexpression and the generation of adenosine is a novel, hitherto unknown, tool in the regulatory repertoire of multipotent bone marrow-derived murine MSCs. The identification of adenosine generation has major implications for a better understanding of MSC functions. The striking similarities to some Treg cell functions support the hypothesis that MSCs are promising candidates for the development of novel therapies to treat autoimmune diseases and the development of immunosuppressive strategies such as local or systemic immunosuppression in allogenic cell or organ transplantations.

Footnotes

Acknowledgments

This work was supported by grants of the BMBF (01GN0523) (J.S.) and the Deutsche Forschungsgemeinschaft (Transregio Research Unit FOR 535, WO 685/10) (J.S.). The results presented in this work are part of the doctoral thesis of Manuela Steinsdoerfer.

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