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The characteristics and immunoregulatory functions of regulatory dendritic cells induced by mesenchymal stem cells derived from bone marrow of patient with chronic myeloid leukaemia

Published:November 30, 2011DOI:https://doi.org/10.1016/j.ejca.2011.11.003

      Abstract

      Dendritic cells (DCs) are specialised antigen-presenting cells that play crucial roles in the initiation and regulation of immune responses. Recently, mesenchymal stem cells (MSCs) have gained further interest after demonstration of immunomodulatory effects on complicated interactions between T cells and even DCs. However, the mechanisms underlying these immunoregulatory effects of MSCs induced DCs are poorly understood. In addition, it is unclear whether the immunoregulatory functions of MSCs are altered in disease states. In this study, we showed that chronic myeloid leukaemia (CML) patients bone marrow derived MSCs (CML–MSC) could differentiate mature DCs (mDCs) into a distinct regulatory DC population, they had lower expression of CD40, CD80, CD83 and CD86. Similar to immature DCs (imDCs), CML–MSC induced DCs (CML–MSC–DCs) displayed powerful phagocytic capacity. Moreover, CML–MSC–DCs had the capacity to induce T cell anergy, another capacity of regulatory DCs. CML–MSC–DCs could inhibit the proliferation of T cells not only through TGF-β1, but also by inducing the production of Treg cells or T-cell anergy. At last, CML–MSC–DCs could efficiently induce more CD4+CD25+Foxp3+Tregs from naive CD4+CD25−Foxp3−T cells than that of normal-MSC–DCs in vitro. CML–MSC–DCs derived TGF-β1 was largely responsible for the increase in CD4+CD25+Foxp3+Tregs based on knockdown studies. The immunoregulatory effects of DCs induced by CML–MSCs enhance the potential use of autologous MSCs in cell therapy.

      Keywords

      1. Introduction

      Mesenchymal stem cells (MSCs) are multipotential cells that reside within the bone marrow and can be induced to differentiate into various components of the marrow microenvironment, including osteoblasts, adipocytes, myoblasts, chondroblasts, neurons and gliacytes under certain conditions.
      • Jiang Y.
      • Jahagirdar B.N.
      • Reinhardt R.L.
      • et al.
      Pluripotency of mesenchymal stem cell derived from adult marrow.
      MSCs are able to support haematopoiesis in long-term bone marrow culture by producing numerous extra-cellular matrix ligands and a number of haematopoietic cytokines. MSCs are able to inhibit T cells proliferation in vitro and mediate a systemic immunosuppressive property in vivo.
      • Bartholomew A.
      • Sturgeon C.
      • Siatskas M.
      • et al.
      Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
      • Le Blanc K.
      • Frassoni F.
      • Ball L.
      • et al.
      Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.
      In addition, Horwitz et al. reports the first use of MSCs for the treatment of children with severe osteogenesis imperfecta based on their ability to differentiate to bone cells.
      • Horwitz E.M.
      • Prockop D.J.
      • Fitzpatrick L.A.
      • et al.
      Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.
      Koc et al. demonstrated that autologous MSCs could be infused along with peripheral blood stem cells in advanced breast cancer patients, the infusion could promote the haematopoietic recovery and was not an adverse reaction.
      • Koc O.N.
      • Gerson S.L.
      • Cooper B.W.
      • et al.
      Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.
      Other studies suggest that cotransplantation of MSCs permits HSC alloengraftment and decreases graft-versus-host disease (GVHD) through the approach of regulatory immune response in vivo.
      • Ball L.M.
      • Bernardo M.E.
      • Roelofs H.
      • et al.
      Cotransplantation of ex vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem-cell transplantation.
      • Lazarus H.M.
      • Haynesworth S.E.
      • Gerson S.L.
      • Rosenthal N.S.
      • Caplan A.I.
      Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.
      • Baron F.
      • Lechanteur C.
      • Willems E.
      • et al.
      Cotransplantation of mesenchymal stem cells might prevent death from graft-versus-host disease (GVHD) without abrogating graft-versus-tumor effects after HLA-mismatched allogeneic transplantation following nonmyeloablative conditioning.
      Taking advantage of their immune suppressive effect as well as the ability to support haematopoiesis, both autologous and allogeneic MSCs are of great therapeutic potential in the context of cell-based therapy.
      Dendritic cells (DCs), the most potent antigen-presenting cells (APCs), are derived from CD34+ BM stem cells and can be generated from monocytes in vitro by incubation with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).
      • Chapuis F.
      • Rosenzwajg M.
      • Yagello M.
      • et al.
      Differentiation of human dendritic cells from monocytes in vitro.
      DCs are key mediators for the initiation and regulation of both innate and adaptive immune responses. DCs exist in at least two different stages: immature and mature. Depending on their different states, DCs possess different functional properties. Mature DCs (mDCs) initiate an immune response depending on its transition from antigen processing to antigen-presenting cell, during which it up-regulates MHC class II and costimulatory molecules (CD80 and CD86) on the cell surface.
      • Mellman I.
      • Steinman R.M.
      Dendritic cells: specialized and regulated antigen processing machines.
      Immature DCs (imDCs) that are deficient of costimulatory molecules can induce T-cell anergy, generate regulatory T cells and promote alloantigen-specific tolerance.
      • Banchereau J.
      • Briere F.
      • Caux C.
      • et al.
      Immunobiology of dendritic cells.
      Thus, the unique capacity of DCs to respond to maturation signals and subsequently to activate naive T cells enables these cells to determine the fundamental outcome of immunity.
      Compared to allogenetic MSCs, autologous MSCs from patients who needed cell-based therapy may be an ideal alternative stem cell source. Our previous studies demonstrated that CML derived MSCs (CML–MSC) were similar to normal adult derived MSCs in phenotype, morphology, multi-differentiation capacity and haematopoiesis supporting function. CML–MSC did not express BCR/ABL gene and Ph chromosome.
      • Zhao Z.G.
      • Tang X.Q.
      • You Y.
      • et al.
      Assessment of bone marrow mesenchymal stem cell biological characteristics and support hemotopoiesis function in patients with chronic myeloid leukemia.
      Because the immunomodulatory effects of MSCs are critical for the clinical application of CML–MSC, we want to understand the immunomodulatory effects and definitive mechanisms of CML–MSC. Although CML–MSC is able to inhibit T cells proliferation in vitro,
      • Zhao Z.G.
      • Li W.M.
      • Chen Z.C.
      • Zou P.
      Immunosuppressive properties of mesenchymal stem cells derived from bone marrow of patients with chronic myeloid leukemia.
      a definitive mechanism underlying this phenomenon is still lacking. In addition, the effects and mechanisms of CML–MSC on the development of DCs are not clear.
      Recently, Zhang et al. showed that MSCs could induce mDCs into a subset of DC population with immunoregulatory property.
      • Zhang B.
      • Liu R.
      • Shi D.
      • et al.
      Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.
      However, the mechanisms underlying these immunoregulatory effects of MSCs induced DCs are still poorly understood. Moreover, it is unclear whether the immunoregulatory functions of MSCs are altered in disease states. In this study, we showed that CML patients bone marrow derived MSCs could differentiate mDCs into a distinct regulatory DC population. Compared with mDCs, they had lower expression of CD40, CD80, CD83 and CD86, but higher expression of CD11b. CML–MSC induced DCs could efficiently inhibit the proliferation of T-lymphocyte not only through TGF-β1, but also through the induction of Tregs or T cell anergy. In addition, CML–MSC–DCs could efficiently induce more CD4+CD25+Foxp3+Tregs from naive CD4+CD25−Foxp3−T cells than that of normal-MSC–DCs in vitro. CML–MSC–DCs derived TGF-β1 was largely responsible for the increase in CD4+CD25+Foxp3+Tregs based on knockdown studies.

      2. Materials and methods

      2.1 Isolation and culture of MSCs derived from CML patients

      Sixteen patients with CML (aged from 23 to 56; median age: 41) were investigated in this study. For CML patients, 13 patients were in their initial chronic phase and 3 patients were in accelerated phase. Thirteen patients were untreated at the time of study. Three patients were receiving hydroxyurea at the time of marrow collection. Eight normal adults (aged from 19 to 35; median age: 32; 4 males and 4 females) were investigated as control. Institutional Review Board approval was obtained for the use of the human bone marrow. Demography and diagnosis of the patients were listed in Table 1. Mononuclear cells (MNCs) were separated by a Ficoll-Paque gradient centrifugation (specific gravity 1.077 g/ml; Sigma Diagnostics, St. Louis, MO, USA), and cultured in an expansion medium at 37 °C with 5% CO2 in fully humidified atmosphere. After being cultured for 24–48 h, the culture medium was replaced and non-adherent cells were removed. Once cells were more than 80% confluent, they were detached with 0.25% trypsin–EDTA (Sigma), then CD14 positive cells were depleted using CD14 micromagnetic beads (Miltenyi Biotec, Auburn, USA) and CD14 negative cells were replated. MSCs expressed CD105, CD29 and CD44; they did not express haematopoietic markers CD34, CD45 and endothelial markers CD31, vWF. Moreover, CD14 and HLA-DR were also negative. They had the ability to differentiate into, at least, adipocyte, osteoblast, endothelial and neural in vitro.
      • Zhao Z.G.
      • Tang X.Q.
      • You Y.
      • et al.
      Assessment of bone marrow mesenchymal stem cell biological characteristics and support hemotopoiesis function in patients with chronic myeloid leukemia.
      Table 1Presenting features of patients at diagnosis.
      PatientAgeSexDiagnosisWCC (109/L)Ph+ (%)BCR/ABL
      BCR/ABL gene was detected by RT-PCR
      Treatment
      146FCML-CP148100+None
      248MCML-CP247100+None
      337FCML-CP178100+None
      434FCML-CP11588+None
      545FCML-AP355100+Hydroxyurea
      641MCML-CP125100+None
      736FCML-CP97100+None
      843MCML-CP11889+None
      944MCML-CP217100+None
      1039MCML-AP159100+Hydroxyurea
      1146FCML-CP295100+Hydroxyurea
      1241MCML-CP135100+None
      1336FCML-CP137100+None
      1442MCML-AP12875+None
      1541MCML-CP87100+None
      1638MCML-CP129100+None
      WCC represents white blood cell count; CP indicates chronic phase; AP indicates accelerated phase.
      low asterisk BCR/ABL gene was detected by RT-PCR

      2.2 Differentiation of DCs

      Human peripheral blood mononuclear cells (hPBMCs) from healthy donors were isolated by centrifugation over Ficoll-Hypaque gradients (Nycomed Amersham, Uppsala, Sweden). CD14+ monocytes were isolated as the adherent fraction after incubation for 1 h in RPMI 1640 (BioWhittaker, Verviers, Belgium) supplemented with 10% foetal calf serum (FCS) (BioWhittaker), 100 U/mL penicillin/streptomycin (Bristol-Myers Squibb, Sermoneta, Italy), and 50 μM 2-mercaptoethanol (Bio-Rad, Segrate, Italy) (DC medium) at 37 °C. After extensive washing, adherent monocytes were differentiated into DCs by culture in 10 ng/mL recombinant human IL-4 (rhIL-4) (R&D Systems, Minneapolis, MN) and 100 ng/mL recombinant human GM-CSF (rhGM-CSF) (Schering-Plough, Kenilworth, NJ) in DC medium. To induce maturation of monocyte-derived cells, lipopolysaccharide (LPS) (1 μg/mL) was added for another 48 h of culture with GM-CSF and IL-4.

      2.3 Isolation of CD4+T cells and their subsets

      hPBMC was isolated by Ficoll-Paque (1.077 g/mL) density gradient centrifugation. CD4+T lymphocytes were isolated from hPBMC by using CD4 micromagnetic beads (Miltenyi Biotec, Auburn, USA) according to the manufacturer’s instructions. CD4+ cell purity was 97 ± 2%. Subsequently, CD4+CD25−T cells and CD4+CD25+T cells were obtained by using the CD25 beads (Miltenyi Biotec).

      2.4 Coculture experiment

      Once MSCs reached 50% confluence, mDCs were seeded onto MSC monolayers at a density of 106 per 5 mL per well in 6-well plates, and the culture medium was replaced with RPMI 1640 supplemented with 5% FCS. DCs cultured on MSCs for at least 10 days were washed off with 0.1% trypsin and 5 mM EDTA, and were purified with CD11b magnetic microbeads and either were analysed or used in further experiments.
      APCs (either mDCs or CML–MSC–DCs or normal-MSC–DCs) and T cells (either CD4+CD25−T cells or CD4+CD25+T cells) were cocultured at a different ratio in RPMI 1640 with 5% FCS.

      2.5 Fluorescence activated cell sorter (FACS) analysis

      For immunophenotype analysis, cultured cells were washed with PBS containing 0.5% bovine serum albumin (BSA, Sigma), and incubated with primary antibodies (10–20 ng/ml) for 30 min at 4 °C. Primary antibodies included mAb against CD4, CD11b, CD25, CD40, CD80, CD83, CD86 and Foxp3 (BD Biosciences Pharmingen, San Diego, CA, USA). We used same-species, same-isotype irrelevant antibody as negative control. Cell analysis was performed with FACS Calibur system using Cellquest software.

      2.6 Cytokine analysis

      Cytokines produced in culture supernatants at day 5 from mixed lymphocyte reaction (MLR) were detected by using ELISA kits (R&D Systems) for IL-2 and IFN-γ. For detection of IL-12, IL-10, and transforming growth factor β1 (TGF-β1) produced by APCs, cells were cultured for 24 h with or without LPS (1.0 μg/mL) stimulus.

      2.7 Endocytosis assay

      Endocytosis was measured as the cellular uptake of fluorescein isothiocyanate (FITC)–dextran and was quantified by flow cytometry. Approximately 5 × 105 cells per sample were incubated in medium containing FITC–dextran (1 mg/mL; molecular weight 40,000; Sigma, St. Louis, MO) for 60 min. After incubation, cells were washed twice with cold phosphate buffered saline (PBS) to stop endocytosis and remove excess dextran and were then fixed in cold 1% formalin. The quantitative uptake of FITC–dextran by the cells was determined by FACS. At least 10,000 cells per sample were analysed.

      2.8 Mixed leucocyte reaction

      CD4+T cells resuspended at 1 × 105–1 × 106 cells/well were added to wells containing or lacking irradiated (15 Gy) allogeneic suppressive cells. The culture was continued and 3H-thymidine was added 18 h before the end of the 120-h culture. The T-cell proliferation was represented as the incorporated radioactivity in cpm and shown as mean ± SD of triplicate values.
      To examine the ability of MSC–DCs to induce T cell anergy, human naive CD4+T cells (5 × 106) were cultured with allogeneic MSC–DCs (5 × 105) or LPS stimulated MSC–DCs (1 × 105) or imDCs (1 × 105) or LPS stimulated imDCs (1 × 105) or mDCs (1 × 105) for 3 days. These CD4+T cells were then rescued, and cells (1 × 105) were subsequently restimulated with allogeneic mDCs (1 × 104) or Phytohaemagglutinin (PHA) in a second coculture. The culture was continued and 3H-thymidine was added 18 h before the end of the 120-h culture.
      To identify soluble factors responsible for the inhibitory effect of MSC–DCs, neutralising monoclonal antibodies directed against cytokines were added in MLR. In these experiments, MLR containing PHA-stimulated T cells without neutralising monoclonal antibodies was set up as a control. The following neutralising monoclonal antibodies were used: monoclonal antibody anti-rhTGF-β1, anti-rhIL-10, anti-rhIL-2 and anti-rhIFNγ. In some experiments, 1-methyltryptophan, indomethacin and 1,4 Phenylene-bis-isothiourea (PBIT) were added at the beginning of the culture.

      2.9 TGF-β1 knockdown in MSCs induced DCs

      TGF-β1 siRNA duplexes were used to knock down this respective gene in MSCs induced DCs. Briefly, MSCs induced DCs (106) were seeded in 75-cm2 flasks, after 24 h, 100 nM siRNA was delivered via DharmaFECT Transfection Reagent (Dharmacon, USA). TGF-β1 siRNA duplex sequence was selected as follows: 5′-gca aca auu ccu ggc gau a-3′ and 5′-uau cgc cag gaa uug uug c-3′. All siRNA duplexes were obtained from Takara Biotech (Dalian, China). Knockdown of TGF-β1 was confirmed by Western blot.

      2.10 Western blotting

      For Western blotting, equivalent amount of protein lysates, obtained from induced cells, were loaded per lane. After SDS–PAGE, proteins were electrophoretically transferred onto nitrocellulose membrane (Amersham Pharmacia Biotech; Uppsala, Sweden). After blocking, blots were incubated with mouse polyclonal antibodies against TGF-β1 (Santa Cruz Biotechnology) at 1:200. Expression of the β-actin was used as an internal control. Immunodetection using the enhanced chemiluminescence method (ECL kit; Amersham, Piscataway, NJ) was performed according to the manufacturer’s instructions.

      2.11 Statistical analysis

      The results were statistically analysed by using the SPSS11.0 statistical package (SPSS Inc., Chicago, IL). The Student t test for paired data (two-tail) was used to test the probability of significant differences between samples.

      3. Results

      3.1 CML–MSC induce mDCs into a distinct DC subset

      To investigate the influence of CML–MSC on the development of mDCs, mDCs derived from monocytes were cultured with CML–MSC for at least 10 days before analysis. CML–MSC treated mDCs expressed high levels of the myeloid lineage marker CD11b and low levels of the functional markers of DCs, such as CD40, CD80, CD83 and CD86 (Fig. 1A). The immunophenotype of CML–MSC treated mDCs was similar to that of imDCs. However, in contrast to imDCs, such expression of these markers at low levels was not increased when the cells were stimulated by LPS, indicating a stable immature-like phenotype for these cells. In addition, similar to imDCs, CML–MSCs treated mDCs displayed high endocytic capacity (Fig. 1B). Analysis of cytokine profile revealed that CML–MSC treated mDCs spontaneously secreted more TGF-β1 and IL-10 but less IL-12 (Fig. 1C). This profile was significantly enhanced after LPS stimulation, suggesting that CML–MSC induced DC may be involved in immune regulation. These results implied that CML–MSC could induce DC to differentiate into a distinct DC subset (CML–MSC–DCs).
      Figure thumbnail gr1a
      Fig. 1Characteristic profile of CML–MSC–DCs. Mature DCs (mDCs) derived from monocytes were cultured with CML–MSC or normal mesenchymal stem cells (MSCs) for 2 weeks. (A) Phenotype of CML–MSC–DCs. The immature DCs (imDCs), mDCs, CML–MSC–DCs, and normal-MSC–DCs were analysed by FACS. Grey lines indicate background staining. Numbers in histograms indicate the mean fluorescence of each DC population. (B) The phagocytic ability of imDCs, mDCs, CML–MSC–DCs, and normal-MSC–DCs was tested by measuring fluorescein isothiocyanate (FITC)–dextran phagocytosis using FACS. Filled histograms represent imDCs, mDCs, CML–MSC–DCs or normal-MSC–DCs cultured with 1 mg/mL FITC–dextran for 60 min and were marked with geometric mean fluorescence. (C) Cytokine secretion profile of mDCs, imDCs, CML–MSC–DCs, and normal-MSC–DCs was measured by ELISA. Data are expressed as mean ± SD of triplicates of six separate experiments. P ⩽ 0.05.
      Figure thumbnail gr1bc
      Fig. 1Characteristic profile of CML–MSC–DCs. Mature DCs (mDCs) derived from monocytes were cultured with CML–MSC or normal mesenchymal stem cells (MSCs) for 2 weeks. (A) Phenotype of CML–MSC–DCs. The immature DCs (imDCs), mDCs, CML–MSC–DCs, and normal-MSC–DCs were analysed by FACS. Grey lines indicate background staining. Numbers in histograms indicate the mean fluorescence of each DC population. (B) The phagocytic ability of imDCs, mDCs, CML–MSC–DCs, and normal-MSC–DCs was tested by measuring fluorescein isothiocyanate (FITC)–dextran phagocytosis using FACS. Filled histograms represent imDCs, mDCs, CML–MSC–DCs or normal-MSC–DCs cultured with 1 mg/mL FITC–dextran for 60 min and were marked with geometric mean fluorescence. (C) Cytokine secretion profile of mDCs, imDCs, CML–MSC–DCs, and normal-MSC–DCs was measured by ELISA. Data are expressed as mean ± SD of triplicates of six separate experiments. P ⩽ 0.05.
      In order to understand whether the immunoregulatory functions of MSCs will be altered with disease state, normal MSCs induced DCs (normal-MSC–DCs) are obtained and compared to CML–MSC–DCs. Our results showed that CML–MSC–DCs were similar to normal-MSC–DCs in phenotype, morphology and endocytic capacity. However, we found that CML–MSC–DCs spontaneously secreted TGF-β1 at higher levels than normal-MSC–DCs (Fig. 1C).

      3.2 CML–MSC–DCs induce T cell anergy

      To investigate the ability of CML–MSC–DCs to stimulate T lymphocyte proliferation, CD4+T cells were cocultured with MSC–DCs or LPS stimulated MSC–DCs or imDCs or LPS stimulated imDCs or mDCs. The results demonstrated that CML–MSC–DCs could hardly stimulate T-cell proliferation even when CML–MSC–DCs were stimulated by LPS. Although imDCs did not stimulate T-cell proliferation, after stimulated by LPS, imDCs could promote T-cell proliferation as that of mDCs. Similar to CML–MSC–DCs, normal-MSC–DCs also could not promote T-cell proliferation (Fig. 2A). Previous studies demonstrated that regulatory DCs possessed the capacity of inducing T cell anergy.
      • Schwartz R.H.
      T cell anergy.
      So, we examined the ability of allogeneic CML–MSC–DCs in the induction of T-cell anergy. As shown in Fig. 2B, we observed that the primed CD4+T cells with CML–MSC–DCs exhibited a weaker response to the allogeneic mDCs or PHA in a second culture than those primed with allogeneic mDCs. In addition, we found that imDCs possessed the function of inducing T cell anergy, but, mDCs or PHA could efficiently promote the proliferation of the primed CD4+T cells with LPS stimulated imDCs in a second culture, indicating imDCs failed to induce T cell anergy after stimulated by LPS. Moreover, we also observed that normal-MSC–DCs could induce T cell anergy. The function of inducing T cell anergy of normal-MSC–DCs was similar to that of CML–MSC–DCs, but stronger than that of imDCs. These results imply that CML–MSC–DCs may be a distinct subset of DCs with regulatory function.
      Figure thumbnail gr2
      Fig. 2CML–MSC–DCs are not immunogenic and can induce T-cell anergy. (A) CD4+T cells were cocultured with irradiated (15 Gy) allogeneic suppressive cells for 5 days in the presence of PHA. T-lymphocyte proliferation was assessed by [3H]-thymidine incorporation. Data are expressed as mean ± SD of triplicates of four separate experiments. P ⩽ 0.05. (B) Human naive CD4+T cells were cultured with allogeneic suppressive cells for 3 days. These CD4+T cells were then rescued, and cells were subsequently restimulated with irradiated (15 Gy) allogeneic mDCs or PHA in a second coculture. The proliferative response was measured on day 5. Data are expressed as mean ± SD of triplicates of five separate experiments. P ⩽ 0.05.

      3.3 Inhibitory effect of CML–MSC–DCs on T cell proliferation

      To explore the immune regulatory function of CML–MSC–DCs, we added CML–MSC–DCs into mDCs/lymphocytes or PHA/lymphocytes coculture system to see whether CML–MSC–DCs could inhibit lymphocyte proliferation. As shown in Fig. 3A, there was a significant reduction in T-cell proliferation when mixed culture of T lymphocytes stimulated by PHA or mDCs was performed in the presence of CML–MSC–DCs. Moreover, we found that normal-MSC–DCs could inhibit the proliferation of T-cell, but the immunosuppression rate of CML–MSC–DCs on T-cell proliferation was higher than that of normal-MSC–DCs (Fig. 3A).
      Because previous studies demonstrated that MSCs inhibition of T-lymphocyte proliferation was mediated by cell-to-cell contact or soluble factors,
      • Krampera M.
      • Glennie S.
      • Dyson J.
      • et al.
      Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.
      • Aggarwal S.
      • Pittenger M.F.
      Human mesenchymal stem cells modulate allogeneic immune cell responses.
      we next investigated T-lymphocyte proliferation in a transwell system in which T cells, plated in the lower chamber, were physically separated from CML–MSC–DCs or normal-MSC–DCs plated in the upper chamber. As shown in Fig. 3B, when CML–MSC–DCs or normal-MSC–DCs were separated from effectors by using the transwell system, a significant reduction in T-lymphocyte proliferation was observed. The rate of T-lymphocyte inhibition was lower than that observed when MSC–DCs were in direct contact with T-lymphocyte stimulated by PHA or mDCs. Moreover, we used the conditioned supernatant obtained from CML–MSC–DCs or normal-MSC–DCs cocultured and then passed through filter units of 0.22-μm pore size. In this case, the conditioned supernatant could suppress the proliferation of T cells proliferation stimulated by PHA or mDCs (data not shown). These results demonstrated that soluble factors were indeed involved in suppressive effects of MSC–DCs but not in a dependent manner, because the rate of T-lymphocyte inhibition was different between cell-to-cell contact system and transwall system, suggesting cell-to-cell contact was also involved in the regulatory function of MSC–DCs.
      Figure thumbnail gr3
      Fig. 3CML–MSC–DCs inhibit T-lymphocyte proliferation. (A) Irradiated (15 Gy) CML–MSC–DCs or normal-MSC–DCs were cultured for 5 days with CD4+T-lymphocyte in the presence of PHA or irradiated (15 Gy) allogeneic mDCs, then assessed by [3H]-thymidine incorporation. Data are expressed as mean ± SD of triplicates of five separate experiments. P ⩽ 0.05. (B) T-lymphocyte stimulated with PHA was cultured in the transwell plates to avoid cell–cell contact with irradiated (15 Gy) allogeneic CML–MSC–DCs or normal-MSC–DCs. As a control, irradiated (15 Gy) allogeneic CML–MSC–DCs or normal-MSC–DCs and T-lymphocyte were cocultured in non-transwell plates at the same ratio for the same time. Results are expressed as mean ± SD of triplicates of six separate experiments. P ⩽ 0.05. (C) anti-rhIL-10 mAb, or anti-rhIL-2 mAb, or anti-rhIFN-γ mAb, or 1-methyltryptophan (1 mM), indoleamine 2,3-dioxygenase (IDO) inhibitor, or indomethacin (40 μM), PGE2 inhibitor, or PBIT (40 μg), NO inhibitor was added at the beginning of MSC–DCs/mDCs/CD4 coculture. T-lymphocyte proliferation was assessed by [3H]-thymidine incorporation. Data are expressed as mean ± SD of triplicates of five separate experiments. P ⩽ 0.05. (D) anti-rhTGF-β1 mAb was added at the beginning of MSC–DCs/mDCs/CD4 coculture. T-lymphocyte proliferation was assessed by [3H]-thymidine incorporation. P ⩽ 0.05.
      To investigate inhibitory cytokines potentially suppressing T-cell proliferation, monoclonal antibodies neutralising distinct cytokines that were known to be produced by MSC–DCs were evaluated for their capacity to restore T-lymphocyte proliferation. Our results demonstrated that anti-rhIL-10, or anti-rhIL-2 or anti-rhIFN-γ failed to restore T-lymphocyte proliferation suppressed by CML–MSC–DCs or normal-MSC–DCs. It had been reported that prostaglandin E2 (PGE2), NO and indoleamine 2,3-dioxygenase (IDO; a tryptophan-catabolising enzyme) could inhibit T-cell proliferation,
      • Santoli D.
      • Phillips P.D.
      • Colt T.L.
      • Zurier R.B.
      Suppression of interleukin 2-dependent human T cell growth in vitro by prostaglandin E (PGE) and their precursor fatty acids. Evidence for a PGE-independent mechanism of inhibition by the fatty acids.
      • Munn D.H.
      • Sharma M.D.
      • Lee J.R.
      • et al.
      Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase.
      • Meisel R.
      • Zibert A.
      • Laryea M.
      • et al.
      Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation.
      • Sato K.
      • Ozaki K.
      • Oh I.
      • et al.
      Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells.
      so we added indomethacin, an inhibitor of PGE2 biosynthesis, or 1,4 PBIT, an inhibitor of iNOS or 1-methyltryptophan, a specific IDO inhibitor, into the coculture system. But, we found that the inhibitory effects of MSC–DCs were not substantially reversed (Fig. 3C).
      The high levels of TGF-β1 secreted by CML–MSC–DCs or normal-MSC–DCs suggest that TGF-β1 may be involved in the inhibition of T cell proliferation. We added series of concentrations of anti-rhTGF-β1 into MRL, and found that T-lymphocyte proliferation suppressed by CML–MSC–DCs or normal-MSC–DCs could be significantly restored by anti-rh TGF-β1 (⩾0.5 μg/mL) (Fig. 3D).

      3.4 CML–MSC–DCs generate CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells

      CD4+CD25+Foxp3+Tregs have been shown to critically regulate self- and allograft tolerance in several model systems.
      • Sakaguchi S.
      Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self.
      Previous studies had reported that different subsets of DCs could expand CD4+CD25+Foxp3+Tregs cells in vivo and in vitro.
      • Sato K.
      • Yamashita N.
      • Yamashita N.
      • Baba M.
      • Matsuyama T.
      Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse.
      • Sato K.
      • Yamashita N.
      • Baba M.
      • Matsuyama T.
      Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells.
      In this study, we cocultured CML–MSC–DCs or normal-MSC–DCs with CD4+CD25+T cells, and found that CML–MSC–DCs or normal-MSC–DCs could not expand CD4+CD25+T cells in vitro. Although mDCs could significantly expand CD4+CD25+T cells, the number of CD4+CD25+Foxp3+Tregs had no change, suggesting that mDCs could not expand or generate CD4+CD25+Foxp3+Tregs. Then, we wondered whether CML–MSC–DCs could generate CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells, we cocultured CML–MSC–DCs with CD4+CD25−T cells. The results showed that CML–MSC–DCs could efficiently generate CD4+CD25+Foxp3+Tregs from CD4+CD25−T cells, whereas mDCs failed to induce CD4+CD25+Foxp3+Tregs from CD4+CD25−T cells. Compared to CML–MSC–DCs, our results demonstrated that normal-MSC–DCs could generate CD4+CD25+Foxp3+Tregs from CD4+CD25−T cells, the inducible rate was lower than that of CML–MSC–DCs (Fig. 4A). In addition, our results demonstrated that CML–MSC–DCs induced CD4+CD25+Foxp3+Tregs or normal-MSC–DCs induced CD4+CD25+Foxp3+Tregs were able to inhibit T-cell proliferation. Moreover, we found that the addition of CML–MSC–DCs induced CD4+CD25+Foxp3+Tregs or normal-MSC–DCs induced CD4+CD25+Foxp3+Tregs to T-lymphocyte stimulated with PHA suppressed the mitogenic response in a dose-dependent fashion (Fig. 4B).
      Figure thumbnail gr4
      Fig. 4CML–MSC–DCs generate CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells. (A) CD4+CD25−T cells (5 × 106) were cultured with mDCs or CML–MSC–DCs (5 × 105) or normal-MSC–DCs (5 × 105) for 5 days, and CD4+T cells were collected. The expression of CD25 and Foxp3 on CD4+T cells was analysed by FACS. Results are expressed as mean ± SD of triplicates of four separate experiments. P ⩽ 0.05. (B) CD4+T cells were cocultured with CML–MSC–DCs generated CD4+CD25+T cell or normal-MSC–DCs generated CD4+CD25+T cell or mDCs generated CD4+CD25+T cell in the presence of PHA, and the T-lymphocyte proliferation was measured on day 5 by [3H]-thymidine incorporation. Results are expressed as mean ± SD of triplicates of six separate experiments. P ⩽ 0.05.

      3.5 Role of TGF-β1 in the generation of CD4+CD25+Foxp3+Tregs

      As shown in Fig. 1C, high levels of TGF-β1, which were shown to be important in the induction of tolerance and Tregs,
      • Peng Y.
      • Laouar Y.
      • Li M.O.
      • Green E.A.
      • Flavell R.A.
      TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes.
      • Chen W.
      • Jin W.
      • Hardegen N.
      • et al.
      Conversion of peripheral CD4+CD25-naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.
      • Yamazaki S.
      • Bonito A.J.
      • Spisek R.
      • et al.
      Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+CD4+ regulatory T cells from peripheral Foxp3 precursors.
      were secreted by CML–MSC–DCs or normal-MSC–DCs. To investigate whether CML–MSC–DCs or normal-MSC–DCs derived TGF-β1 could induce CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells, we performed knockdown of TGF-β1 in CML–MSC–DCs or normal-MSC–DCs before adding them to cocultures. Knockdown CML–MSC–DCs or normal-MSC–DCs were studied for TGF-β1 production. Western blot showed nearly undetectable TGF-β1 in TGF-β1siRNA-transfected CML–MSC–DCs or normal-MSC–DCs (Fig. 5A). In addition, our results demonstrated that in the absence of TGF-β1 in CML–MSC–DCs or normal-MSC–DCs, there were no significant differences in the generation of CD4+CD25+Foxp3+Tregs (4.8 ± 0.4% for T cell alone, 5.6 ± 0.5% for CML–MSC–DCs, and 6.4 ± 0.6% for normal-MSC–DCs) (Fig. 5B). These results contrast with studies using untransfected CML–MSC–DCs or normal-MSC–DCs (Fig. 4A) or mutant siRNA, which showed a significant increase in the generation of CD4+CD25+Foxp3+Tregs (4.6 ± 0.5% for T cell alone, 15.7 ± 1.5% for CML–MSC–DCs, and 10.4 ± 0.9% for normal-MSC–DCs) (Fig. 5C). Moreover, anti-rhTGF-β1 was added in cocultures of CD4+CD25−T cells and untransfected CML–MSC–DCs or normal-MSC–DCs. We found that there were no significant differences in the generation of CD4+CD25+Foxp3+Tregs (5.1 ± 0.4% for T cell alone, 5.7 ± 0.6% for CML–MSC–DCs, and 6.4 ± 0.8% for normal-MSC–DCs) (Fig. 5D). Together, the results indicate a significant role for TGF-β1 secreted by CML–MSC–DCs or normal-MSC–DCs in the generation of CD4+CD25+Foxp3+Tregs.
      Figure thumbnail gr5
      Fig. 5Role of TGF-β1 in the generation of CD4+CD25+Foxp3+Tregs. (A) Western blot confirmed efficient knockdown of TGF-β1. (B) CD4+CD25−T cells (5 × 106) were cultured with TGF-β1 knockdown CML–MSC–DCs (5 × 105) or normal-MSC–DCs (5 × 105) for 5 days, and CD4+T cells were collected. The expression of CD25 and Foxp3 on CD4+T cells was analysed by FACS. Results are expressed as mean ± SD of triplicates of four separate experiments. P ⩽ 0.05. (C) CD4+CD25−T cells (5 × 106) were cultured with mutant siRNA transfected CML–MSC–DCs (5 × 105) or normal-MSC–DCs (5 × 105) for 5 days, and CD4+T cells were collected. The expression of CD25 and Foxp3 on CD4+T cells was analysed by FACS. Results are expressed as mean ± SD of triplicates of four separate experiments. P ⩽ 0.05. (D) anti-rhTGF-β1 mAb was added at the beginning of coculture of CD4+CD25−T cells (5 × 106) and untransfected CML–MSC–DCs (5 × 105) or normal-MSC–DCs (5 × 105) for 5 days, and CD4+T cells were collected. The expression of CD25 and Foxp3 on CD4+T cells was analysed by FACS. Results are expressed as mean ± SD of triplicates of five separate experiments. P ⩽ 0.05.

      4. Discussion

      In this study, we demonstrated that CML–MSC could induce mDCs into a new subset of DCs that were characterised by high expression of CD11b and low expression of CD40, CD80, CD83 and CD86. This phenotype remained unchanged when these cells were stimulated by LPS, indicating a stable regulatory DCs phenotype for these cells. Further research displayed that CML–MSC–DCs secreted less IL-12, but more TGF-β1 and IL-10, a characteristic of regulatory DCs population.
      • Sato K.
      • Yamashita N.
      • Baba M.
      • Matsuyama T.
      Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells.
      Similar to the regulatory DCs reported by others, our induced DCs displayed powerful phagocytic capacity. In addition, our results showed that CML–MSC–DCs had the capacity to induce T cell anergy, another capacity of regulatory DCs. Moreover, CML–MSC–DCs could inhibit the proliferation of T cells stimulated by mDCs or PHA. These results showed that CML–MSC–DCs were a novel DC population with low immunogenicity and high immunoregulatory potential, and we referred to CML–MSC–DCs as regulatory DCs.
      The regulatory DCs reported until now have similar functions but different phenotypes and immunoregulatory mechanisms. Tang et al. found that the inhibition of T-lymphocyte proliferation of endothelial splenic stroma cells induced regulatory DCs was mediated by soluble factor of NO.
      • Tang H.
      • Guo Z.H.
      • Zhang M.H.
      • et al.
      Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10.
      However, Zhang et al. demonstrated that mouse MSCs induced DCs might exert their inhibitory effects through direct contact between cells rather than by soluble factors.
      • Zhang B.
      • Liu R.
      • Shi D.
      • et al.
      Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.
      TGF-β1 was one of important immunoregulatory cytokines in regulation of T cell mediated immune responses. Our results found that CML–MSC–DCs secreted high levels of TGF-β1, suggesting that TGF-β1 might be involved in the inhibition of T cell proliferation. Our results showed that T-lymphocyte proliferation suppressed by CML–MSC–DCs could not be restored by low dose of anti-rhTGF-β1, but high dose of anti-rhTGF-β1 (⩾0.5 μg/mL) could efficiently restore the inhibitation of T-lymphocyte proliferation, indicating the inhibitory effects of CML–MSC–DCs were mediated by soluble factor of TGF-β1. Therefore, we thought that the high level of TGF-β1 secreted by CML–MSC–DCs could explain, at least in part, the increase in the function of inhibition of T-lymphocyte proliferation. Moreover, our results also found that soluble factors were not the only mechanism of inhibitory function of CML–MSC–DCs, because the rate of T-lymphocyte inhibition was lower in transwell system than that observed in cell-to-cell contact system. So, we suggested that other mechanisms also got be involved in the regulatory function of CML–MSC–DCs.
      The induction of tolerance is critical for the maintenance of immune homeostasis.
      • Steinman R.M.
      • Nussenzweig M.C.
      Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance.
      The mechanisms of tolerance include clonal deletion and anergy as well as active suppression by regulatory T cells, such as CD4+CD25+Foxp3+Tregs and inducible interleukin-10 producing Tr1 cells.
      • Weaver C.T.
      • Harrington L.E.
      • Mangan P.R.
      • Gavrieli M.
      • Murphy K.M.
      Th17: an effector CD4 T cell lineage with regulatory T cell ties.
      • Morelli A.E.
      • Thomson A.W.
      Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction.
      In this study, firstly, our results demonstrated that CML–MSC–DCs had the capacity to induce T cell anergy. Secondly, CD4+CD25+Foxp3+Tregs have been shown to critically regulate self- and allograft tolerance in several model systems. Our results found that CML–MSC–DCs could efficiently generate CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells. Thirdly, CD4+CD25+Foxp3+Tregs induced by CML–MSC–DCs could efficiently inhibit the proliferation of T cell, suggesting that CD4+CD25+Foxp3+Tregs induced by CML–MSC–DCs were involved in the regulatory function of CML–MSC–DCs. Taken together, our results demonstrated that CML–MSC–DCs could efficiently inhibit the proliferation of T-lymphocyte not only through TGF-β1, but also by inducing the production of Treg cells or T-cell anergy.
      Moreover, we examined whether the immunoregulatory functions of MSCs are altered in disease states. Our results showed that MSCs derived from normal adult could differentiate mDCs into a distinct regulatory DC population (normal-MSC–DCs). Normal-MSC–DCs were similar to CML–MSC–DCs in phenotype, morphology and endocytic capacity. In addition, similar to normal-MSC–DCs induced CD4+CD25+Foxp3+Tregs, CML–MSC–DCs induced CD4+CD25+Foxp3+Tregs could efficiently inhibit T-lymphocyte proliferation in a dose-dependent manner. But, there were several differences between CML–MSC–DCs and normal-MSC–DCs as follows: first, we found that CML–MSC–DCs spontaneously secreted TGF-β1 at higher levels than normal-MSC–DCs; second, although normal-MSC–DCs could efficiently inhabit the proliferation of T-lymphocyte though TGF-β1, the immunosuppression rate of CML–MSC–DCs on T-cell proliferation was higher than that of normal-MSC–DCs; at last, normal-MSC–DCs could generate CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells, but the inducible rate was lower than that of CML–MSC–DCs.
      TGF-β is a critical factor in the regulation of T cell–mediated immune responses and in the induction of immune tolerance.
      • Gorelik L.
      • Flavell R.A.
      Transforming growth factor-beta in T-cell biology.
      • Chen W.
      • Wahl S.M.
      TGF-beta: receptors, signaling pathways and autoimmunity.
      It has been reported that TGF-β1 has been linked to the expansion of Tregs.
      • Peng Y.
      • Laouar Y.
      • Li M.O.
      • Green E.A.
      • Flavell R.A.
      TGF-beta regulates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsible for protection against diabetes.
      • Chen W.
      • Jin W.
      • Hardegen N.
      • et al.
      Conversion of peripheral CD4+CD25-naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3.
      • Yamazaki S.
      • Bonito A.J.
      • Spisek R.
      • et al.
      Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+CD4+ regulatory T cells from peripheral Foxp3 precursors.
      Patel et al. demonstrated that MSCs derived TGF-β1 could expand CD4+CD25+Foxp3+Tregs.
      • Patel S.A.
      • Meyer J.R.
      • Greco S.J.
      • et al.
      Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta.
      In addition, Yamazaki et al. found that DCs from the intestinal environment were able to differentiate Foxp3+Tregs through TGF-β.
      • Yamazaki S.
      • Bonito A.J.
      • Spisek R.
      • et al.
      Dendritic cells are specialized accessory cells along with TGF- for the differentiation of Foxp3+CD4+ regulatory T cells from peripheral Foxp3 precursors.
      Consistent with this, our results showed that T-lymphocyte proliferation suppressed by CML–MSC–DCs or normal-MSC–DCs could be restored by high dose of anti-rhTGF-β1 (⩾0.5 μg/mL), indicating the inhibitory effects of CML–MSC–DCs or normal-MSC–DCs were mediated by soluble factor of TGF-β1. Moreover, our results also demonstrated that MSC–DCs derived TGF-β1 was largely responsible for the increase in CD4+CD25+Foxp3+Tregs based on knockdown studies.
      Abundant studies demonstrated that MSCs could effectively inhibit T-lymphocyte proliferation induced by allogeneic cells or mitogens in vitro.
      • Krampera M.
      • Glennie S.
      • Dyson J.
      • et al.
      Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide.
      • Aggarwal S.
      • Pittenger M.F.
      Human mesenchymal stem cells modulate allogeneic immune cell responses.
      • Beyth S.
      • Borovsky Z.
      • Mevorach D.
      • et al.
      Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness.
      • Le Blanc K.
      • Tammik L.
      • Sundberg B.
      • Haynesworth S.E.
      • Ringdén O.
      Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.
      • Gieseke F.
      • Böhringer J.
      • Bussolari R.
      • et al.
      Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells.
      Furthermore, infusion of allogeneic MSCs into baboons was well tolerated in most animals and prolonged the survival of allogeneic skin graft.
      • Bartholomew A.
      • Sturgeon C.
      • Siatskas M.
      • et al.
      Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo.
      Based on this, human MSCs have been administered in vivo to improve the outcome of allogeneic transplantation by promoting haematopoietic engraftment and to hamper GVHD. Lazarus et al. and others reported that autologous MSCs, derived from haematologic malignancies bone marrow, were infused intravenously into those patients, and could reduce acute and chronic GVHD without toxicity.
      • Lazarus H.M.
      • Haynesworth S.E.
      • Gerson S.L.
      • Rosenthal N.S.
      • Caplan A.I.
      Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use.
      • Lazarus H.
      • Curtin P.
      • Devine S.
      • et al.
      Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.
      • Weng J.Y.
      • Du X.
      • Geng S.X.
      • et al.
      Mesenchymal stem cell as salvage treatment for refractory chronic GVHD.
      Le Blanc et al. demonstrated that allo-MSCs transplantation could treat patients with severe treatment-resistant grace IV acute GVHD.
      • Le Blanc K.
      • Frassoni F.
      • Ball L.
      • et al.
      Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study.
      • Le Blanc K.
      • Rasmusson I.
      • Sundberg B.
      • et al.
      Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.
      Moreover, our previous findings demonstrated that CML–MSC were similar to normal adult derived MSCs in phenotype, morphology and multi-differentiation capacity, CML–MSC possessed the haematopoiesis support function and immunoregulatory effects.
      • Zhao Z.G.
      • Tang X.Q.
      • You Y.
      • et al.
      Assessment of bone marrow mesenchymal stem cell biological characteristics and support hemotopoiesis function in patients with chronic myeloid leukemia.
      • Zhao Z.G.
      • Li W.M.
      • Chen Z.C.
      • Zou P.
      Immunosuppressive properties of mesenchymal stem cells derived from bone marrow of patients with chronic myeloid leukemia.
      In this study, CML–MSC could differentiate mDCs into a distinct regulatory DC population. CML–MSC–DCs could generate more CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells than that of normal-MSC–DCs. Both CML–MSC–DCs and CML–MSC–DCs induced CD4+CD25+Foxp3+Tregs could inhibit the proliferation of T cells. Taken together, our data suggested that CML–MSC could be preferentially used in the control of GVHD in future clinical trials.
      To conclude, we investigate for the first time the characteristics and immunoregulatory functions of one kind of regulatory DCs induced by CML–MSC. In addition, our results show that CML–MSC–DCs have the capacity to induce T cell anergy. Moreover, CML–MSC–DCs can efficiently inhibit the proliferation of T-lymphocytes not only through TGF-β1, but also by inducing the production of Treg cells or T-cell anergy. CML–MSC–DCs can generate more CD4+CD25+Foxp3+Tregs from CD4+CD25−Foxp3−T cells than that of normal-MSC–DCs though TGF-β1.

      Conflict of interest statement

      All authors declare that we have no conflict of interest.

      Acknowledgments

      The authors thank all staff of the Department of haematology of the Oncology hospital and the Union hospital for the donation and collection of bone marrow samples. This work is supported by grants from National Natural Science Foundation (No. 30801051).

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