Mesenchymal Stem Cells Inhibit the Differentiation of CD4+ T Cells into Interleukin-17-Secreting T Cells

Mesenchymal stem cells (MSCs) are a subset of stem cells which reside principally in bone marrow and can be isolated from a wide variety of adult and fetal tissues. MSCs can be differentiated into multiple mesodermal tissues, including osteoblasts, chondrocytes and adipocytes, providing promising cellular resources for the repair of tissues. In addition to their differentiation potential, MSCs have been reported to exhibit immunoregulatory functions via the suppression of T cells, B cells, NK cells and dendritic cell functions [1, 2]. As MSCs evidence such unique immunoregulatory properties, it may be possible to employ MSCs as therapeutic tools for immune-mediated diseases. The immunomodulatory effect of MSCs on T cells is involved in the inhibition of proliferation and cytokine production, as well as the enhancement of MSC immunosuppressive activity via T cell-derived cytokines. T helper 17 (Th17) cells were identified as a new arm of an effector T cell lineage which can be induced by transforming growth factor (TGF)-β and interleukin (IL)-6 stimulation and can generate IL-17 [3]. TGF-β and IL-6 induce Th17 cells, and IL-23 may be crucial for the maintenance of Th17 pools in chronic inflammation. Although IL-17 may be involved in the recruitment of neutrophils, eradicating extracellular bacteria and chronic inflammation, it has also been reported that IL-17A enhanced the proliferation of MSCs [4]. From these fundamental facts, we hypothesized that MSCs might exert an effect on the differentiation of Th17 cells and might possibly be one of the immunoregulatory mechanisms exploited by MSCs.Six- to eight-week-old C57BL/6 female mice were killed by cervical dislocation and limbs were removed. The bone marrow was flushed from the medullary cavities of both the femurs and tibias with serum-free RPMI 1640 (Gibco BRL, Carlsbad, Calif., USA) medium using a 25-gauge needle, filtrated through Nylon meshes and centrifuged for 5 min at 1,200 rpm. Isolated bone marrow cells were then incubated in RBC lysis solution (0.15 M NH4Cl, 10 mM NaHCO3, 10 mM EDTA) and washed twice with phosphate-buffered saline. The cells were then plated at 1 × 107 cells/100-mm culture dishes in Iscove’s modified Dulbecco’s medium (Sigma, St. Louis, Mo., USA) with 10% heat-inactivated fetal bovine serum. After 48 h, nonadherent cells were removed via aspiration and the medium, consisting of MesenCult™ basal medium and 10% MSC stimulatory supplement (Stemcell Technologies, Vancouver, Canada), was replenished. We cultured plastic adherent cells for more than 3 weeks in MSC medium. After 14 days, colony-forming units of fibroblasts were observed and confirmed after Giemsa staining (fig. 1). For adipogenic differentiation, MSCs were cultured for 3 weeks in MesenCult basal medium containing 10% adipogenic stimulatory supplement (Stemcell Technologies). Total CD4+ cells were prepared from the lymph nodes and spleen (purity >98%, 3 × 106 cells/mouse) using magnetic beads (clone L3T4, Miltenyi Biotec, Germany). Antigen-presenting cells (APCs) were prepared by plastic adherence from a collagenase-digested splenocyte suspension. The isolated CD4+ cells (105 cells/well) were cocultured with APCs (105 cells/well) in 200 µl/well complete medium plus 1 µg/ml anti-CD3 antibody (clone 145-2C11, R&D Systems, Minneapolis, Minn., USA), 2 ng/ml TGF-β (R&D Systems) and 10 ng/ml IL-6 (R&D Systems). Afterward, 5 × 104 MSCs were added and cultured for 3 days. The differentiation of Th17 cells was determined via intracellular staining using phycoerythrin-conjugated anti-mouse IL-17 (TC11-18H10.1, Biolegend, San Diego, Calif., USA) and allophycocyanin-conjugated anti-mouse interferon (IFN)-γ (XMG1.2, Biolegend) antibodies, and the Th17 cells were identified as IL-17+/IFN-γ–/CD4+ cells via flow cytometry analysis [5]. Anti-mouse CD4 antibody (GK1.5) was from BD Pharmingen (San Jose, Calif., USA). The percentage of CD4+/IL-17+ cells was 1.55 ± 0.43 (mean ± SD) in CD4+ T cells only, 9.87 ± 0.64 in APC-cocultured cells and 1.90 ± 0.27 in APC + MSC-cocultured CD4+ T cells (n = 6 per group). The percentage of IFN-γ–/ IL-17+ cells was 4.23 ± 1.71 in CD4+ T cells only, 16.51 ± 2.27 in APC-cocultured cells and 6.88 ± 1.79 in APC + MSC-cocultured CD4+ T cells (n = 6 per group). APC + MSC-cocultured CD4+ T cells showed a significantly decreased percentage of CD4+/IL-17+ cells as well as IFN-γ–/IL-17+ cells compared with APC-cocultured groups (p < 0.05 by Student’s t test).It has been reported that Th17 cells are profoundly proinflammatory and that Th17 cells specific for self-antigens might possibly induce autoimmune responses in animal models [6]. In addition, IL-17 expression was detected in human tissues of multiple sclerosis, rheumatoid arthritis and psoriasis [7]. In this study, however, we determined that MSCs inhibited the induction of CD4+ T cells into IL-17-secreting cells (fig. 2). This result indicates that the additional immunoregulatory mechanisms of MSCs might inhibit inflammation via the suppression of CD4+ T cell differentiation into Th17 cells.This work was supported by a Korea Research Foundation Grant funded by the Korean Government (KRF-2007-531-E00061, KOEHRD, Basic Research Promotion Fund).

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