Galectin-1 and Semaphorin-3A Are Two Soluble Factors Conferring T-Cell Immunosuppression to Bone Marrow Mesenchymal Stem Cell

Introduction

B one marrow (BM) mesenchymal stem cells (MSCs) are multipotent progenitors able to differentiate into various mesenchymal cell subtypes in vitro and in vivo [1]. MSCs do not express hematopoietic or endothelial specific surface markers (CD31, CD34, and CD45). MSCs are usually identified by the expression of adhesion molecules (CD29, CD44, CD73, CD105, CD106, and CD166) [2]. Moreover, MSCs have been previously described to be involved in hematopoiesis through extracellular matrix proteins expression and cytokines secretion [3]. Furthermore, MSCs promote cell–cell interactions through the expression of adhesion molecules [3].

Several studies reported the immunosuppressive properties of MSCs in vitro and in vivo. Recently, MSCs have been shown to exert suppressive effect on T-cell proliferation [4,5], on NK cell activities [6,7], on B-cell functions [8,9], and on dendritic cell differentiation and maturation [10]. While this immunosuppression is well established, the mechanism of action is still unclear and controversial. Several studies have previously demonstrated the implication in this process of cell–cell contacts and soluble factors such as cytokines (TGF-b, IL-10), nitric oxide, or PGE2/IDO [11]. Nevertheless, the precise mechanism and the factors which govern these MSCs immune regulatory functions remain largely elusive and are still a matter of debate.

A recent study has shed light on the constitutive presence of Galectin-1 in MSCs [12]; however, its function in this model is elusive. Galectin-1 is a homodimeric galactose-binding lectin with a single carbohydrate-recognition domain that is able to selectively bind neuropilin-1 (NP-1) [13]. NP-1, a neuronal receptor, is constitutively expressed on T-cell surface [14,15] and is also involved in the regulation of T-cell proliferation through its main ligand Semaphorin-3A (Sema-3A) [16] that arrests T cells in G0/G1 cell cycle phase [17]. Given the immunomodulatory properties of both Galectin-1 and Sema-3A, we have tested the probable impact of NP-1 ligands in MSCs functions.

Here, we show that both Galectin-1 and Sema-3A are highly expressed and secreted by MSCs. Moreover, addition of both recombinant immunosuppressive factors (Galectin-1 or Sema-3A) inhibits T-cell proliferation. Furthermore, anti-Sema-3A or anti-Galectin-1-blocking antibodies and recombinant NP-1 neutralized MSCs immunosuppressive properties on T cells. Therefore, Galectin-1 and Sema-3A seem to be crucial immunoregulatory proteins involved in MSCs functions.

Materials and Methods

Results and Discussion

In this study, we have investigated the mechanisms involved in the immunosuppressive functions of MSCs. In accordance with previous report [19], BM MSCs used here did not express hematopoietic antigens, such as CD14, CD34, and CD45, or endothelial markers like CD31. Expanded MSCs expressed CD13, CD73, CD90, CD29 (b1-integrin), CD44 and CD166 (adhesion molecules), and CD105 (endoglin receptor) antigens (Fig. 1A). Furthermore, MSCs differentiated into adipogenic (defined by the accumulation of lipid-rich vacuoles) and osteogenic (characterized by ALP activity and deposition of a calcium-rich mineralized extracellular matrix) lineages [1] (Fig. 1B and 1C, respectively).

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FIG. 1.

Human bone marrow (BM) mesenchymal stem cells (MSCs) immunosuppression depends on soluble neuronal receptor (NP)-1 ligands. (A) Immunophenotyping by flow cytometry analysis. Cells were labeled with FITC or PE-coupled specific antibodies (red line) or isotype control antibodies (green line) (B and C) In vitro adipogenic and osteogenic differentiations. MSCs differentiate into adipocytes characterized by lipid vacuoles (phase contrast 200×) (B) and in osteocytes characterized by Alizarin red staining (upper left: in the well, right: 400×) and ALP activity (bottom left: in the well, right: 400×) (C). (D) MSCs block peripheral blood mononuclear cell (PBMCs) proliferation. PBMCs were cultured in the presence of increasing number of MSCs at ratio ranging from 1/1,000 to 1/5. (E) TGF-b do not control MSCs function. TGF-b-blocking antibodies (10–40 μg/mL) were used to neutralize the immunosuppressive activity of MSCs on PBMCs. (F) Western blot (WB) and immunoprecipitation (IP) analysis of Sema-3A and Galectin-1 expression and secretion by MSCs. (G) Neuropilin-1 expression on cultured MSCs and PBMC. (H) Galectin-1 and Sema-3A are efficient to neutralized T-cell proliferation. (I–K) Neutralization of Sema-3A and Galectin-1 restores PBMCs proliferation. PBMCs and MSCs were cultured (ratio 1/5) in the presence of increasing concentration of Sema-3A (I) or Galectin-1 (J) mAb (0–40 μg/mL) or recombinant Fc-NP-1 (0–5 μg/mL) (K). PBMCs proliferation was evaluated by ³H-thymidine incorporation. Data are means ± SD of triplicates from 1 representative of at least 12 independent experiments. P values from Student's paired t-test are indicated (not significant: NS, *P < 0.5, ^** P < 0.01, ^*** P < 0.001).

As MSCs from BM, synovium, or adipose tissues are able to inhibit T-cell proliferation in a mixed-lymphocyte reaction, we tested their immunosuppressive activity on allogenic T-cell proliferation at different MSCs:T cells ratios (Fig. 1D), in accordance with the literature [5,20 –22]. The immunosuppression of T-cell proliferation by MSCs was more efficient at higher MSCs:T cells ratio (Fig. 1D). Many studies have pointed out the role of soluble factors such as TGF-b1 in the mechanism by which MSCs inhibit T-cell proliferation [5]. Consistently with previous reports [23], in our hands, no significant reversion of MSCs immunosuppressive effect was observed on T-cell proliferation in the presence of TGF-b1-blocking antibodies (Fig. 1E), while MSCs secreted TGF-b1 (1,787 ± 652 pg/mL). Recent work also pointed to the presence and the potential role of Galectin-1 in MSCs functions [12]. However, no experimental results have proved yet this concept. Since Galectin-1 as well as Sema-3A [15,16] are both immunosuppressive proteins acting through NP-1 [13] expressed on T cells, we investigated whether these soluble factors were involved in the MSCs immunosuppressive properties. We first demonstrated the expression of both molecules and their receptor neuropilin-1 in MSCs (Fig. 1F and 1G, respectively). We next assessed whether Sema-3A and Galectin-1 were directly involved in T-cell immunosuppression mediated by MSCs, since both ligands are capable to specifically inhibit T-cell proliferation (Fig. 1H). To this aim, MSCs and PBMCs were co-cultured in the presence of neutralizing Galectin-1 or Sema-3A antibodies. The blockade of Sema-3A and Galectin-1 secreted by MSCs restored the proliferation of PBMCs in a dose-dependent manner (Fig. 1I and 1J, respectively). To reinforce this result, we also used recombinant soluble NP-1 that bound and blocked both Sema-3A and Galectin-1 proteins, allowing the MSCs immunosuppressive activity neutralization (Fig. 1K). Taken together, these results demonstrated the implication of both Galectin-1 and Sema-3A in the immunosuppressive properties of MSCs.

Sema-3A is a member of a large protein family, which is phylogenetically conserved secreted protein originally implicated in axon guidance [24]. These proteins have been extensively studied in the nervous system, but accumulated evidence showed that they have important functions on nonneuronal cell system including angiogenesis and tumor biology. More recently, we and others have shown that they are also involved in the complex system of interactions between epithelial cells and thymocytes within the thymus as well as between T and DC cells in the periphery [14,15,25]. More consistently, Galectin-1 is overexpressed in regulatory T cells and its neutralization significantly reduced the inhibitory effects of human and mouse CD4⁺CD25⁺ T regulatory cells [26]. These results suggest that Galectin-1 is a key effector of the regulation mediated by immunosuppressive cells. Here, we show for the first time that MSCs also expressed another immunosuppressive protein, Sema-3A. Taken together, expression of both Sema-3A and Galectin-1 by MSCs may explain their immunosuppressive activities, in accordance with MSCs molecular signature [27]. Finally, these results also provided new insights in the physiopathology and treatment of various immune and neoplastic disorders.