Immunomodulatory properties and therapeutic application of mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) are multi-potent progenitor cells that are isolated from the bone marrow and several adult organs and tissues. These cells possess remarkable immunosuppressive properties and can inhibit the proliferation and function of the major immune cell populations, including T cells, B cells and natural killer (NK) cells; modulate the activities of dendritic cells (DCs); and induce regulatory T cells both in vivo and in vitro. These unique properties make MSCs ideal candidates for clinical application as immunosuppressants. The immunomodulatory effect of MSCs is mediated by a non-specific anti-proliferative action of these cells, which is dependent on cell–cell contact or secreted soluble factors such as indoleamine 2,3-dioxygenase (IDO), prostaglandin E₂ (PGE₂), nitric oxide (NO), histocompatibility leucocyte antigen-G (HLA-G), transforming growth factor (TGF)-β, interferon (IFN)-γ and interleukin (IL)-1β. Considerable progress has been obtained in preclinical studies on MSCs, including those on their ability to activate allogeneic cells. This review examines the current understanding of the immunomodulatory properties of MSCs and its therapeutic implication for immune-mediated diseases and transplant rejection.

Introduction

Mesenchymal stem cells (MSCs) are multi-potent progenitors constituting a small proportion of the bone marrow and are present in both adult and fetal tissues [1,2], including adipose tissue, umbilical cord blood, amniotic fluid and fetal lung [3–6]. They were first characterized by Friedenstein and colleagues more than 30 years ago, and were described as fibroblast-like cells with the property of adhering to plastic when cultured [7,8]. This property can be used to purify these cells and enables them to expand several-fold in vitro without losing their differentiation capacity. MSCs are able to differentiate into both mesenchymal and non-mesenchymal cell lineages such as adipocytes, osteoblasts, chondrocytes, tenocytes, skeletal myocytes, neurones and cells of the visceral mesoderm, both in vitro and in vivo[9,10].

Because a specific marker for human MSCs has not been identified, it is difficult to recognize these cells. The International Society for Cellular Therapy has recommended the following minimum criteria for defining multi-potent human MSCs [11,12]: (i) adherence to plastic under standard culture conditions; (ii) positive for expression of CD105, CD73 and CD90 and negative for expression of the haematopoietic cell surface markers CD34, CD45, CD11a, CD19 or CD79a, CD14 or CD11b and histocompatibility locus antigen (HLA)-DR; and (iii) under a specific stimulus, differentiation into osteocytes, adipocytes and chondrocytes in vitro. Because of their unique regenerative potential, MSCs exhibit potential for use in tissue regeneration and repair for conditions such as cardiac anomalies or injury, bone disorders and metabolic diseases. One of the most intriguing features of MSCs is that they escape immune recognition and can inhibit immune responses [13].

In this review, we discuss the in vivo and in vitro immunomodulatory properties of MSCs, the possible mechanisms underlying the expression of these properties and the potential clinical use of MSCs in vivo as modulators of immune responses.

MSCs modulate T cell proliferation and function

Numerous studies have demonstrated that MSCs can suppress the T lymphocyte proliferation induced by alloantigens, mitogens and anti-CD3 and anti-CD28 antibodies in vitro in humans, baboons and mice [14–20]. MSCs have a similar effect on memory and naive T cells [20], as well as CD4⁺ and CD8⁺ T cells [21], of a murine model. In addition, this suppressive effect did not require major histocompatibility complex (MHC) restriction and could also be mediated by allogeneic MSCs [15,20]. This effect may be attributed to the inhibition of cell division, which is evidenced by the accumulation of cells in the G0/G1 phase of the cell cycle [21]. At the molecular level, cyclin D2 expression is down-regulated, whereas p27 expression is up-regulated; this may explain why T cell proliferation, rather than activation, and interferon (IFN)-γ production are affected by MSCs [21]. Inhibition of T cell proliferation by MSCs appears to be mediated by both cell–cell interaction [17,22,23] and release of soluble factors such as IFN-γ and interleukin (IL)-1β[24,25]. Some studies have indicated that soluble factors are essential for enhancing the suppressive effect of human MSCs, while the effect of rodent MSCs is mediated by cell–cell contact [14,17,20,26]. Transforming growth factor (TGF)-β1, hepatocyte growth factor (HGF) [14], indoleamine 2,3-dioxygenase (IDO) [27] and prostaglandin E₂(PGE₂) [28] represent MSC-derived molecules that are believed to have immunomodulatory activity on T cell responses. Neutralizing antibodies against TGF-β and HGF can restore the MSC-induced suppression of T cell proliferation [14]. Treatment with IFN-γ causes MSCs to express the protein IDO and exhibit functional activity of IDO, which in turn degrades essential tryptophan and results in kynurenine synthesis and thereby suppresses lymphocyte proliferation [27]. Co-culturing T cells with MSCs resulted in elevated levels of PGE₂, and treatment with inhibitors of PGE₂ production mitigated the MSC-mediated immune modulation [28]; however, the mechanism underlying the immunosuppressive effect of PGE₂ is poorly understood. The production of nitric oxide (NO) by MSCs has also been implicated as a potential mechanism by which MSCs inhibit T cell proliferation [29]. NO inhibits the proliferation of T cells by suppressing the phosphorylation of signal transducer and activator of transcription-5 (STAT5), a transcription factor crucial for T cell activation and proliferation [30] (Fig. 1). Ding et al. reported that matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9 secreted by MSCs, mediate the suppressive activity of MSCs via reduction of CD25 expression on responding T cells in a model of allogeneic islet transplant [31]. The secretion of human leucocyte antigen-G5 (HLA-G5) by MSCs is reported to be essential for the following effects of MSCs: suppression of T cell and NK cell function, shift of the allogeneic T cell response to a T helper type 2 (Th2) cytokine profile [32] and induction of CD4⁺CD25^highforkhead box P3 (FoxP3⁺) regulatory T cells (T_regs) [33].

Fig. 1.

Effects of mesenchymal stem cells (MSCs) on immunocytes. MSCs modulate the immune response by their interaction with a wide range of immune cells, including T cells, B cells, dendritic cells (DCs), regulatory T cells (T), natural killer (NK), NK T and γδT cells. Inhibitory role by MSCs is dependent on cell–cell contact and soluble factors released by MSCs. HGF: hepatocyte growth factor; iDC: immature dendritic cell; IDO: indoleamine 2, 3-dioxygenase; IL-10: interleukin-10; mDC: mature dendritic cell; NO: nitric oxide; PGE₂: prostaglandin E₂; TGF-β: transforming growth factor β.

MSCs do not express MHC class II and co-stimulatory molecules such as CD80, CD86 or CD40 [9,17,34], and it is believed that T cell activity may result in anergy, which is reflected as immune tolerance. Le Blanc and co-workers reported that when MSCs are treated with IFN-γ, which is up-regulated in inflammation, they express MHC class II [15]. In an experimental arthritis model, MSCs decreased antigen-specific Th1/Th17 cell expansion and decreased the production of cytokines released by Th1/Th17 cells, such as IFN-γ and IL-17, and caused the Th2 cells to increase production of IL-4 [20,28,35] and IL-10 in lymph node joints [36]. T cell inhibition by MSCs is not due to the induction of apoptosis, but by the inhibition of cell division and probably by the production of soluble factors [14]. However, a recent study reported that MSCs could induce apoptosis in activated T cells [CD3(+) and bromodeoxyuridine (BrdU)(+)], but not in the resting T cells [CD3(+) and BrdU(−)]; this leads to marked attenuation of delayed-type hypersensitivity (DTH) response in vivo by inducing NO production [37]. Moreover, MSCs can inhibit the cytotoxic effects of antigen-primed cytotoxic T cells (CTLs) [16] by suppressing the proliferation of CTLs, rather than by direct inhibition of cytolytic activity [26,38].

A recent study showed that the negative co-stimulatory molecule B7-H4 was involved in the immunosuppressive effect of MSCs on T cell activation and proliferation via induction of cell cycle arrest and inhibition of the nuclear translocation of nuclear factor (NF)-kappa B [39]. Some studies revealed that the absence of T cell response in the presence of MSCs was transient and could be restored after the removal of MSCs [14,20]; however, others reported that T cell tolerance was induced by MSCs in murine models [35].

Although some of the mechanisms underlying the immunosuppressive effects of MSCs on T cells have been elucidated previously, the molecular mechanisms underlying this effect remain controversial. It is believed that the mechanisms underlying the suppressive effect of MSCs may differ by species. Ren and colleagues demonstrated that mouse MSCs and human MSCs utilize different effector molecules in suppressing immune reactions [40]. Immunosuppression by human- or monkey-derived MSCs is mediated by IDO, whereas mouse MSCs exert their effect via NO under the same culture conditions. Immunosuppression by human MSCs was not intrinsic, but was induced by inflammatory cytokines and was chemokine-dependent, as it is in mouse [40]. The degree of the suppressive effect depends on the concentration of the MSCs. The high MSC/lymphocyte ratio is associated with the inhibitory effect of MSCs, while a low MSC/lymphocyte ratio is often accompanied by enhanced proliferation [41]. In this setting, MSCs may act synergistically with HLA-DR antigens upon mitogenic stimulation. However, the exact mechanisms need to be investigated further.

MSCs modulate B cell proliferation and function

In murine models, MSCs have been shown to inhibit the proliferation of B cells when stimulated with anti-CD40L and IL-4 [21] or pokeweed mitogen [42]. Similarly, in humans, MSCs have also been shown to inhibit the proliferation of B cells activated with anti-immunoglobulin (Ig) antibodies, anti-CD40L antibody and cytokines (IL-2 and IL-4) [43]. In addition, the B cell functions of antibody production and secretion of the chemokine receptors CXCR4, CXCR5 and CCR7, which are responsible for chemotaxis to CXCL12 and CXCL13, were impaired by MSCs; however, the expression of B cell co-stimulatory molecules and cytokine production were not affected by MSCs [43]. MSCs inhibited the proliferation of B cells only in the presence of IFN-γ, which probably implies that IFN-γ causes MSCs to produce IDO, which in turn suppressed the proliferation of effector cells through the tryptophan pathway [24] (Fig. 1).

The nature of the mechanism involved in this inhibitory effect of MSCs has not yet been elucidated completely. Thus far, the major mechanism of B cell suppression by MSCs is attributed partly to the physical contact between MSCs and B cells and in part to the soluble factors released by MSCs; this leads to the blocking of B cell proliferation in the G0/G1 phase of the cell cycle with no apoptosis [21,42,43], unlike the case with T cells. Deng et al. reported that allogeneic MSCs inhibited the activation, proliferation and IgG secretion of B cells in BXSB mouse models of human systemic lupus erythematosus (SLE) [44]. In addition, MSCs enhanced the CD40 expression and CD40 ligand ectopic hyperexpression on the B cells of BXSB mice [44].

MSCs modulate the functions of natural killer cells

Numerous studies have shown that MSCs suppress NK cell proliferation and IFN-γ production driven by IL-2 or IL-15, but only partially inhibit the proliferation of activated NK cells [22,26,28,38,45]. Rasmusson et al. reported that MSCs did not inhibit the lysis of freshly isolated NK cells [26] and that MSCs were not lysed by allogeneic NK cells [26]. Conversely, Krampera et al. reported that NK cells cultured for 4–5 days with IL-2 in the presence of MSCs showed reduced cytolytic potential against K562 target cells and that this suppressive effect might be attributed to the IFN-γ produced by NK cells [24]. Exposure to IFN-γ did not ablate MSC-induced inhibition of T cell proliferation, but triggered the expression of HGF and TGF-β1 secreted by MSCs at concentrations that suppressed alloresponsiveness [45] (Fig. 1). Furthermore, a study indicated that MSCs suppressed NK cell cytotoxicity against HLA class I-positive cells more effectively than HLA class I-negative cells [22]. Low HLA class I expression makes allogeneic as well as syngeneic MSCs more susceptible to lysis by activated NK cells [22,46,47]. Incubation of MSCs with IFN-γ decreased their susceptibility to NK cell-mediated lysis because of the up-regulation of HLA class I expression on MSCs [46]. The mechanisms underlying the immunosuppressive effects of MSCs are still unclear and several different, sometimes contradictory, theories have been proposed. Soluble factors such as TGF-β1 and PGE₂ are believed to play a role in the MSC-mediated suppression of NK cell proliferation [22].

The physiological interactions between MSC and NK cells would be the reciprocal effects exerted by the two cell types, in particular the ability of activated NK cells to kill MSC. Some studies showed that IL-2-activated NK cells can effectively lyse MSCs [22,46,47] because MSCs express ligands (ULBP, PVR and nectin-2) that are recognized by activated NK receptors (NKp30, NKG2D and DNAM-1), which in turn trigger NK cell alloreactivity [46,48].

Recently, Prigione et al. found that the inhibitory effect of MSCs on the proliferation of invariant NK T (iNK T, Vα24⁺Vβ11⁺) and γδT (Vδ2⁺) cells in the peripheral blood is mediated by releasing PGE₂, rather than IDO and TGF-β1 (Fig. 1); however, cytokine production and cytotoxic activity of the cells were only partially affected by MSCs [49]. Vδ2⁺ cells also serve as professional antigen-presenting cells for naive CD4⁺ T cell response, and MSCs did not inhibit antigen processing/presentation by activated Vδ2⁺ T cells to CD4⁺ T cells [49].

Interaction between MSCs and dendritic cells (DCs)

MSCs impaired the differentiation of monocytes or CD34⁺ haematopoietic stem cells into dendritic cells (DCs) by inhibiting the response of the former to maturation signals, reducing the expression of co-stimulatory molecules and hampering the ability of the former to stimulate naive T cell proliferation and IL-12 secretion [23,50,51]. In addition, this inhibitory effect might be mediated via soluble factors and may be dose-dependent [50]. Spaggiari et al. showed that MSCs strongly inhibited the maturation and functioning of monocyte-derived DCs by interfering selectively with the generation of immature via inhibitory mediator of MSC-derived PGE₂, but not IL-6 [52] (Fig. 1). However, the mechanism underlying the up-regulation of PGE₂ in monocyte–MSC co-cultures remains unclear. Ramasamy et al. reported that the cell cycle in DCs was arrested in the G0/G1 phase upon interaction with MSCs [53]. A recent study reported that MSCs isolated from human adipose tissue were more potent immunomodulators for the differentiation of human DCs than MSCs derived from the bone marrow [54].

T_regs induced by MSCs

MSCs may also modulate immune responses via the induction of T_regs. MSC can induce the generation of CD4⁺CD25⁺ cells displaying a regulatory phenotype (FoxP3⁺) in mitogen-stimulated cultures of peripheral blood mononuclear cells [28,38], although the functional properties of these cells have not yet been elucidated. For example, depletion of CD4⁺CD25⁺ T_regs had no effect on the inhibition of T cell proliferation by MSCs [20]. However, a recent study reported that MSCs could induce kidney allograft tolerance by inducing the generation of CD4⁺CD25⁺FoxP3⁺ T_regsin vivo[55]. Additionally, MSCs have been reported to induce the formation of CD8⁺ T_regs that are responsible for the inhibition of allogeneic lymphocyte proliferation [19].

In a recent study, Ghannam et al. found that under inflammatory conditions, MSCs prevented the differentiation of naive CD4⁺ T cells into Th17 cells and inhibited the function of Th17 cells in vitro by secreting PGE₂. Moreover, MSCs could induce the T_reg phenotype in Th17 cells, which can inhibit the proliferative responses of activated CD4⁺ T cells in vitro[56]. Tipnis et al. reported that umbilical cord-derived MSCs (UC-MSCs) constitutively express B7-H1, which is a negative regulator of T cell activation. In addition, B7-H1 expression was increased and IDO expression was induced in UC-MSCs after IFN-γ treatment. Furthermore, UC-MSCs inhibited the differentiation and maturation of monocyte-derived DCs and augmented the generation of T_regs[57]. These immunosuppressive effects of UC-MSCs are mediated largely by cell–cell contact [57].

The induction of T_regs by MSCs involves not only involves direct contact between MSCs and CD4⁺ cells, but also the secretion of soluble factors such as PGE₂ and TGF-β1 [58]. Human gingiva-derived MSCs (GMSCs) can induce IL-10, IDO, inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) and thereby serve as immunomodulatory components in the treatment of experimental inflammatory diseases [59] (Fig. 1). A study has shown that the immunosuppressive effect of MSCs is mediated by the secretion of galectin-3, a protein known to modulate T cell proliferation, gene expression, cell adhesion and migration [60]. Madec et al. reported that MSCs prevent autoimmune B cell destruction and subsequent diabetes in NOD mice by inducing T_regs[61]. The effect of MSCs in the treatment of autoimmune diseases may be through the induction of de novo generation of antigen-specific CD4⁺CD25⁺FoxP3⁺ T_reg cells [36,62].

However, a recent study reported that MSCs could sustain or suppress T cell proliferation depending on their concentration, and a low MSC/T-cell ratio might support T cell proliferation [63]. A recent study indicated that MSCs could stimulate the activation and proliferation of resting T cells and generate T_regs[64]. These data suggested that the culture conditions play an important role in the clinical application of MSCs [63].

Clinical application of MSCs for immune-mediated diseases

Over the past 3 decades, numerous efforts have been made to explore the therapeutic applicability of MSCs. In pathological conditions, MSCs migrate preferentially into lymphoid organs, allografts, injured and/or inflammatory tissue sites after systemic transfusion, where MSCs interact with the activated immune cells and modulate their function [65,66]. The in vivo immunomodulatory properties of MSCs were first described in a baboon model of skin transplantation [18]. The therapeutic potential of MSCs in immunomodulation is being explored currently in several Phase I, II and III clinical trials [67], many of which have recently been completed or are under way, as reported in the clinical trials website of the United States sponsored by the National Institutes of Health (http://clinicaltrials.gov). Because of their immunosuppressive properties, MSCs are believed to play a role in the maintenance of peripheral tolerance and the induction of transplantation tolerance, and they are considered potential candidates in cellular therapy for graft-versus-host disease (GVHD) and autoimmune diseases and in protecting solid-organ grafts from being rejected [39]. Table 1 presents data on the several applications of MSCs as immunosuppressants as studied in clinical trials (data available at the time of the preparation of this manuscript). Recently, Le Blanc et al. reported that MSCs obtained from HLA-identical sibling donors, haploidentical donors and third-party HLA-mismatched donors infused in 55 patients with steroid-refractory acute GVHD elicited a response in more than half the patients; the study showed that MSCs exerted their therapeutic effect in the case of both HLA-matched and HLA-unmatched donors. However, for GVHD, the use of MSCs is a double-edged sword, because the prevention of GVHD was associated with a high incidence of leukaemia relapse, which is the result of the non-specific immunosuppressive effect of MSCs on graft-versus-leukaemia [68,69]. Liang et al. reported that allogeneic MSC transplantation in patients with refractory SLE resulted in the amelioration of disease activity, improvement in the levels of serological markers and stabilization of renal function without the occurrence of serious adverse events [70]. For solid organ transplantation, the beneficial effect of MSC-based immunosuppressive therapy is debatable. The application of calcineurine inhibitors (CNIs) would abrogate the immunosuppressive effect of MSC therapy. In addition, CNIs cause renal failure, hypertension and hyperglycaemia and increase the risk of malignancy; therefore, efforts have been made to minimize the use of CNIs treatment in organ transplantation protocols. Consequently, it may be worthwhile to compare the usefulness of combining CNI treatment and MSC therapy in organ transplantation [71]. Conversely, non-selective immunosuppression would have affected patients' antiviral immunity equally [70].

Table 1.

Clinical trials using mesenchymal stem cells (MSCs) as immunosuppressants

Clinical trial	Disease	Cell type/source	Status	Sponsor	ClinicalTrials.gov identifier
Autologous mesenchymal stem cells from adipose tissue in patients with secondary progressive multiple sclerosis	Multiple sclerosis	Adipose tissue-derived autologous MSCs	Recruiting	Fundacion Progreso y Salud, Spain	NCT01056471
Mesenchymal stem cell infusion as prevention for graft rejection and GVHD	GVHD	BM-derived autologous MSCs	Recruiting	University Hospital of Liege, Belgium	NCT00504803
Mesenchymal stem cell transplantation in the treatment of chronic allograft nephropathy	Chronic allograft nephropathy	BM-derived autologous MSCs	Not yet recruiting	Fuzhou General Hospital, China	NCT00659620
Mesenchymal stem cells and subclinical rejection	Renal transplantation	BM-derived allogenic MSCs	Not yet recruiting	Leiden University Medical Center, the Netherlands	NCT00734396
Safety and efficacy study of umbilical cord blood-drived mesenchymal stem cells to promote engraftment of unrelated haematopoietic stem cell transplantation	GVHD	Human umbilical cord blood-derived MSCs	Not yet recruiting	Medipost Co. Ltd, Korea	NCT00823316
Safety and efficacy study of allogenic mesenchymal stem cells to treat extensive chronic GVHD	Chronic GVHD	BM-derived allogenic MSCs	Not yet recruiting	Guangdong General Hospital, China	NCT00972660
Evaluation of the role of mesenchymal stem cells in the treatment of GVHD	Steroid-resistant GVHD	BM-derived autologous MSCs	Recruiting	Christian Medical College, Vellore, India	NCT00314483
Mesenchymal stem cell infusion as treatment for steroid-resistant acute GVHD or poor graft function	GVHD, poor graft function	BM-derived allogenic MSCs	Recruiting	University Hospital of Liege, Belgium	NCT00603330
Safety and efficacy study of adult human mesenchymal stem cells to treat acute GVHD	GVHD	BM-derived allogenic MSC (prochymal)	Completed	Osiris Therapeutics, USA	NCT00136903
Treatment of refractory (acute or chronic) GVHD by the infusion of expanded in-vitro allogenic mesenchymal stem cell	GVHD	BM-derived allogenic MSCs	Recruiting	University of Salamanca, Spain	NCT00447460
Mesenchymal stem cells for treatment of amyotrophic lateral sclerosis	Amyotrophic lateral sclerosis	Adipose tissue-derived autologous MSCs	Not yet recruiting	Mayo Clinic, USA	NCT01142856
Mesenchymal stem cells under basiliximab/low-dose RATG to induce renal transplant tolerance	Kidney transplant	BM-derived autologous MSCs	Recruiting	Mario Negri Institute for Pharmacological Research, Italy	NCT00752479
Mesenchymal stem cells transplantation for refractory systemic lupus erythematosus	Refractory systemic lupus erythematosus	BM-derived allogenic MSCs	Recruiting	Nanjing Medical University, China	NCT00698191
Efficacy and safety of adult human mesenchymal stem cells to treat patients who have failed to respond to steroid treatment for acute GVHD	GVHD	BM-derived allogenic MSC (prochymal)	Completed	Osiris Therapeutics, USA	NCT00366145
Donor mesenchymal stem cell infusion in treating patients with acute or chronic GVHD after undergoing a donor stem cell transplant	GVHD	BM-derived allogenic MSCs	Not yet recruiting	Case Comprehensive Cancer Center	NCT00361049
Safety and efficacy of prochymal for the salvage of treatment-refractory acute GVHD patients	GVHD	BM-derived allogenic MSCs	Completed	Osiris Therapeutics, USA	NCT00284986
Safety and efficacy of human mesenchymal stem cells for treatment of liver failure	Liver failure	Human umbilical cord-derived MSCs	Recruiting	Beijing 302 Hospital, China	NCT01218464
Umbilical cord mesenchymal stem cells for immune reconstitution in HIV-infected patients	HIV	Human umbilical cord-derived MSCs	Recruiting	Beijing 302 Hospital, China	NCT01213186

BM: bone marrow; GVHD: graft-versus-host disease; HIV: human immunodeficiency virus; RATG: rabbit anti-human thymocyte globulin.

In the future, well-designed preclinical trials should be conducted to explore the clinical applicability of MSCs. Thereafter, randomized trials comparing treatment with infusions of MSCs and conventional drug-based therapies should be undertaken to confirm the therapeutic potential of these cells, as it is important not to overestimate the potential therapeutic effects of MSCs. Information gathered over such studies would help to develop innovative cell-based therapies for the treatment of diseases characterized by exaggerated immune responses.

Disclosure

The authors have no financial conflict of interest.