Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells

Cytotherapy, 2016; 18: 160–171

REVIEW ARTICLES

Mesenchymal stromal cells and immunomodulation: A gathering of regulatory immune cells

MEHDI NAJAR, GORDANA RAICEVIC, HUSSEIN FAYYAD-KAZAN, DOMINIQUE BRON, MICHEL TOUNGOUZ & LAURENCE LAGNEAUX Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles, Campus Erasme, Brussels, Belgium Abstract Because of their well-recognized immunomodulatory properties, mesenchymal stromal cells (MSCs) represent an attractive cell population for therapeutic purposes. In particular, there is growing interest in the use of MSCs as cellular immunotherapeutics for tolerance induction in allogeneic transplantations and the treatment of autoimmune diseases. However, multiple mechanisms have been identified to mediate the immunomodulatory effects of MSCs, sometimes with several ambiguities and inconsistencies. Although published studies have mainly reported the role of soluble factors, we believe that a sizeable cellular component plays a critical role in MSC immunomodulation. We refer to these cells as regulatory immune cells, which are generated from both the innate and adaptive responses after co-culture with MSCs. In this review, we discuss the nature and role of these immune regulatory cells as well as the role of different mediators, and, in particular, regulatory immune cell induction by MSCs through interleukin-10. Once induced, immune regulatory cells accumulate and converge their regulatory pathways to create a tolerogenic environment conducive for immunomodulation. Thus, a better understanding of these regulatory immune cells, in terms of how they can be optimally manipulated and induced, would be suitable for improving MSC-based immunomodulatory therapeutic strategies. Key Words: immunomodulation, MSCs, regulatory immune cells, tolerance

Introduction Mesenchymal stromal cells: generalities Mesenchymal stromal cells (MSCs) are stromalderived adult progenitor cells that were originally identified as precursors for cells of the osteogenic lineage by Friedenstein and colleagues in 1970 [1]. Later on, Pittenger et al. reported the multilineage potential of MSCs based on their ability to differentiate into distinct mesenchymal cell lineages, including the chondrogenic, adipogenic and osteogenic lineages [2]. Although initially isolated from the bone marrow, MSCs were subsequently obtained from other sources, which include adult and fetal tissues. Currently, MSCs are isolated from adipose tissue and the umbilical cord, representing major alternative sources to bone marrow [3].The presence of heterogeneous populations within MSCs, isolated by traditional plastic adherence, have

led to the discovery of potentially novel markers and the development of a critical panel for future investigation [4]. Despite significant advances, numerous misconceptions regarding the nature and function of MSCs persist that may significantly impede the advancement of MSC-based therapies. Six prevalent misconceptions have been identified, and a great deal of work has been conducted to clarify or rectify these misconceptions [5]. Moreover, because MSCs did not meet the specified stem cell criteria, the International Society for Cellular Therapy (ISCT) had stated that these fibroblast-like plastic-adherent cells, regardless of the tissue of origin, should be termed “multipotent mesenchymal stromal cells” and retain the acronym “MSCs” [6]. Caplan et al. [7] suggested that MSCs should be named “medicinal signaling cells” because they serve as drugstores that secrete diverse bioactive molecules with immunomodulatory and trophic

Correspondence: Mehdi Najar, PhD, Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles, Campus Erasme, Bâtiment de Transfusion (Level +1), Route de Lennik no. 808, 1070 Brussels, Belgium. E-mail: [email protected] (Received 3 July 2015; accepted 13 October 2015) ISSN 1465-3249 Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.10.011

Large-scale expansion of MSCs with the use of Quantum activities. To be consistent in characterizing human MSCs and to facilitate the exchange of data among investigators, the Mesenchymal and Tissue Stem Cell Committee of the ISCT proposed a minimum set of criteria to define MSCs [8]. First, MSCs must be plastic-adherent during culture and present a fibroblast-like shape. Second, MSCs must present a specific immunophenotype by the expression of surface molecules CD105, CD73 and CD90, and not CD45, CD34, CD14 (or CD11b), CD79alpha (or CD19) or human leukocyte antigen (HLA)-DR molecules.Third, MSCs must differentiate in vitro into osteoblasts, adipocytes and chondroblasts. Properties of MSCs include high self-renewal, multilineage potential and immunomodulatory capability [9]. Interest in MSCs is due to their ability to differentiate into different cell lineages, which can potentially be used for regenerative medicine [10]. By producing several extracellular matrix proteins, cytokines, growth factors and chemokines, MSCs provide a specialized microenvironment that supports hematopoiesis, which is conducive for hematopoietic stem cell transplantation [11]. However, during the past few years the ability of MSCs to modulate the immune response has emerged and creates immense hope for the immunotherapy field. Immunomodulation: checkpoints Several in vitro studies, as well as pre-clinical models and phase 1/2 clinical trials, have demonstrated that MSCs possess diverse immunomodulatory activities and they can efficiently treat immunological disease [12]. Several points that address how to efficiently use MSCs for immunotherapy are discussed in this section. Immunological guidelines Within the scientific community, there is some controversy about the effects and mechanisms related to MSC immunomodulation. To resolve this, the ISCT has proposed standardized guidelines and protocols for the immunological characterization of MSCs.This should allow for reproducibility and consistency that will ultimately significantly validate and strengthen the various MSC-based clinical approaches to treat immunological diseases [13]. In parallel, an immunopotency assay (IPA) measuring MSC-mediated T-cell inhibition has been developed for in vitro quality control, but additional studies are needed to correlate IPA with MSC inhibition efficiency in vivo [14]. Immunological plasticity During an immune response, MSCs are known to communicate with the inflammatory microenvironment. MSCs actively interact and crosstalk with innate

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and adaptive immune cells [15,16]. Indeed, MSCs express a large number of surface molecules [17] that include members of the integrin family and adhesion molecules that promote cellular interactions via receptor binding on immune cells [18]. In parallel to these interactions, MSCs are also sensitive to the inflammatory milieu that determines the biological fate of MSCs. Indeed, immunomodulation by MSCs is not constitutive but is induced by inflammatory cytokines mainly secreted by activated immune cells. Cytokines critically influence the immunomodulatory effects of MSCs depending on the type of cytokine and its concentration, indicating that MSC function is plastic [19]. This point has to be considered before any clinical application and is important to understand for the success of MSC therapeutics. Immunological requirements To fulfill their roles as immunomodulatory cells, MSCs must be phenotypically acceptable by the recipient as well as being properly functional. 1.The allogeneic state. Importantly, the potential therapeutic benefit of MSCs relies, in part, on their allogeneic state. Although autologous MSCs have been most commonly used for clinical trials, they display disease and age-related impairments [20]. Therefore, the use of “off-the-shelf ” MHC-mismatched MSCs is becoming prevalent because of several advantages, two of which are that cells are immediately available and that they originate from young nondiseased individuals. However, their immunogenic state can be viewed as a limitation to their use. In vitro, both allogeneic and autologous MSCs display the same immunomodulatory potential, indicating that these effects are independent of major histocompatibility complexes (MHC) [21]. MSCs do not constitutively express MHC class II and costimulatory molecules, such as CD40, CD80 or CD86. Interestingly, some studies reported MHC class II upregulation on MSCs upon priming by inflammatory mediators in the absence of T-cell activation [22]. Under inflammatory conditions, only CD40 expression was increased in MSCs.This may be counteracted by the parallel induction of B7-H1 (PD-L1; CD274), an inhibitory molecule for T cells, thereby leading to lymphocyte inhibition [23]. Preclinical and some clinical studies on the immunogenicity state of MSCs are contradictory. Ryan et al. [24] showed that MSCs are in an immuno-privileged state, allowing for their safe application. Others have presented clear evidence that MSCs can create alloimmunity and even stimulate graft rejection [22]. Thus, the immunogenicity of MSCs is of special interest and needs to be investigated in more detail. The immunophenotype of MSCs is an important criterion to be considered before their clinical use,

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and, most important, in vivo immuno-monitoring after injection should be taken into account [20]. 2.The processing of MSCs. Although MSCs represent a promising tolerogenic immunotherapeutic strategy, many challenges remain for their clinical application—in particular, the preparation and processing of MSCs may affect their potency in patients. A major issue for the use of MSCs as immunotherapeutic cells is the manufacture of clinical cell products that are compliant with Good Manufacturing Practice. An article describing standard quality requirements for MSC production has been released, and a key requirement is the preservation of the immunomodulatory potential of MSC products, among other important criteria [25]. Indeed, the production of MSCs by using a variety of clinicalgrade processes resulted in differences in their immunomodulatory properties [26]. Recently, an optimized MSC expansion protocol that preserves both MSC safety and immunomodulatory performance was developed for therapeutic purposes [27]. Gammairradiation and hypoxia allow MSCs to retain their immunomodulatory functions, whereas high passage number, long cryopreservation time and lack of a postthaw equilibration period adversely affect such functions.To restore the weakened immunomodulatory activity of MSCs resulting from long-term culture, recent findings have suggested the usage of Substance P (SP) as a stimulatory agent to promote MSCmediated immunosuppression [28]. This study demonstrated that SP pretreatment of MSCs at late passages facilitates the retention of their immunomodulatory capacity, probably by enhancing their secretion of transforming growth factor (TGF)-β1. Another major issue for the use of MSCs in the clinic is their expression or the absence of ABO blood group antigens, which are known to be one of the major immunogenic barriers that hamper tissue transplantation. MSCs do not inherently express ABO antigens but can potentially absorb immunogenic ABO substances from undefined human serum or plasma that are used for cell processing. To maximize the cell product quality and functionality in vivo, classical serum should be substituted with a more defined and nonimmunogenic one, particularly when the cells are given to immunocompetent patients [29]. Immunomodulation: a gathering of regulatory immune cells Several studies have found that MSCs have immunomodulatory functions and can be applied to a wide range of immune-mediated conditions [30]. However, the main pathway underlying these effects remains unclear. A number of tolerogenic mecha-

nisms have been reported that involve cell-to-cell contact, secretion of soluble factors, induction of anergy, apoptosis and regulatory immune cells [31]. In this sense, MSCs do not fit the definition of an immune cell but should rather be defined as coordinators of the immune system [12]. In the immune system, both immune and non-immune cells are tightly linked together in a complex network of cytokine expression and responses. This would create a platform of cellular communication and interactions that organize the biological outcome of the inflammatory response [32]. Although MSCs are known to modulate the immune response by interacting with cells of both innate and adaptive immunity, the exact mechanism governing these effects are not yet clear. A huge number of studies have focused on the key role played by soluble mediators in MSC-dependent immunomodulation. As discussed previously, many factors and cytokines were reported to take part in these effects, with divergent mechanistic results [33]. Moreover, the expression profile, as well as the role of these regulatory mediators, is not the same in different species [34]. In parallel, little is known about the cellular components that contribute to these immunomodulatory effects. Indeed, there is accumulating evidence suggesting that MSCs by themselves may not be directly active as immuno-regulators after administration. MSCs may hamper T-cell-, B-cell-, antigen-presenting cell (APCs)- and natural killer (NK) cell–mediated immune responses. By inducing critical changes in their immunobiology [35–37], MSCs can participate in immune homeostasis. It is likely that MSCs re-educate immune cells to induce the generation of regulatory immune cells with tolerogenic properties [12]. These regulatory immune cells, such as regulatory T-cells (Tregs), regulatory B-cells (Bregs), regulatory APCs and NK cells, will gather to create a tolerogenic environment suitable for modulating the immune response (Figure 1). For that, MSCs use multiple regulatory pathways with interleukin (IL)-10 having a central role. This review discusses the nature and role of these immune regulatory cells, as well as the role of different mediators, and, in particular, induction of IL-10 by MSCs. We think that this gathering of regulatory immune cells with converging regulatory pathways will efficiently permit immunomodulation. Regulatory immune cells induced by MSCs represent therapeutic targets for various indications, and thus, they deserve further research. Collectively, there are growing interests in using MSCs as an immunomodulatory strategy for tolerance induction in several immune-related diseases or disorders. We need to increase our knowledge and comprehension of the immunobiology of these regulatory immune cells, especially when associated with MSCs in way to improve immunomodulation-based strategies.

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Figure 1. MSCs re-educate the immune cells to induce the generation of regulatory immune cells with tolerogenic properties. These regulatory immune cells such as Tregs, Bregs, regulatory APC and NK cells will gather to create a tolerogenic environment suitable to modulate the immune response. Multiple regulatory pathways with a central role for IL-10 could then be used by these cells to finally establish immunomodulation.

Regulatory immune cells To date, published studies have not addressed the immunomodulatory effects of MSCs as a result of complex and global interactions with APCs, lymphocytes and NK cells. MSCs, by using different pathways, can interfere with the biology and generation of distinct regulatory immune cells. A gathering of these regulatory immune cells will establish a tolerogenic state that can modulate both innate and adaptive responses. Antigen-presenting cells. As reviewed by Spaggiari et al., most studies that address the interaction between MSCs and APCs have demonstrated that MSCs modulate dendritic cells (DCs) at multiple levels. Overall, MSCs alter the phenotype, cytokine release, differentiation and maturation of DCs and compromise their antigen presentation ability [38]. Recently, a study investigated the influence of MSCs on different monocyte subpopulations (classical, intermediate and nonclas-

sical) [39]. MSCs differentially modulate the cytokine secretion profile of these monocytes subsets. As a result, DCs are unable to efficiently prime T cells for a robust immune response. However, there are no overarching conclusions on the effects that are elicited by MSCs, and it is still in question whether these modulated DCs are impaired or newly tolerogenic, which can stimulate the expansion of Tregs. Moreover, these prevailing observations support a major role for regulatory factors during DC immunomodulation by MSCs and, to a lesser extent, by cell contact [40]. PGE2 is a potent inducer of IL-10, and both factors have a pivotal role in the cross-regulation of DCs. Thus, PGE2-induced IL-10 is a key regulator of the bone marrow-DC (BMDC) pro-inflammatory response [41]. Additionally, as we have postulated for T cells, the IL-10/IL-10R axis may serve as a relevant immunomodulatory loop for DC regulation with important consequences for the immune response [41,42]. MSCs have the ability to induce regulatory DCs (MSC-DCs) with

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T-cell-suppressing properties. These MSC-DCs are characterized by their low immunogenicity, impaired capacity to stimulate T cells and the capacity to induce Tregs. Most important, MSC-DCs are accompanied by elevated IL-10 secretion [43,44]. Induction of these MSC-DCs involves different molecules, such as STAT3 [45], Notch [46], SOCS1 [47] and MSC-derived PGE2 play a central role in such immunomodulation [48,49]. STAT3 is considered a negative regulator of inflammatory responses. It regulates APC biology as well as IL-10 expression. Conversely, by increasing STAT3 activity in APCs, MSCs induce regulatory DCs that secrete IL-10 resulting in impaired T-cell responses. It is worth noting that although IL-10 gene expression is regulated by STAT3, IL-10 itself can also strongly activate STAT3. However, according to GurWahnon et al., this happens rarely, and the contactdependent activation of STAT3 is privileged [45]. Interestingly, STAT3 might also be modulated by different PGE2 signaling pathways and may consequently modulate DCs and IL-10 [50]. Previous findings defined a role for Notch as a molecular switch between pro-inflammatory and anti-inflammatory Th1 cell functions [51]. Similarly, plasmacytoid DCs induce IL10 production in T-cells by exploiting the Notch pathway [52]. Li et al. suggested that MSC-DCs are able to induce the generation of Tregs through Notch activation [46]. According to Deng et al., adoption of a tolerogenic phenotype by MSC-DCs requires the activation of SOCS1, which is primarily mediated by IL-6 [47]. Finally, SOCS1 was previously shown to be a key determinant of M2 macrophage polarization, activation and function [53]. Macrophages can be derived from circulating inflammatory or resident monocytes, which are recruited to tissue sites of infection and inflammation. In response to environmental signals, they can display high plasticity and adapt their physiology [54]. Thus, different populations of macrophages with distinct functions may arise, including the pro-inflammatory subtype (M1, classically activated macrophages) and the anti-inflammatory subtype (M2, alternatively activated macrophages) [55]. François et al. reported that IDO activity in MSCs is involved in the generation of anti-inflammatory M2 macrophages. These monocyte-derived M2 cells are involved in T-cell inhibition in an IL-10–independent manner, thereby amplifying the immunosuppressive effects of MSCs [56]. Interestingly, through the constitutive secretion of IL-6, MSCs induce M2 macrophages that are characterized by concomitant enhanced production of IL-10 [57]. As underlined by Melief et al., the roles of IL-6 and PGE2 on monocytes are not considered contradictory but work in a synergistic manner. IL-6 is involved in M2 macrophage generation, whereas

PGE2 selectively interferes with the generation of immature DCs. Conversely, IL-6–dependent PGE2 secretion is correlated with the immunosuppressive activity of MSCs [58]. Indeed, PGE2 regulates the production of a wide array of cytokines. PGE2 up-regulates both IL-10 and IL-6 in activated murine macrophages [59]. As shown by Bouffi et al., IL-6 is a central player because it is induced by PGE2, and IL-6 is able to positively regulate COX2 [58]. In a different model, PGE2 is able to stimulate IL-6 production, which, in turn, induces increased PGE2 secretion, COX-2 and EP4/EP2 expression in bone cells [60]. However, the molecular pathways governing the increased secretion of macrophage-derived IL10 and IL-6 in response to PGE2 are different.Whereas the synthesis of IL-10 is dependent on p38 MAP kinase activity [59], IL-6 is regulated by protein kinase C [61]. Collectively, these findings provide evidence for the interaction of PGE2 and IL-6 signaling pathways in monocyte modulation by MSCs. Another mechanism has been highlighted by which MSCs induce regulatory monocytes that can be involved in the modulation of T cells. Mechanistically, MSCs, through the secretion of HGF, induce monocytes to produce high levels of IL-10 [62]. Tregs. Allograft rejection is the result of a complex series of interactions between the innate and adaptive immune system with T cells playing a major role [63]. MSCs are able to alter lymphocyte activation, proliferation and differentiation, and MSCs can generate T cells that have regulatory properties [35]. The role of Tregs in MSC immunomodulation is controversial. Several studies have suggested that MSCs indirectly modulate the immune response via Treg induction, whereas others see no role for Tregs [64]. In general, MSCs preferentially cause the generation and/ or expansion of T-cell subsets harboring different regulatory phenotypes. Additionally, Treg phenotype and function are preserved upon MSC recruitment [65,66]. Other findings have indicated that MSCs induce the generation of Tregs from CD4+ or CD8+ lymphocytes, and these Tregs have powerful immunoregulatory effects that strongly inhibit lymphocytes [67,68]. In addition, MSCs generate induced Tregs (iTregs) that suppress activated T cells [69]. As reviewed by Burr et al. [70], several regulatory molecules are involved in the recruitment, expansion and induction of Tregs. Thus, an increase in the secretion/ expression levels of CCL1/I-309 [71], HLA-G5 [72], LIF [73], PGE2, TGF-β [74], HO-1 [75], IL-2 [76], IDO-1, ILT-3, ILT-4 [77] and IL-1β/CCL-1 [78] during the co-culture of MSCs and T cells have been associated with Treg generation [69,79]. However, according to Prevosto et al. [67], soluble factors, such as IL-10, TGF-β and PGE2, did not appear to be involved in Treg generation nor in their

Large-scale expansion of MSCs with the use of Quantum immunoregulatory function. Interestingly, recent results have indicated that glucocorticoid (GC)-induced leucine zipper (GILZ) plays a critical role in mediating the crosstalk between MSCs and Tregs as well as in MSC immune suppression [80]. Enhanced expression of GILZ promotes DC differentiation into regulatory cells that drive Treg expansion [81]. In addition to classical CD4+ Tregs, CD8+ Tregs are emerging as an important subset of T-suppressor cells. These Tregs have the capacity to inhibit T-cell responses and suppress autoimmunity as well as alloimmunity and thus are of clinical importance [82]. BM-MSCs increase the frequency and suppressive function of CD8+CD28− Tregs by modulating IL-10 and FasL expression [83]. Importantly, during Treg generation, IL-10 is considerably up-regulated in parallel with other factors. We have previously discussed the interplay and synergy that exist between IL-10 and several regulatory factors that ultimately lead to T-cell modulation by MSCs. As reported for IL-10, other interactions between these factors can occur. TGF-β in association with the COX-2/PGE2 network promotes the development of Tregs as both of these players share signaling pathways [84]. LIF is suggested to participate in up-regulating PGE2 production as well as in the expression of its receptor [85]. LIF expression itself may be increased by TGF-β as demonstrated in human thymic epithelial cells (TECs), and thus may indirectly affect T-cell development [86]. Secretion of PGE2 by cancer cells induces IDO expression and kynurenine production in stromal fibroblasts, revealing the interplay between cancer and stromal cells [87].TGF-β and IDO also have interconnected signaling pathways that promote the activation of their own genes and thus amplify their expression [88]. These molecular insights suggest a synergistic potential for TGF-β and IDO in creating a tolerogenic state that regulates the immune response [89]. Both HLA-G and IDO are key molecules involved in immune tolerance, but there is no clear link between them. Depending on the target cell, the function and expression of IDO and HLA-G5 could be mutually influenced, but they are complementary for the induction and maintenance of immune tolerance through two independent pathways [90,91]. In addition, recombinant HLA-G5 and HLA-G6 molecules induce a significant increase in TGF-β production in myelomonocytic cells [92]. According to Liu et al., IL-6, TGF-β, IDO and PGE2 are not involved in the expression and modulation of IL-10 or FasL in CD8+CD28− T-cells after their co-culture with MSCs [83]. Until now, no study has shown a direct and critical role for IL-10 in Treg induction by MSCs. IL10 is not involved in Treg generation by MSCs or in Treg suppressive function [67]. Although IL-10 is not essential for the expansion of Tregs, its production may

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be critical for T-cell inhibition [93]. In a model of transplantation, the maintenance of an immune hyporesponsive state, as well as graft function, is associated with increased IL-10 production but not Treg expansion [94]. A growing body of evidence suggests that functionally distinct subsets of Tregs are induced with different phenotypes exist. These Treg subsets have suppressive/regulatory properties that tightly control immune responses. However, the mechanisms involved in Treg development or regulatory function are more complex and require diverse signals [95]. A novel approach to induce mixed chimerism and permanent tolerance through combinatorial cellbased immunomodulation of MSCs andTregs has been proposed as a clinically applicable strategy to induce the potent inhibition of host immune responses [96]. Moreover, co-transplantation of Tregs with MSCs dramatically enhanced the survival rate, proliferation and angiogenesis potential of MSCs, thus demonstrating cross-regulation between the two populations, which is beneficial for cell-based therapy [97]. Although sometimes contradictory, the literature suggests that MSCs may indirectly favor Treg generation via different pathways to modulate T-cell immunobiology, and the presence of high IL-10 plays a role that is not yet clear. Further studies are needed to fully understand the mechanisms and to identify key regulatory molecules involved in these effects. Investigation of the microRNA (miR) expression profiles of MSCs and Tregs and their impact on immune regulation may provide new targets for immunotherapy. Recent studies have shown an important interplay between IL-10 and miRNAs with reciprocal feedback loops. IL-10 can be directly post-transcriptionally regulated by several miRNAs. In turn, some miRNAs are regulated by IL10, and thus they may be important for the balance between pro- and anti-inflammatory responses [98]. Bregs. B-cells and humoral immune responses are increasingly acknowledged as crucial mediators of chronic allograft rejection. As reviewed by Franquesa et al., published papers on the effects of MSCs on B-cell immunobiology have disparities in their approaches and results [36]. MSCs were reported to alter the immunobiology of B-lymphocytes and to target the humoral immune response. Indeed, proliferation, differentiation and immunoglobulin production of B cells are significantly affected by MSCs [99]. Like T helper cells, B cells can be classified into subsets according to their cytokine secretion profiles. Bregs, a new B-cell subset with regulatory properties, contribute to the maintenance or induction of tolerance by suppressing and/or regulating immune responses [100]. The lack or loss of Bregs is linked to several autoimmune diseases (mouse models) [101]. Although the frequency of naturally existing Bregs is extremely low, these cells can be expanded in vitro for

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further characterization. Several studies have suggested that Bregs are heterogeneous, consisting of cells that are differentiated from each other under the appropriate stimulatory, temporal and spatial microenvironment. Regarding the regulatory/suppressive function of Bregs, a variety of immune cells are targeted by Bregs. Several direct and indirect mechanisms underlying Breg effects during immune responses have been reported [102]. Briefly, Bregs play an important role in T-cell plasticity by modulating the Th1/Th2 balance in favor of a Th2 response. Bregs can also convert Th1 effector cells into regulatory Tr1 cells, suppress Th17 cell differentiation, and promote Treg expansion. Furthermore, Bregs induce tolerogenic DCs or invariant NK cells that further influence T-cell plasticity. These effects are mediated by the release of regulatory factors, such as IL-10, TGF-β and semaphorin3A (Sema3A), through cell-to-cell contact and by inducing apoptotic cell death or anergy [103,104].The cytokine IL-35 is both an inducer and a mediator of Breg function. Given that Bregs can induce Tregs, there may be a feedback loop between these two populations that depends on IL-35 [105]. Few studies have addressed the impact of MSCs on Bregs. Guo et al. reported the first observation of Breg modulation by MSCs in a mouse model of multiple sclerosis [106]. Treatment with MSCs suppressed the severity of the disease by increasing the frequency and activity of Bregs along with increased secretion of IL10. In vitro, MSCs abrogate plasmablast formation and induce Bregs by producing IL-10 independently of T cells [107]. Park et al. observed that MSCs ameliorated autoimmunity in a murine model of systemic lupus erythematosus (SLE) by inducing the expansion of Bregs and secretion of IL-10 in vivo [108].They also observed that MSCs induced the expansion of marginal zone B-cell populations including Bregs. Interestingly, in a prospective clinical study of refractory chronic graft-versus-host disease (cGVHD), patients infused with MSCs had clinical improvements that were associated with increased survival and Breg proliferation as well as increased IL-10 production. These effects on Bregs were partially mediated by MSC expression of IDO [109].The expansion of Bregs with increased secretion of IL-10 is one of the regulatory mechanisms involved in the combinatorial cellbased immunomodulation strategy for facilitating the induction of mixed chimerism and permanent tolerance [96]. In this context, IL-10 is pivotal in mediating Breg regulatory effects. Considering that Bregs are valuable inhibitors of the immune response and inflammation, MSCs may be a promising therapeutic strategy to target B-cell-mediated autoimmune diseases. As proposed by Vadasz et al., the potentiation of Breg function should be the aim of immunomodulatory drugs, contributing to better

control of autoimmune diseases [104]. In this context, compared with conventional drugs, MSCs may be more beneficial and more efficient because they are well-tolerated and non-toxic as potent immunosuppressive cells. NK cells. NK cells are granular cytotoxic lymphocyte effector cells and are one of the main cellular components of innate immunity. NK cells play an important role in both innate and adaptive immune responses against the allograft because they can distinguish between self and non-self and are capable of lysing activated targets [110]. In addition, they can produce a number of cytokines, such as tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)γ. Thus, NK cells can regulate the activities of other immune cells, such as T cells and APCs. Importantly, the effector function of NK cells is influenced by the balance between activating and inhibitory signals. Most of the data regarding MSC and NK cell interactions derive from in vitro studies, and few in vivo studies are available [22]. The MSC:NK cell interaction is interesting to investigate as modulation of NK cell function might improve transplant outcome. MSCs and NK cells interact in a complex manner with bidirectional regulatory effects [111]. Different authors have showed that MSCs are able to inhibit the cytotoxic potential, proliferation and cytokine production of activated NK cells. Furthermore, MSCs alter the phenotype of NK cells by decreasing the expression of activation markers, such as NKp44, NKp30, NKG2D and CD132 [112,113]. These inhibitory effects were mediated by soluble factors such as IDO, PGE2 and soluble HLA-G5 [72]. Other factors including IL-10, TGF-β and HGF may play additional roles. However, impairment of NK-cell cytotoxicity by MSCs required cell–cell contact [114]. Chatterjee et al. demonstrated that a subset of NK cells acquired CD73 expression after co-culture with MSCs, but the expression of CD39 remained unchanged [115].These new CD73+ NK cells are able to convert adenosine 5′-monophosphate into adenosine, which is known for its immunoregulatory effects, and modulate NK cell biology in an autocrine or paracrine manner. Thus, CD73+ NK cells may represent a new subset of regulatory NK cells that are generated after co-culture with MSCs, which lead to their immunomodulation effects. MSCs are reported to be susceptible to NK-cellmediated lysis. Remarkably, both allogeneic and autologous MSCs are killed to the same degree by activated NK cells [111,112,116]. Depending on their origin, MSCs can be killed by different mechanisms. TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways preferentially lyze fetal and adult MSCs, respectively [117]. Among several strategies to rescue MSCs from NK-cell-mediated

Large-scale expansion of MSCs with the use of Quantum cytotoxicity [118], IFN-γ priming of MSCs leads to decreased NK cytotoxicity through up-regulation of MHC class I expression while promoting the suppressive function of MSCs [119,120]. Moreover, it was recently shown that the stimulation of specific toll-like receptors may protect MSCs from NK cell lysis [121]. Interestingly, the effects of MSCs on invariant NK T (iNKT) cells and reciprocal interactions between the two populations were reported. iNKT cells are an unconventional T-cell subset expressing an invariant T-cell receptor α chain (Vα24-Jα18) paired with a limited T-cell receptor Vβ chain repertoire (Vβ11) and NK-cell-related markers. This unique subset bridges innate and adaptive immunity and plays an important role in both protective and regulatory immune responses [122]. iNKT cells regulate a broad range of immune responses by recognizing glycolipids presented by the non-classical MHC molecule CD1d.The regulatory cascade initiated by regulatory iNKT cells is essential for the maintenance of tolerance as well as for preventing autoimmune disease development [123]. MSCs inhibited iNKT cell expansion, proliferation and IFN-γ secretion, and we speculate that PGE2 may have been involved. However, MSCs were not lysed by activated iNKT cells, which is consistent with the absence of CD1d expression on MSCs because the latter is known to be the restrictive element for iNKT cell–mediated antigen recognition and activation [124]. A better understanding of the mechanisms underlying MSC:NK crosstalk remains an important research goal. Previously, NK cells were only recognized as killers. However, they are currently thought to have important functions that modulate adaptive immune responses and play immunoregulatory roles. To ensure a therapeutic benefit, optimization of MSCbased immunotherapy is important and may involve the reduction of NK cell activity. Thus, re-educating NK cells by MSCs to become more regulatory (such as CD73+ NK) or tolerogenic will pave the way for new immunotherapeutic approaches. These new regulatory NK-cell subsets may create a tolerogenic environment that control inflammation and maintain immune homeostasis [125]. Conclusions Our review introduces novel concepts regarding the immunomodulatory properties of MSCs. During coculture, crosstalk between MSCs and immune cells influences the biology of the latter. Of importance, different regulatory immune cells, such as Tregs, Bregs, regulatory APCs and NK cells, are induced and expanded. By doing so, MSCs create a gathering of regulatory immune cells that actively compete in the

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