IntroductionIn recent years, mesenchymal stem cells (MSCs) have become a promising tool for novel therapeutic approaches aimed at inhibition of the immune responses. [1][2][3] In particular, MSCs may be used to prevent/suppress graft-versus-host disease (GVHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) 4 or for the treatment of certain autoimmune diseases. 5,6 So far, results of phase 1 trials have revealed the feasibility of MSC isolation, in vitro expansion, and infusion with no reports of major adverse reactions. 7,8 The first in vitro evidences of an effective MSC-mediated immunoregulatory activity rapidly evolved into clinical use of these cells in novel protocols of adoptive immunotherapy. Therefore, it is particularly important to clarify the mechanism(s) underlying the inhibitory effect exerted by MSCs on immunocompetent cell populations.Natural killer (NK) cells are major effector cells of the innate immunity and are generally thought to play a fundamental role in antiviral and antitumor responses. 9,10 As first described by Ruggeri and colleagues 11,12 and subsequently confirmed by other groups, 13 donor-derived NK cells would be responsible for eradication of leukemic cells in acute myeloid leukemia (AML) patients who received haploidentical HSCT. Remarkably, such a graft-versusleukemia (GVL) effect was evident only in donor-recipient couples in which a killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch was present. Therefore, after selection of the most suitable donor, NK cells could be used in novel HSCT-associated immunotherapeutic strategies either as cells originating directly from transplanted CD34 ϩ hematopoietic precursors or as mature NK cells that had been highly purified from peripheral blood and infused intravenously.MSCs and NK cells have been shown to interact in vitro. [14][15][16][17] The outcome of this interaction may depend on the state of NK-cell activation and/or on the cytokines present in the milieu. Thus, it may result in altered cell function and/or survival of either one or the other cell type. We previously described that the cytokineinduced proliferation of freshly isolated, resting NK cells is highly susceptible to MSC-mediated inhibition. 14 We asked whether such inhibitory effects could be exerted also on NK-cell effector functions, such as cytotoxic activity and cytokine production. These NK-cell functions are regulated by a series of surface receptors that can transduce either inhibitory or activating signals. [18][19][20] Exposure of resting NK cells to activating cytokines, such as interleukin-2 (IL-2), induces either de novo expression or increase of surface density of the activating receptors NKp44, CD69, NKp30, and NKG2D. As the levels of surface expression of activating NK receptors are positively correlated with NK-cell function, 21,22 we analyzed whether MSCs could exert an inhibitory effect on the IL-2-induced upregulation of the major activating receptors. Thus, in parallel with the phenotypic analysis, we perf...

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Various studies analyzed the inhibitory effect exerted by mesenchymal stem cells (MSCs) on cells of the innate or acquired immunity. Myeloid dendritic cells (DCs) are also susceptible to such inhibition. In this study, we show that MSCs strongly inhibit DC generation from peripheral blood monocytes. In the presence of MSCs, monocytes supplemented with granulocytemacrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) did not acquire the surface phenotype typical of immature (CD14 ؊ , CD1a ؉ ) or mature (CD80 ؉ , CD86 ؉ , CD83 ؉ ) DCs, failed to produce IL-12, and did not induce T-cell activation or proliferation. Analysis of the molecular mechanism(s) responsible for the inhibitory effect revealed a major role of prostaglandin E 2 (PGE 2 ). Thus, addition of the PGE 2 inhibitor NS-398 restored DC differentiation and function. Moreover, PGE 2 directly added to cultures of monocytes blocked their differentiation toward DCs in a manner similar to MSCs.Although IL-6 has been proposed to play a role in MSC-mediated inhibition of DC differentiation, our data indicate that PGE 2 and not IL-6 represents the key inhibitory mediator. Indeed, NS-398 inhibited PGE 2 production and restored DC differentiation with no effect on IL-6 production. These data emphasize the role of MSCs in inhibiting early DC maturation and identifying the molecular mechanisms responsible for the inhibitory effect. (Blood. 2009; 113:6576-6583) IntroductionHuman mesenchymal stem cells (MSCs) represent a relatively rare stromal cell population that resides primarily in the bone marrow 1 but can be isolated also from other adult and fetal tissues, including adipose tissue, 2 umbilical cord blood, 3 amniotic fluid, 4 and fetal lung. 5 MSCs do not express specific markers but can be phenotypically identified on the basis of the absence of hemopoietic cell markers, namely, CD45, CD34, CD3, CD14, and by the expression of markers, such as CD29, CD90, CD73, CD105, and CD106. 6 MSCs represent multipotent cells capable of differentiating into various lineages, including adipose, osteogenic, and chondrogenic tissues. 7 They secrete several cytokines, growth factors, and extracellular matrix molecules that play an important role in the proliferation and maturation of hematopoietic stem cells (HSCs), thus revealing their potential usefulness for promoting engraftment of HSC transplantation. 8,9 Recently, MSCs have gained attraction not only in regenerative medicine but also in the prevention and treatment of graft-versus-host disease (GVHD) 10-14 thought to reflect their capability (documented in humans, rodents, and primates) of suppressing allogeneic T-cell responses. [15][16][17][18] Subsequent studies further investigated other possible cellular targets of the MSC-mediated immunosuppression. MSCs were shown to exert a strong inhibitory effect on other cells belonging to either innate or adaptive immunity, including natural killer (NK) cells, 19,20 dendritic cells (DCs), 21-23 B cells, 24 and unconventional T cells, including NKT and ␥␦ ϩ cel...

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Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR þ innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-a, IL-8 and IL-2, and activates endothelial cells. Tumour NCR þ ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR þ ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR þ ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (M MSC ), through a prostaglandin E2-dependent mechanism. M MSC were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-b. These cytokines contributed to the immunoregulatory properties of M MSC , which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, M MSC inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNg, while M2 affected only IFNg production. Moreover, M MSC inhibited the proliferation of CD8 1 T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of M MSC to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches. STEM CELLS 2016;34:1909-1921 SIGNIFICANCE STATEMENTMesenchymal stromal cells (MSCs) display various immunoregulatory activities and they are also present in the stroma of different tumors. Emerging evidences revealed that the cellular interactions occurring between tumor cells, stromal cells and immune effectors in the tumor microenvironment play a key role in the establishment of tumor immune escape. In the present study, we show that MSCs induce the polarization of macrophages toward a novel M2-like phenotype (M MSC ). M MSC are able to block natural killer cell activation and induce the expansion of Tregs, thus affecting both innate and adaptive immune responses. These data dissect a novel mechanism through which MSCs may exert their immunoregulatory activity, and may offer new clues for target-based cancer immunotherapy.

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