In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cellcultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.

The cross-talk among cells of the innate immunity can greatly affect both innate and adaptive responses. Here we analyzed the molecular interactions between human natural killer (NK) cells and autologous macrophages. Activated NK cells killed M0 and M2, whereas M1 macrophages were more resistant to lysis because of their higher expression of HLA class I molecules. Following exposure to LPS or bacillus Calmette-Guérin, M0 and M2, but not polarized (endotoxin tolerant) M1 macrophages, induced strong activation of resting NK cells. The expression of CD69 and CD25 activation markers and the acquisition of cytotoxicity against tumor cells and immature dendritic cells required soluble factors being mostly contact independent. On the contrary, IFN-γ production was contact dependent and required the interaction of DNAM-1 and 2B4 (on NK) with their ligands on macrophages as well as IL-18. IL-18 was involved also in the acquisition of CCR7 by NK cells. Interestingly, M0 and M2 cells expressed a membrane-bound form of IL-18, which was released in small amounts after LPS treatment. Our data indicate that, upon interaction with M0 macrophages exposed to microbial products, NK cells may amplify classical type 1 immune responses. In addition, M1-polarizing stimuli can rescue M2 macrophages from their immunomodulatory state and shape their functional behavior toward NK stimulatory capability.cytolityc activity | proinflammatory cytokines | lypopolissacaride | molecular interactions | nectins' family

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.

In the course of inflammatory responses in peripheral tissues, NK cells may be exposed to cytokines such as IL-12 and IL-4 released by other cell types that may influence their functional activities. In the present study we comparatively analyzed purified human peripheral blood NK cells that had been exposed to either IL-12 or IL-4 during short (overnight) incubation. We show that although IL-12-cultured NK cells produced abundant IFN-γ, TNF-α, and GM-CSF in response to stimuli acting on the NKp46-activating receptor, IL-4-cultured NK cells did not release detectable levels of these cytokines. In contrast, IL-4-cultured NK cells produced significant levels of TNF-α and GM-CSF only when stimulated with PMA and ionomycin. In no instance could the production of IL-5 and IL-13 be detected. Importantly, IL-12-cultured, but not IL-4-cultured, NK cells displayed strong cytolytic activity against various tumor cells or immature dendritic cells (DCs). Moreover, only NK cells that had been cultured in IL-12 were able to induce substantial DC maturation. Our data suggest that NK cells exposed to IL-12 for a time interval compatible with in vivo responses may favor the selection of appropriate mature DCs for subsequent Th1 cell priming in secondary lymphoid organs. On the contrary, NK cells exposed to IL-4 do not exert DC selection, may impair efficient Th1 priming, and favor either tolerogenic or Th2-type responses.

While during the first trimester of pregnancy natural killer (NK) cells represent the most abundant lymphocyte population in the decidua, their actual function at this site is still debated. In this study we analyzed NK cells isolated from decidual tissue for their surface phenotype and functional capability. We show that decidual NK (dNK) cells express normal surface levels of certain activating receptors, including NKp46, NKG2D, and 2B4, as well as of killer cell immunoglobulinlike receptors (KIRs) and CD94/NKG2A inhibitory receptor. In addition, they are characterized by high levels of cytoplasmic granules despite their CD56 bright CD16 ؊ surface phenotype. Moreover, we provide evidence that in dNK cells, activating NK receptors display normal triggering capability whereas 2B4 functions as an inhibitory receptor. Thus, cross-linking of 2B4 resulted in inhibition of both cytolytic activity and interferon-␥ (IFN-␥) production. Clonal analysis revealed that, in the majority of dNK cell clones, the 2B4 inhibitory function is related to the defi- IntroductionNatural killer (NK) cells represent a lymphocyte population capable of recognizing and lysing not only tumor cells but also virus-infected cells in the absence of previous sensitization. 1,2 NK-cell function is regulated by a balance between activating and inhibitory signals. 3 Among peripheral-blood NK (pNK) cells, 2 subsets can be identified on the basis of CD56 surface expression: those with low/intermediate levels of CD56 (CD56 dim ) and those with high levels of CD56 (CD56 bright ). 4 CD56 dim NK cells represent the majority (Ͼ 90%) of pNK cells, contain abundant cytoplasmic granules, are highly cytotoxic, and mediate antibody-dependent cell cytotoxicity (ADCC) against IgG-coated target cells through the engagement of CD16. 5 The phenotypically and functionally distinct CD56 bright subset, representing less than 10% of pNK cells, does not express CD16 molecules or expresses low-density CD16 molecules, does not contain cytoplasmic granules, and is poorly cytolytic but has greater cytokine-production capacity. 4 In addition, the 2 pNK-cell subsets differ in their expression of killer-cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A receptor. Thus, CD56 bright pNK cells have low to absent expression of KIRs, whereas they express high levels of CD94/NKG2A inhibitory receptor. In contrast, most CD56 dim pNK cells are KIR positive and express low levels of CD94/NKG2A. 4 During the first trimester of pregnancy, NK cells constitute 50% to 90% of the lymphocytes present in the decidua, a tissue in which B and T lymphocytes are infrequent. It is currently believed that in the early stages of gestation, decidual NK (dNK) cells may play an important role in the control of trophoblastic growth, differentiation, and invasion. Decidual NK cells are phenotypically similar to the CD56 bright pNK cell subset. However, transcriptional geneexpression profiling has shown that dNK cells express both perforin and granzymes at a similar or even higher level than CD56 di...