Alessandra Dondero

Natural Killer Cell-Mediated Killing of Freshly Isolated Neuroblastoma Cells Critical Role of DNAX Accessory Molecule-1–Poliovirus Receptor Interaction

In the present study, we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor–ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts, similar to neuroblastoma cell lines, involves NKp46 and NKp30 (natural cytotoxicity receptors). However, freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover, a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably, susceptibility to lysis directly correlated with the surface expression, on neuroblasts, of poliovirus receptor [PVR (CD155)], a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed, PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover, monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus, assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing.

NK Cells Recognize and Kill Human Glioblastoma Cells with Stem Cell-Like Properties

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 cell-cultured 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.

Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors

NKG2D and natural cytotoxicity receptors (NCR) are essential recognition structures that mediate NK cell activation. NKG2D and NCR signaling is achieved through membrane association with signaling adaptors. The adaptors that associate with NCR — such as CD3ζ, FcRγ and KARAP/DAP12 — bear intracytoplasmic immunoreceptor tyrosine‐based activation motifs that activate Syk protein tyrosine kinases. Human NKG2D associates with the DAP10 transmembrane adaptor, which bears a YxxM motif and activates the phosphatidylinositol 3‐kinase pathway. In the mouse, a short NKG2D‐S isoform, generated by Nkg2d alternative splicing, can associate with either DAP10 or KARAP/DAP12. Here, we report that neither short human NKG2D alternative transcripts nor NKG2D association with KARAP/DAP12 was detected in activated human NK cells. Despite these results, NK cell triggering by both recombinant soluble NKG2D ligands (MICA and ULBP‐1) and anti‐NCR cross‐linking antibodies inducedsimilar CD25 expression, NK cell proliferation and cytokine production. In contrast, NKG2D triggering by anti‐NKG2D antibodies did not lead to any detectable activation signals. These data thus show that target recognition via NKG2D or NCR triggers all aspects of NK activation, and pave the way for further dissection of the signaling pathways induced by NK cell recognition of ULBP‐1 and MICA.

The interaction of human natural killer cells with either unpolarized or polarized macrophages results in different functional outcomes

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.

Both CD133+ and CD133- medulloblastoma cell lines express ligands for triggering NK receptors and are susceptible to NK-mediated cytotoxicity.

Adoptive cellular immunotherapy has been proposed as an additional treatment of medulloblastoma, an intracranial tumor characterized by a particularly poor prognosis. However, little is known on the ability of the immune system to effectively attack this tumor. In this study, we show that activated human NK cells efficiently kill medulloblastoma cell lines in vitro. NK‐mediated killing involved different activating receptors (including NKp46, NKp30, DNAM‐1 and NKG2D) and correlated with the presence of their specific ligands on tumor cells. In contrast, the absence of major adhesion interactions, such as LFA‐1/ICAM did not impair the NK‐mediated cytotoxicity. Medulloblastoma expressed a number of tumor‐associated molecules including CD146 and CD133, considered a marker for cancer stem cells. Remarkably, both CD133‐positive and CD133‐negative cell lines were susceptible to lysis. Tumor cells also expressed molecules that are currently used as diagnostic tools for neuroblastoma cell identification. In particular, B7 homolog 3 (B7‐H3) was expressed by all the medulloblastoma cell lines analyzed, while the presence of GD2 and NB84 was restricted to given cell lines and/or marked a defined tumor cell subset.

Neuroblastoma-Derived TGF-β1 Modulates the Chemokine Receptor Repertoire of Human Resting NK Cells

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56dim subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-β1, and rTGF-β1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-β1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-β2–conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

Human NK cell infusions prolong survival of metastatic human neuroblastoma-bearing NOD/scid mice

AimSeveral lines of evidence suggest that NK cell immunotherapy may represent a successful approach in neuroblastoma (NB) patients refractory to conventional therapy. However, homing properties, safety and therapeutic efficacy of NK cell infusions need to be evaluated in a suitable preclinical murine NB model.Materials and methodsHere, the therapeutic efficacy of NK cell infusions in the presence or absence of NK-activating cytokines have been evaluated in a NB metastatic model set up in NOD/scid mice, that display reduced functional activity of endogenous NK cells.ResultsIn NOD/scid mice the injected NB cells rapidly reached all the typical sites of metastatization, including bone marrow. Infusion of polyclonal IL2-activated NK cells was followed by dissemination of these cells into various tissues including those colonized by metastatic NB cells. The early repeated injection of IL2-activated NK cells in NB-bearing NOD/scid mice significantly increased the mean survival time, which was associated with a reduced bone marrow infiltration. The therapeutic effect was further enhanced by low doses of human recombinant IL2 or IL15.ConclusionOur results indicate that NK-based adoptive immunotherapy can represent a valuable adjuvant in the treatment of properly selected NB patients presenting with metastatic disease, if performed in a minimal residual disease setting.

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells

Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G-mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56brigh) and CD56dim NK cells. Such sHLA-G-mediated down-modulations were reverted by adding anti-HLA-G or anti-ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G-treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G-mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G-mediated inhibition of NK-cell functions.

TLR activation of tumor-associated macrophages from ovarian cancer patients triggers cytolytic activity of NK cells

We analyzed the functional outcome of the interaction between tumor‐associated macrophages (TAMs) and natural killer (NK) cells. TAMs from ascites of ovarian cancer patients displayed an alternatively activated functional phenotype (M2) characterized by a remarkably high frequency and surface density of membrane‐bound IL‐18. Upon TLR engagement, TAMs acquired a classically activated functional phenotype (M1), released immunostimulatory cytokines (IL‐12, soluble IL‐18), and efficiently triggered the cytolytic activity of NK cells. TAMs also induced the release of IFN‐γ from NK cells, which however was significantly lower compared with that induced by in vitro‐polarized M2 cells. Most tumor‐associated NK cells displayed a CD56bright, CD16neg or CD56bright, CD16dim phenotype, and very poor cytolytic activities, despite an increased expression of the activation marker CD69. They also showed downregulation of DNAM‐1, 2B4, and NTB‐A activating receptors, and an altered chemokine receptor repertoire. Importantly however, when appropriately stimulated, NK cells from the patients, including those cells isolated from ascites, efficiently killed autologous TAMs that expressed low, “nonprotective” levels of HLA class I molecules. Overall, our data show the existence of a complex tumor microenvironment in which poorly cytolytic/immature NK cells deal with immunosuppressive tumor‐educated macrophages.

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

ABSTRACT The prognosis of high-risk neuroblastoma (NB) remains poor, although immunotherapies with anti-GD2 antibodies have been reported to provide some benefit. Immunotherapies can be associated with an IFNγ storm that induces in tumor cells the “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls). Tumor cells can also constitutively express PD-Ls in response to oncogenic signaling. Here, we analyze the constitutive and the inducible surface expression of PD-Ls in NB cells. We show that virtually all HLA class Ipos NB cell lines constitutively express PD-L1, whereas PD-L2 is rarely detected. IFNγ upregulates or induces PD-L1 both in NB cell lines in vitro and in NB engrafted nude/nude mice. Importantly, after IFNγ stimulation PD-L1 can be acquired by NB cell lines, as well as by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interestingly, in one patient NB cells were poorly responsive to IFNγ stimulation, pointing out that responsiveness to IFNγ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we document the presence of lymphocytes expressing the PD-1 receptor in NB-infiltrated bone marrow of patients. PD-1pos cells are mainly represented by αβ T cells, but also include small populations of γδ T cells and NK cells. Moreover, PD-1pos T cells have a higher expression of activation markers. Overall, our data show that a PD-L1-mediated immune resistance mechanism occurs in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches.