Natural Killer Cell-mediated Antitumor Research Articles

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Increased CaMKK2 Expression Is an Adaptive Response That Maintains the Fitness of Tumor-Infiltrating Natural Killer Cells.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been interest in developing CaMKK2 inhibitors as potential anticancer therapeutics. One impediment to clinical development of these agents is that the roles of CaMKK2 in other cellular compartments within the tumor immune microenvironment remain to be established. We report herein that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. NK cell-intrinsic deletion of CaMKK2 increased metastatic progression in several murine models, establishing a critical role for this enzyme in NK cell-mediated antitumor immunity. Ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK-cell survival. These results indicate an important scaffolding function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDD) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactic acid-rich tumor microenvironment. The findings of this study should inform strategies to manipulate the CaMKK2-signaling axis as a therapeutic approach in cancer.

Epigenetic Regulation of NK Cell-Mediated Antitumor Immunity.

Natural killer (NK) cells are critical innate lymphocytes that can directly kill target cells without prior immunization. NK cell activation is controlled by the balance of multiple germline-encoded activating and inhibitory receptors. NK cells are a heterogeneous and plastic population displaying a broad spectrum of functional states (resting, activating, memory, repressed, and exhausted). In this review, we present an overview of the epigenetic regulation of NK cell-mediated antitumor immunity, including DNA methylation, histone modification, transcription factor changes, and microRNA expression. NK cell-based immunotherapy has been recognized as a promising strategy to treat cancer. Since epigenetic alterations are reversible and druggable, these studies will help identify new ways to enhance NK cell-mediated antitumor cytotoxicity by targeting intrinsic epigenetic regulators alone or in combination with other strategies.

Exosome-Containing Preparations From Postirradiated Mouse Melanoma Cells Delay Melanoma Growth In Vivo by a Natural Killer Cell–Dependent Mechanism

To investigate the ability of radiation to stimulate exosome release from melanoma cells and to characterize the resulting exosome-containing vesicle preparations for their ability to promote a host antitumor immune response. Cultured B16F10 murine melanoma cells or tumors were irradiated, and secreted extracellular vesicles were isolated and characterized. The exosome-containing vesicle preparations were injected into fresh tumors in syngeneic mice, and tumor growth and infiltrating T cells and natural killer (NK) cells were characterized. Irradiation stimulated exosome release from B16F10 murine melanoma cells. Exosome preparations from irradiated cell culture supernatants were biologically active, as demonstrated by uptake into recipient cells and by the ability to induce dendritic cell maturation and activation in vitro. Intratumoral injection significantly delayed tumor growth in vivo, whereas injection of similar preparations from non irradiated cells had no effect. The antitumor effect was correlated to an increase in interferon gamma-producing tumor-infiltrating NK cells. Pretreatment of the host mice with anti-NK cell antibodies abolished the effect, whereas pretreatment with anti-CD8+ T-cell antibodies did not. Exosomes from irradiated cells, or synthetic mimics, might provide an effective strategy for potentiation of NK cell-mediated host antitumor immunity.

Myeloid deletion of phosphoinositide-dependent kinase-1 enhances NK cell-mediated antitumor immunity by mediating macrophage polarization

ABSTRACT A large number of heterogeneous macrophages can be observed in solid tumor lesions. Classically activated M1 macrophages are a powerful killer of cancer cells. In contrast, tumor-associated macrophages (TAMs) are often referred to as M2 phenotype and usually impair tumor immunity mediated by cytotoxic lymphocytes, natural killer (NK) cells and CD8+ T cells. Therefore, orchestrating M2 to M1 reprogramming will provide a promising approach to tumor immunotherapy. Here we used a PyMT-induced spontaneous breast cancer model in which M2-polarized macrophages were abundant. This M2 phenotype was closely related to tumor progression and immune dysfunction of NK cells and CD8+ T cells. We then found that these TAMs showed increased energy expenditure and over-activation of two kinases, Akt and mammalian target of rapamycin (mTOR). Myeloid inactivation of phosphoinositide-dependent kinase-1 (PDK1), the upstream regulator for Akt and mTOR signaling, significantly reduced excessive metabolic activation of macrophages. Notably, the loss of PDK1 significantly led to regression of breast cancer and prevented lung metastasis. Mechanistically, PDK1 deficiency mainly inhibited the activation of mTOR complex 1 (mTORC1), transforming TAMs into M1 phenotype, thereby reversing tumor-related dysfunction of T cells and NK cells. Therefore, targeting PDK1 may be a new approach for M2 macrophage-enriched solid tumor immunotherapy.

IL-21 Signaling in the Tumor Microenvironment.

IL-21 is an immunomodulatory cytokine produced by natural killer (NK) cells and T cells that has pleiotropic roles in immune and nonimmune cells. IL-21 can modulate innate and specific immunity activities. It is a potent stimulator of T and natural killer cell-mediated antitumor immunity but also has pro-inflammatory functions in many tissues and is involved in oncogenesis. It is important to understand IL-21 biology in these different situations to ensure the maximal benefit of therapeutic strategies targeting this cytokine. This chapter summarizes IL-21 characteristics and signaling, its role in immune system components, and its use in cancer immunotherapies.

Administration of Gemcitabine After Pancreatic Tumor Resection in Mice Induces an Antitumor Immune Response Mediated by Natural Killer Cells

Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D× p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon. We co-delivered a transposon encoding a constitutively active form of Akt2 (myrAkt2). Carcinogenesis and histopathologic features of tumors were examined. Metastasis was monitored by bioluminescence imaging. Tumors were resected and mice were given gemcitabine, and tumor recurrence patterns and survival were determined. Immune cells were collected from resection sites and analyzed by flow cytometry and in depletion experiments. After electroporation of oncogenic plasmids, mice developed a single pancreatic tumor nodule with histopathologic features of human PDAC. Pancreatic tumors that expressed myrAkt2 infiltrated the surrounding pancreatic tissue and neurons and became widely metastatic, reflecting the aggressive clinical features of PDAC in patients. Despite early tumor resection, mice died from locally recurring and distant tumors, but adjuvant administration of gemcitabine after tumor resection prolonged survival. In mice given adjuvant gemcitabine or vehicle, gemcitabine significantly inhibited local recurrence of tumors, but not metastasis to distant organs, similar to observations in clinical trials. Gemcitabine inhibited accumulation of CD11b+Gr1intF4/80int myeloid-derived suppressor cells at the resection margin and increased the number of natural killer (NK) cells at this location. NK cells but not Tcells were required for gemcitabine-mediated antitumor responses. Gemcitabine administration after resection of pancreatic tumors in mice activates NK cell-mediated antitumor responses and inhibits local recurrence of tumors, consistent with observations from patients with PDAC. Transgenic mice with resectable pancreatic tumors might be promising tools to study adjuvant therapy strategies for patients.

Natural Killer Cell-Mediated Antitumor Effect of Syngeneic Hematopoietic Stem Cell Transplantation

Introduction: Autologous hematopoietic stem cell transplantation (HSCT) has been thought to be important in tumor eradication induced by high-dose chemotherapy with hematopoietic stem cell rescue. It was reported recently that autologous HSCT can also induce a strong antitumor immunity by homeostatic proliferation (HP) of T cells following preconditioning-induced lymphopenia. It is known that T cell HP is driven by the recognition of self antigens, and that the availability of tumor antigens during HP leads to effective antitumor autoimmunity with specificity and memory. The effect is mediated by a reduction in the activation threshold of low-affinity tumor-specific T cells, leading to their preferential engagement and expansion. However, the mechanisms of antitumor immunity induced by the autologous HSCT are not fully understood. Especially, in addition to adaptive immunity, the role of innate immunity is unclear. In this study, we examined the role of natural killer (NK) cells on the antitumor effect in HSCT recipients using mouse xenograft models. Furthermore, since neutrophils have been reported to be required for homeostasis, maturation, and function of NK cells in human and mice (Jaeger et al. J. Eep. Med. 209:565, 2012), we focused on the interaction between neutrophils and NK cells in tumors after syngeneic HSCT.Results: BALB/c mice were injected subcutaneously with CT26 murine colon cancer cells shortly after lethal irradiation (9Gy), and then bone marrow cells (5 x 106 cells) and splenocytes (4 x 106 cells) from syngeneic donor mice were infused into the recipient mice. Tumor growth was significantly suppressed in the HSCT recipients as previously reported (Narumi et al. Gene Ther 19:34, 2012, Udagawa et al. J. Immunol 191:3440, 2013). To clarify the contribution of NK cells to antitumor effects after syngeneic HSCT, we depleted NK cells by intraperitoneal injection of anti-asialo GM1 antibody in the HSCT recipients. The tumor growth inhibition was largely cancelled by the depletion of NK cells especially during an early period after HSCT (Figure 1a: 2303 mm3 vs. 1383 mm3 at day26; P = 0.0042). Flow cytometry showed that the frequency of surface CD107a+ NK cells, which is an activation marker, was significantly higher in tumors of HSCT recipients (24.4% vs. 6.3%; P = 0.0022) than that in non-HSCT mice, indicating that syngeneic HSCT induces NK cell activation which contributes to the antitumor effect.Next, we examined what factor influences the activation of NK cells in tumors after syngeneic HSCT. We found that a large number of neutrophils accumulated in tumors after HSCT by immunostaining. Thus, we focused on the relationship between neutrophils and NK cells in tumors after HSCT. The cytokine antibody array showed that lungkine (CXCL15), which is one of mouse ELR+ CXC chemokine and recruits neutrophils to lung airspaces during pneumonia, was markedly up-regulated in tumor at day14 after HSCT as compared with non-HSCT tumor. The real-time quantitative polymerase chain reaction confirmed that the lungkine mRNA level was higher in HSCT-tumor at day 6 than that in non-HSCT tumor (Figure 1b: relative expression: 4.0 vs. 0.3; P = 0.0075).Then, to investigate the effects of neutrophils on NK cells in tumors, we depleted neutrophils by an intraperitoneal injection of anti-Ly6G antibody from days 6 to 12 after syngeneic HSCT. Neutrophils were almost depleted in the spleens and tumors. The tumor growth was significantly suppressed in HSCT recipients as previously shown, whereas the depletion of neutrophil significantly increased tumor volumes in HSCT recipients but not in non-HSCT mice at day 10 (Figure 1c: 143 mm3 vs. 84 mm3; P = 0.024). Flow cytometry revealed that the neutrophil-depletion did not change the frequency of NK cells within live cells in tumors at days 14 and 21, however, neutrophil-depletion increased the fraction of dead NK cells at day 14 (Figure 1d: 32.2% vs. 19.2%; P = 0.042). Thus, neutrophils in tumors may prevent NK cells from cell death induction during HP, leading to the significant antitumor effect.Conclusions: NK cells play a major role in the antitumor effect in the early period after syngeneic HSCT. The neutrophils in tumors may support a sustained antitumor effect of NK cells. This novel relationship reveals an important aspect of antitumor immunity induction in HSCT recipients and may contribute to the development of effective therapeutic strategy for cancer using HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Maraba MG1 Virus Enhances Natural Killer Cell Function via Conventional Dendritic Cells to Reduce Postoperative Metastatic Disease

This study characterizes the ability of novel oncolytic rhabdoviruses (Maraba MG1) to boost natural killer (NK) cell activity. Our results demonstrate that MG1 activates NK cells via direct infection and maturation of conventional dendritic cells. Using NK depletion and conventional dendritic cells ablation studies in vivo, we established that both are required for MG1 efficacy. We further explored the efficacy of attenuated MG1 (nonreplicating MG1-UV(2min) and single-cycle replicating MG1-Gless) and demonstrated that these viruses activate conventional dendritic cells, although to a lesser extent than live MG1. This translates to equivalent abilities to remove tumor metastases only at the highest viral doses of attenuated MG1. In tandem, we characterized the antitumor ability of NK cells following preoperative administration of live and attenuated MG1. Our results demonstrates that a similar level of NK activation and reduction in postoperative tumor metastases was achieved with equivalent high viral doses concluding that viral replication is important, but not necessary for NK activation. Biochemical characterization of a panel of UV-inactivated MG1 (2-120 minutes) revealed that intact viral particle and target cell recognition are essential for NK cell-mediated antitumor responses. These findings provide mechanistic insight and preclinical rationale for safe perioperative virotherapy to effectively reduce metastatic disease following cancer surgery.

Intergroup ALFA/GOELAMS randomized phase II trial of lirilumab anti-KIR monoclonal antibody (IPH2102/BMS986015) as maintenance treatment in elderly patients with acute myeloid leukemia (EFFIKIR trial).

TPS3117^ Background: Inhibitory killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK cell-mediated antitumor efficacy and increased survival in patients with acute myeloid leukemia (AML) upon haploidentical stem cell transplantation from KIR-mismatched donors(Ruggeri, Blood 2007). Anti-KIR antibody treatment resulted in long-term survival in SCID mice inoculated with lethal autologous AML cells (Romagne, Blood 2009). Lirilumab is a second generation fully human monoclonal antibody targeting the major inhibitory KIR on NK cells. The objectives of this study are to determine if maintenance therapy with lirilumab can improve leukemia-free survival (LFS) of elderly patients in first complete remission (CR1) of AML and to assess two dose schedules leading to either intermittent or continuous KIR occupancy. Methods: EFFIKIR is a randomized double-blind 3-arm placebo controlled trial of lirilumab in elderly patients in CR1 of AML. Patients aged 60 to 80 in CR1 of AML following standard induction and consolidation programs are randomly allocated to receive placebo or lirilumab given at either 0.1 mg/kg q 12 weeks or 1 mg/kg q 4 weeks according to a minimization algorithm adjusting for center, primary vs. secondary AML, no. of consolidation cycles (1 vs. 2) and cytogenetics (intermediate vs. high risk). Patients are to receive up to 2 yrs of therapy. ECOG performance status of 0-1, adequate hematologic, liver and renal function, and recovery from toxicities of prior chemotherapies are required. Patients are excluded if they are eligible for bone marrow transplantation and if the time interval since last consolidation exceeds 3 mos. The primary endpoint is LFS based on independent central review. The trial will accrue 50 patients in each arm and is powered (80%) to detect an improvement in LFS with a hazard ratio of 0.60 and a one-sided alpha of 0.05. Each dose schedule will be compared to placebo using a Hochberg procedure. The first patient was randomized on 12/11/2012. Clinical trial information: NCT01687387.

Clinical-Scale Isolation of Interleukin-2-Stimulated Liver Natural Killer Cells for Treatment of Liver Transplantation with Hepatocellular Carcinoma

Tumor recurrence is the main limitation of liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) and can be promoted by immunosuppressants. However, there is no prevention or treatment for HCC recurrence after LT. Here we describe a clinical-scale method for an adoptive immunotherapy approach that uses natural killer (NK) cells derived from deceased donor liver graft perfusate to prevent tumor recurrence after LT. Liver mononuclear cells (LMNCs) that were extracted from deceased donor liver graft perfusate contained a high percentage of NK cells (45.0 ± 4.0%) compared with peripheral blood mononuclear cells (PBMCs) (21.8 ± 5.2%) from the same donor. The CD69 activation marker and the natural cytotoxicity receptors, NKp44 and NKp46, were expressed at high levels in freshly isolated liver NK cells. Furthermore, interleukin-2 (IL-2)-stimulated NK cells showed greater upregulation of activation markers and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is critical for NK cell-mediated antitumor cell death and increased production of interferon. Moreover, IL-2 stimulation induced LMNCs to exhibit a strong cytotoxicity against NK-susceptible K562 target cells compared with PBMCs (p < 0.01). Finally, we also showed that the final product contained a very low T-cell contamination (0.02 ± 10(6) cells/kg(-1)), which reduces the risk of graft-versus-host disease (GVHD). Collectively, our results suggest that the adoptive transfer of IL-2-stimulated NK cells from deceased donor liver graft perfusate could be a promising treatment for LT patients with HCC.

Fusion Proteins of Ligands for NKG2D and CD20-Directed ScFvs Sensitize Lymphomas for NK Cell-Mediated Lysis

AbstractAbstract 2843Natural killer group 2 member D (NKG2D) is an important activating receptor controlling cytotoxicity of natural killer (NK) cells and T cells and plays an important role in immune surveillance against tumors. For redirecting NK cells to B-lymphoid tumor cells two recombinant bifunctional antibody-based fusion proteins were designed in order to coat malignant cells with ligands for NKG2D and attract NK cells. Therefore, a human CD20-directed single-chain fragment variable (scFv) was fused to NKG2D-specific ligands, either MHC class I chain-related protein A (MICA) or unique long 16-binding protein 2 (ULBP2). These two fully human fusion proteins, designated MICA:CD20 and ULBP2:CD20, respectively, were expressed in eukaryotic cells and purified to homogeneity. Size exclusion chromatography revealed that both purified proteins predominantly formed monomers. MICA:CD20 and ULBP2:CD20 specifically and simultaneously bound to CD20 and NKG2D and efficiently mediated lysis of lymphoma cell lines with mononuclear cells from healthy donors as effector cells. Analysis of the activation status of NKG2D-positive T cells and NK cells revealed that MICA:CD20 and ULBP2:CD20 activated resting NK cells, but not T cells, indicating that NK cells were the relevant effector cell population for the two molecules. In cytotoxicity assays using human NK cells from healthy donors, both agents sensitized lymphoma cell lines as well as fresh tumor cells for NK cell-mediated lysis. MICA:CD20 and ULBP2:CD20 induced lysis at low nanomolar concentrations with half maximum effective concentrations between 1 and 4 nM depending on target cells. Interestingly, ULBP2:CD20 exhibited a higher cytolytic potential than MICA:CD20 in terms of maximum lysis. Importantly, MICA:CD20 and ULBP2:CD20 induced lysis of 13/13 tested primary tumor cell samples from patients with different B cell malignancies including chronic lymphocytic leukemia, mantle cell lymphoma and marginal zone lymphoma. Interestingly, cell surface expression of endogenous MICA and ULBP2 was low or not detectable on fresh tumor cells. In addition, ULBP2:CD20 was also capable of inducing lysis of tumor cells in cytotoxicity experiments using autologous patient-derived NK cells as effector cells, indicating that the triggering signal was sufficient to overcome inhibition by interactions between killer cell immunoglobulin-like receptors and MHC class I molecules. Moreover, both MICA:CD20 and ULBP2:CD20 synergistically enhanced antibody-dependent cellular cytotoxicity (ADCC) by the monoclonal antibody daratumumab directed against CD38 which is co-expressed together with CD20 on certain B cell lymphomas. This approach of simultaneously triggering ADCC and natural cytotoxicity by these bifunctional fusion proteins may represent a promising strategy to achieve stronger NK cell-mediated antitumor responses. Disclosures:de Weers:Genmab : Employment. van De Winkel:Genmab: Employment. Parren:Genmab: Employment.

Expression of WSX1 in Tumors Sensitizes IL-27 Signaling-Independent Natural Killer Cell Surveillance

It is well known that the interleukin (IL)-27 receptor WSX1 is expressed in immune cells and induces an IL-27-dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared with normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenicity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL-27 signaling but is mainly dependent on natural killer (NK) cell surveillance. Deficiency of either the IL-27 subunit EBV-induced gene 3 or the IL-27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, whereas the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on up-regulation of NKG2D ligand expression. Our results reveal an IL-27-independent function of WSX1: sensitizing NK cell-mediated antitumor surveillance via a NKG2D-dependent mechanism.

Dendritic cell-mediated NK cell activation is controlled by Jagged2–Notch interaction

Natural killer (NK) cells regulate various immune responses by exerting cytotoxic activity or secreting cytokines. The interaction of NK cells with dendritic cells (DC) contributes to NK cell-mediated antitumor or antimicrobial responses. However, the cellular and molecular mechanisms for controlling this interaction are largely unknown. Here, we show an involvement of Jagged2-Notch interaction in augmenting NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which was abrogated by depleting NK cells. Moreover, Jag2-A20 cells exerted a suppression on the growth of nonmanipulated A20 cells in SCID mice in an NK-dependent manner. Consistently, coinoculation of A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the growth of A20 cells in mice. Stimulation of NK cells with Jagged2 directly enhanced their cytotoxicity, IFN-gamma production, and proliferation. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was suppressed by a gamma-secretase inhibitor. These results indicate that the Jagged2-Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may provide an approach to induce potent tumor immunity or to inhibit certain autoimmune diseases caused by NK cell activation.

Induction of Natural Killer Cell-dependent Antitumor Immunity by the Autographa californica Multiple Nuclear Polyhedrosis Virus

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

Local interferon‐α gene therapy elicits systemic immunity in a syngeneic pancreatic cancer model in hamster

The interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis and immunomodulation. We previously examined the two antitumor mechanisms, taking advantage of the fact that IFN-alpha did not show cross-species activity in its in vivo effect. In a nude mouse subcutaneous xenograft model using human pancreatic cancer cells, the expression of human IFN-alpha effectively induced cell death of human pancreatic cancer cells, whereas mouse IFN-alpha augmented antitumor immunity by stimulation of natural killer cells. Here, we extended our investigation to a syngeneic pancreatic cancer model, so that the integrated antitumor activity of local IFN-alpha gene therapy, including the antiproliferative, proapoptotic, antiangiogeneic and immunomodulatory effects, can be evaluated rigorously. When a recombinant hamster IFN-alpha adenovirus was injected into syngeneic subcutaneous tumors of hamster pancreatic cancer (PGHAM-1) cells in Syrian hamster, tumor growth was significantly suppressed due to cell death and T cell- and natural killer cell-mediated antitumor immunity. Moreover, in this case, tumor regression was observed not only for the injected subcutaneous tumors but also for the untreated tumors both in the peritoneal cavity and at distant sites. No significant systemic toxicity was observed in the treated hamsters. Moreover, the subcutaneous rechallenge of PGHAM-1 cells was rejected in three of four cured hamsters from the initial tumor challenge. This study further demonstrated that local IFN-alpha gene therapy is a promising therapeutic strategy for pancreatic cancer, due to its multiple mechanisms of antitumor activity and its lack of significant toxicity.

NK cell-based cancer immunotherapy

The identification of spontaneous antitumor immune responses in cancer patients and the demonstration that an intact immune system can prevent against certain forms of malignancies invigorated research efforts in the development of cancer immunotherapies. To date, numerous and diversified approaches are being investigated, thereby providing new means by which to mobilize the cellular and molecular elements of the immune system in order to destroy established cancers. While it is appreciated that tumor-specific cytotoxic T lymphocytes play a critical role in successful immunotherapy, it is becoming increasingly apparent that cellular components of both the innate and the adaptive arms of the immune system can control tumor growth. In particular, natural killer (NK) cells are emerging as key players in the cross-talk between innate and adaptive immunity. With the uncovering of the regulatory mechanisms governing NK cell biology, new immunotherapies based on the harnessing of NK cell antitumor activity are now being developed. In this review, we summarize the role of NK cells in the control of tumor growth and discuss potential cancer therapies incorporating NK cell-mediated antitumor activity.

Resistance of infant leukemia with MLL rearrangement to tumor necrosis factor-related apoptosis-inducing ligand: a possible mechanism for poor sensitivity to antitumor immunity

Malignant cells generally acquire some immune escape mechanisms for clonal expansion. Immune escape mechanisms also contribute to the failure of graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation (allo-SCT). Infant leukemias with mixed-lineage leukemia (MLL) rearrangement have a remarkably short latency, and GVL effect after allo-SCT has not been clearly evidenced in these leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- and FasL-mediated cytotoxic pathways play important roles in cytotoxic T-lymphocyte- and natural killer cell-mediated antitumor immunity and optimal GVL activity. We investigated the in vitro sensitivity of MLL-rearranged acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) cells to TRAIL- and FasL-mediated cytotoxicity. Most of cell lines and primary leukemia cells were highly resistant to TRAIL primarily owing to low cell-surface expression of death receptors in ALL and simultaneous expression of decoy receptors in AML. Nearly half of cell lines and majority of primary leukemia cells showed low sensitivity to FasL. These results suggest that resistance to death-inducing ligands, particularly to TRAIL, could be one of the mechanisms for a rapid clonal expansion and a poor sensitivity to the GVL effect in infant leukemias with MLL rearrangement.

Natural Killer Cell-Mediated Rejection of Experimental Human Lung Cancer by Genetic Overexpression of Major Histocompatibility Complex Class I Chain-Related Gene A

Studies have highlighted molecular and cell biological mechanisms of the NKG2D-NKG2D ligand system in the activation of natural killer (NK) cell and T cell functions. In this study we explore the potential of genetic overexpression of human major histocompatibility complex class I chain-related gene A (MICA), a powerful NKG2D ligand, for the induction of NK cell-mediated antitumor immunity in a humanized murine model of non-small cell lung cancer. Using tissue microarray technology we detected expression of MICA in only 30% of the samples from patients with lung cancer. Staining was always weak and focal, indicating that expression of MICA is detectable but limited in human lung cancer. Genetic overexpression of MICA by means of adenoviral transduction or transfection of expression plasmids was feasible in cell lines in vitro, primary human cancer tissue ex vivo, and in experimental human cancers in vivo. The presence of MICA on the surface of largely NK cell-resistant human lung cancer cells reestablished NK cell susceptibility and provoked NK cell-mediated antitumor immunity by murine and human NK cells in two different experimental therapy models. In this study we analyze the interaction of human NK cells with MICA-positive human cancer cells in an in vivo setting. Our data demonstrate that MICA overexpression can function as NK cell-mediated immunotherapy in experimental lung cancer.

Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages.

Virulizin, a novel biological response modifier, has demonstrated significant antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. The significant role of macrophages and NK (Natural killer) cells was implicated in the antitumor mechanism of Virulizin where expansion as well as increased activity of macrophages and NK cells were observed in mice treated with Virulizin. Depletion of macrophages compromised Virulizin-induced NK1.1+ cell infiltration into xenografted tumors and was accompanied by reduced antitumor efficacy. In the present study, involvement of macrophages in NK cell activation was investigated further. We found that depletion of NK cells in CD-1 nude mice by anti-ASGM1 antibody significantly compromised the antitumor activity of Virulizin. Cytotoxicity of NK cells isolated from Virulizin-treated mice was enhanced against NK-sensitive YAC-1 cells and C8161 human melanoma cells, but not against NK-insensitive P815 cells. An increased level of IL-12beta was observed in the serum of mice treated with Virulizin. IL-12 mRNA and protein levels were also increased in peritoneal macrophages isolated from Virulizin-treated mice. Moreover, Virulizin-induced cytotoxic activity of NK cells isolated from the spleen was abolished when an IL-12 neutralizing antibody was co-administered. In addition, depletion of macrophages in mice significantly impaired Virulizin-induced NK cell cytotoxicty. Taken together, the results suggest that Virulizin induces macrophage IL-12 production, which in turn stimulates NK cell-mediated antitumor activity.