Cytotoxic Effect Of Natural Killer Cells Research Articles

Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer.

Bladder cancer (BC) is a common malignancy and its most prevalent type is urothelial carcinoma, which accounts for ~90% of all cases of BC. The current treatment options for BC are limited, which necessitates the development of alternative treatment strategies. Hyperthermia (HT), as an adjuvant cancer therapy, is known to improve the efficacy of chemotherapy or radiotherapy. The present study aimed to investigate the anti‑tumor effects of HT on cell survival, invasiveness, chemoresistance and immune evasion in human BC cell lines (5637, T24 and UMUC3). Calcein AM staining was performed to analyze the cytotoxicity of natural killer (NK) cells against human BC cells following HT treatment. Cell migration and invasion affected by HT were analyzed using Transwell migration and invasion assays. It was found that HT inhibited the proliferation of BC cells by downregulating the phosphorylation of protein kinase B. Moreover, HT effectively enhanced the sensitivity of BC cells to the chemotherapy drug cisplatin (DDP) and reduced the chemoresistance of DDP‑resistant cells by downregulating the expression of cadherin‑11. It was further demonstrated that HT inhibited the migration and invasion of BC cells and enhanced the cytotoxic effects of NK cells. In summary, the antineoplastic effects of HT were mediated through three main mechanisms: Enhancement of the chemosensitivity of BC cells and mitigation of DDP‑induced chemoresistance, suppression of the invasive potential of BC cells and reinforcement of the anticancer response of NK cells. Thus, HT appears to be a promising adjunctive therapy for human BC.

Oridonin enhances cytotoxic activity of natural killer cells against lung cancer

BackgroundOridonin is a Chinese herbal medicine exhibiting anti-tumor properties; however, its immune modulation capacity has yet to be elucidated. Our objective in this study was to determine whether oridonin enhances the anti-tumor activity of natural killer (NK) cells against lung cancer cells. MethodsLDH-releasing assays were used to investigate the effects of oridonin on NK-92MI cell activity against lung cancer cells. Flow cytometry and real-time PCR were used to examine the effects of oridonin on degranulation markers, cytotoxic factors, activating receptors on NK-92MI cells, and ligands in lung cancer cells. Western blot analysis provided insight into the mechanisms underlying the observed effects. ResultsOridonin enhanced the cytotoxic effects of NK-92MI cells against A549 lung cancer cells. This effect involved upregulating the expression of the degranulation marker CD107a and IFN-γ as well as activating receptors on NK cells and their ligand MICA/B. Oridonin also inhibited STAT3 phosphorylation in A549 cells and NK-92MI cells. A lung cancer mouse model confirmed the anti-tumor effects of oridonin and NK-92MI cells, wherein both treatments alone suppressed tumor growth. Oridonin was also shown to have a synergistic effect on the anti-tumor activity of NK-92MI cells. ConclusionsThe ability of oridonin to enhance the cytotoxic effects of NK cells indicates its potential as a novel therapeutic agent for the treatment of lung cancer.

Antitumor effects of NK cells expanded by activation pre‑processing of autologous feeder cells before irradiation in colorectal cancer.

Natural killer (NK) cells play a crucial role in early immune defenses against transformed cells and are used in the therapeutic management of cancer. However, it is difficult to sufficiently obtain high purity activated NK cells for clinical application. The function of NK cells is dependent on the balance of activating and inhibitory signals. Strong and diverse stimuli are required to increase the function of NK cells. Radiotherapy modulates the expression of various immunomodulatory molecules that recruit and activate NK cells. NK cell-mediated antibody-dependent cellular cytotoxicity is one of the most potent cytotoxic effects of NK cells against target cancer cells. To generate activated and irradiated autologous peripheral blood mononuclear cells (PBMCs), cytokine and monoclonal antibody stimulation followed by ionizing radiation was performed in the present study. The expanded NK cells were cultured for 21 days using activated/irradiated autologous PBMCs. Colorectal cancer cells (SW480 and HT-29) were used to analyze the expression of NK group 2D ligands and EGFR by radiation. The cytotoxicity of radiation plus NK cell-based targeted therapy against colorectal cancer cell lines was analyzed using flow cytometry. Activated and irradiated PBMCs exhibited significantly increased expression of various activating ligands that stimulated NK cells. In total, >10,000-fold high-purity activated NK cells were obtained, with negligible T-cell contamination. To confirm the antitumor activity of the NK cells expanded by this method, the expanded NK cells were treated with cetuximab, radiotherapy, or a combination of cetuximab and radiotherapy in the presence of human colorectal cancer cells. Expanded NK cells were effective at targeting human colorectal cancer cells, particularly when combined with cetuximab and radiotherapy. Thus, in the present study, a novel method for high-purity activated NK cell expansion was developed using activated and irradiated PBMCs. In addition, combined radiotherapy and antibody-based immunotherapy with expanded NK cells may be an effective strategy to enhance the efficiency of treatment against colorectal cancer.

Abstract 3181: A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice

Abstract Epithelial ovarian cancer (OVC) is the leading cause of death among gynecological malignancies. Natural killer (NK) cells are an attractive platform for immunotherapy owing to their superior safety, multiple mechanisms of cytotoxicity, and reduced risk of alloreactivity. Recently, paradigm-shifting studies have shown that brief activation of NK cells with inflammatory cytokines induces differentiation into cytokine induced memory-like (CIML) feature. CIML NK cells have demonstrated potent activity in preclinical and in recent clinical trial settings, suggesting CIML NK cells as a novel platform for genetically modified chimeric antigen receptor (CAR) cells for OVC therapy. Based on patient samples and patient-derived organoids (PDOs), we confirmed mesothelin (MSLN) as an attractive candidate for NK cells. Here, we designed and tested aMSLN CAR CIML NK cells for their functionality against OVC cell lines, PDOs, and xenograft mice. We designed the aMSLN CAR using the ScFv sequence against the proximal domain of MSLN with 4-1BB and CD3z as co-stimulatory domains. Primary NK cells were purified from peripheral blood, activated using IL-12, IL-18, and IL-15 resulting in CIML NK cells. The CAR gene was transduced into CIML NK cells via our optimized BaEV lentiviral system to achieve high transduction efficiency of 40-60%. The direct cytotoxic effect of NK cells was tested against OVC cell lines where the cells were co-cultured with NK cells for 6 hours and analyzed using apoptotic markers. Compared to CIML NK cells, aMSLN CAR CIML NK cells showed enhanced cytotoxicity against OVCAR3 (47.9% vs 14.8%), SKOV3 (29.3% vs 8.0%) and OVCAR8 (60.7% vs 39.8%) cells at a 10:1 ratio. In addition, the aMSLN CAR CIML NK cells exhibit long-term cytotoxicity when evaluated over a period of 3 days; killing efficiency of 71.7% vs 37.6% and 54.6% vs 32.8% for aMSLN CAR CIML NK cells compared to CIML NK cells in OVCAR8 cells and SKOV3 cells, respectively. To test the therapeutic potential of NK cells in a relevant in vitro model, we used PDOs. After 18-hour co-culture, the cytotoxicity was determined by evaluating apoptotic EpCAM+ cancer cells amongst the organoid cell population. Results showed that aMSLN CAR CIML NK cells caused increased apoptotic cells compared to CIML NK cells; 58.2% vs 11.0%, 4.5% vs 61.0%, and 10.5% vs 32.0% in three different PDOs. Finally, we tested the in vivo therapeutic efficacy of aMSLN CAR CIML NK cells against intraperitoneal OVCAR8-mcLuc model wherein we observed significant tumor control, with 80.86% tumor growth inhibition as measured by BLI after 2 doses of 5 million/animal NK cells. We are currently evaluating the functionality of aMSLN CAR CIML NK cells after their exposure to ascites from OVC patients as well as developing methods to enhance their function in highly immunosuppressive tumor microenvironment. Citation Format: Mubin Tarannum, Khanhlinh Dinh, Juliana Vergara, Oyku Ay, Sarah Hill, Rizwan Romee. A novel memory-like NK cell CAR targeting the proximal mesothelin domain shows promising pre-clinical activity in ovarian cancer using cell lines, patient-derived organoid tumors, and xenograft mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3181.

Hsp70, in Combination with IL-15 and PD-1 Blocker, Interferes with The Induction of Cytotoxic NK Cells in Relapsed Acute Myeloid Leukemia Patients.

Natural killer (NK) cells are critical immune cells for acute myeloid leukemia (AML) targeting. However, little is known about the relationship between using checkpoint inhibitors and heat shock protein 70 (Hsp70) as NK cell activators to control AML. Therefore, the study aims to find the best formulation of Hsp70, human PD-1 (Programmed cell death protein 1) blocker, and interleukin 15 (IL-15) to activate NK cells against AML. In this experimental study, the NK cells were isolated from mononuclear cells (MNCs) by using magnetic activation cell sorting (MACS) and were activated using the different combinations of Hsp70, PD-1 blocker, and IL-15 and then followed by immunophenotyping, functional assays to estimate their killing potential, and evaluation of expression pattern of PRF1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A and B. The expression of PD-1 was significantly (P<0.05) reduced after NK cell activation by the different formulas of IL-15, Hsp70, and PD-1 blocker. The expression of NKG2A in the treated NK cells was reduced particularly in the IL-15 (P<0.01) and IL-15+PD-1 blocker (P<0.05) groups. The addition of Hsp70 increased its expression. The cytotoxic effect of NK cells increased in all groups, especially in IL-15+PD-1 blocker besides increasing interferon-gamma (IFN-γ), Granzymes, and perforin expression (P<0.05). All IL-15+PD-1 blocker group changes were associated with the upregulation of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing, and down-regulation of PIK3CB, AKT-1, Granzymes, and Perforin (P<0.05). We suggested the combination of IL-15 and PD-1 blocker could enhance the killing potential of AMLNK cells. Moreover, Hsp70 in combination with IL-15 and PD-1 blocker interferes activation of AML-NK cells through unknown mechanisms.

Natural killer cell therapy potentially enhances the antitumor effects of bevacizumab plus irinotecan in a glioblastoma mouse model.

Various combination treatments have been considered to attain the effective therapy threshold by combining independent antitumor mechanisms against the heterogeneous characteristics of tumor cells in malignant brain tumors. In this study, the natural killer (NK) cells associated with bevacizumab (Bev) plus irinotecan (Iri) against glioblastoma multiforme (GBM) were investigated. For the experimental design, NK cells were expanded and activated by K562 cells expressing the OX40 ligand and membrane-bound IL-18 and IL-21. The effects of Bev and Iri on the proliferation and NK ligand expression of GBM cells were evaluated through MTT assay and flow cytometry. The cytotoxic effects of NK cells against Bev plus Iri-treated GBM cells were also predicted via the LDH assay in vitro. The therapeutic effect of different injected NK cell routes and numbers combined with the different doses of Bev and Iri was confirmed according to tumor size and survival in the subcutaneous (s.c) and intracranial (i.c) U87 xenograft NOD/SCID IL-12Rγnull mouse model. The presence of injected-NK cells in tumors was detected using flow cytometry and immunohistochemistry ex vivo. As a result, Iri was found to affect the proliferation and NK ligand expression of GBM cells, while Bev did not cause differences in these cellular processes. However, the administration of Bev modulated Iri efficacy in the i.c U87 mouse model. NK cells significantly enhanced the cytotoxic effects against Bev plus Iri-treated GBM cells in vitro. Although the intravenous (IV) injection of NK cells in combination with Bev plus Iri significantly reduced the tumor volume in the s.c U87 mouse model, only the direct intratumorally (IT) injection of NK cells in combination with Bev plus Iri elicited delayed tumor growth in the i.c U87 mouse model. Tumor-infiltrating NK cells were detected after IV injection of NK cells in both s.c and i.c U87 mouse models. In conclusion, the potential therapeutic effect of NK cells combined with Bev plus Iri against GBM cells was limited in this study. Accordingly, further research is required to improve the accessibility and strength of NK cell function in this combination treatment.

IL-18BP Improves Early Graft Function and Survival in Lewis-Brown Norway Rat Orthotopic Liver Transplantation Model.

Interleukin-18 (IL-18) can effectively activate natural killer (NK) cells and induce large concentrations of interferon-γ (IFN-γ). In healthy humans, IL-18 binding protein (IL-18BP) can inhibit the binding of IL-18 to IL-18R and counteract the biological action of IL-18 due to its high concentration and high affinity, thus preventing the production of IFN-γ and inhibiting NK-cell activation. Through previous studies and the phenomena observed by our group in pig-non-human primates (NHPs) liver transplantation experiments, we proposed that the imbalance in IL-18/IL-18BP expression upon transplantation encourages the activation, proliferation, and cytotoxic effects of NK cells, ultimately causing acute vascular rejection of the graft. In this research, we used Lewis-Brown Norway rat orthotopic liver transplantation (OLTx) as a model of acute vascular rejection. AAV8-Il18bp viral vectors as gene delivery vehicles were constructed for gene therapy to overexpress IL-18BP and alleviate NK-cell rejection of the graft after transplantation. The results showed that livers overexpressing IL-18BP had reduced damage and could function longer after transplantation, effectively improving the survival time of the recipients.

Studying the Anticancer Effects of Thymoquinone on Breast Cancer Cells through Natural Killer Cell Activity.

Cancer immunotherapy is quickly growing and can now be viewed as the “fifth column” of cancer treatment. In addition, cancer immunotherapy has shown promising results with different kinds of cancers and may be used as a complementary therapy with various types of treatments. Thus, “immuno-oncology” is showing astounding advantages. However, one of the main challenges that face this type of therapy is that cancer cells can evade immune system elimination through different mechanisms. Many studies were done to overcome this issue including adding immune stimulants to generate synergistic effects or by genetically modifying NK cells themselves to be stronger and more resistant. Nigella sativa, also known as black cumin, is a well-known example of a widely applicable herbal medicine. It can effectively treat a variety of diseases, such as hypertension, diabetes, bronchitis, gastrointestinal upset, and cancer. The anticancer qualities of Nigella sativa appear to be mediated by an immune-modulatory effect that stimulates human natural killer (NK) cells. These are a type of lymphocyte and first line of defense against pathogens. Objectives. In this study, we investigated the therapeutic effect of thymoquinone, a major component of Nigella sativa, on the cytotoxic pathways of NK cells. Methods. NK cells were cultured with breast cancer cell line Michigan Cancer Foundation-7 (MCF-7); and were treated with Thymoquinone. The cytotoxicity of NK cells on cancer cells was measured. The cultured media were then collected and measured via enzyme-linked immunosorbent assay (ELISA) for concentrations of perforin, granzyme B and interferon-α (IFN-α). Results. The cytotoxic effect of NK cells on tumor cells was increased in the presence of thymoquinone, with an increased release of perforin, granzyme B, and IFN-α. Conclusion. Thymoquinone promotes the cytotoxic activity of NK cells against breast cancer MCF-7 cells.

Heme oxygenase 1 overexpression induces immune evasion of acute myeloid leukemia against natural killer cells by inhibiting CD48

BackgroundAcute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Given the high relapse rate, more effective treatments are needed to improve clinical outcomes. We previously demonstrated that heme oxygenase 1 (HO1) is overexpressed in AML, while the functional roles of HO1 remain unclear.MethodsBioinformatics analysis and flow cytometry were conducted to assess the association between HO1 levels and immune cells or immune checkpoint/ligand molecules in AML patients. Primary natural killer (NK) cells were purified and subsequently co-cultured in vitro with transduced AML cells to determine the effects of HO1 expression on NK cell functions. AML mice models were established to investigate the effects of HO1 expression on cytotoxic effects of NK cells in vivo. The molecular mechanism was studied by flow cytometry, quantitative real-time PCR (qRT-PCR), western blotting, and immunoprecipitation.ResultsBioinformatics analysis indicated a correlation between HO1 expression and the AML immune microenvironment. The present study findings indicated that HO1 specifically downregulates the expression of CD48, a ligand of the NK cell-activating receptor 2B4, thus decreasing the cytotoxic effect of NK cells. HO1 overexpression promoted tumor growth and inhibited the cytotoxic effect of NK cells in the AML mice model. Mechanistic investigations found that HO1 directly interacted with Sirt1 and increased its expression and deacetylase activity. With the overexpression of HO1, increased Sirt1 in AML cells enabled histone H3K27 deacetylation to suppress CD48 transcription and expression. Administration of Sirt1 inhibitor restored the expression of CD48.ConclusionsCollectively, HO1 promotes NK cell dysfunction in AML. Therefore, restoring NK cell function by inhibiting HO1 activity is a potential immunotherapeutic approach against AML.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

Jinfukang inhibits lung cancer metastasis by upregulating CX3CL1 to recruit NK cells to kill CTCs

Ethnopharmacological relevanceCirculating tumor cells (CTCs) play an important role in tumor metastasis and may be a target for metastasis prevention. The traditional Chinese medicine Jinfukang functions to improve immunity, prevent metastasis, and prolong lung cancer patient survival periods. Yet, whether Jinfukang prevents metastasis by regulating immune cells to clearance CTCs is still unknown. Aim of the studyTo explore the anti-metastasis mechanism of Jinfukang from the perspective of regulating NK cells to clear CTCs. Materials and methodsCTC-TJH-01 cell was treated with Jinfukang. Cytokine chip was used to detect cytokines in cell culture supernatant. Lymphocyte recruitment assay was detected by Transwell and flow cytometry. Protein expression was analysis by Western blot. LDH kit was used to detect cytotoxicity. NOD-SCID mice used for tail vein injection to study lung metastasis. ResultsJinfukang could promote the expression and secretion of the chemokine CX3CL1 by CTCs. In addition, Jinfukang could promote the recruitment of natural killer (NK) cells by CTCs and significantly increase the cytotoxic effect of NK cells on CTCs. Moreover, Jinfukang could upregulate the expression of FasL and promote the secretion of TNF-α by NK cells and that NK cells could induce the apoptosis of CTCs through the Fas/FasL signaling pathway. Finally, we confirmed that Jinfukang could promote NK cells to kill CTCs and then inhibit lung cancer metastasis in vivo. The above effects of Jinfukang could be partially reversed by an anti-CX3CL1 mAb. ConclusionsThese results suggest that Jinfukang may prevent lung cancer metastasis by enhancing the clearance of CTCs in the peripheral blood by NK cells, providing evidence for the anti-metastasis effect of Jinfukang.

Cell membrane-encapsulated magnetic nanoparticles for enhancing natural killer cell-mediated cancer immunotherapy

Natural killer (NK) cells have exhibited therapeutic potential for various malignant tumors. However, the cytotoxic effect of NK cells is relatively weak and less specific compared to other immunotherapy approaches such as chimeric antigen receptor T-Cell (CART) therapy, constituting a great challenge for adoptive immunotherapy. Here, we report cell membrane-encapsulated magnetic nanoparticles for activating NK cells and enhancing anti-tumor effects. Magnetic nanoparticles were coated with silicon dioxide (SiO2), and cancer cell membranes were mixed with Fe3O4@SiO2 to construct cancer cell membrane coated Fe3O4@SiO2 magnetic nanoparticles (CMNPs). The functionalized nanoparticles bearing cancer-specific antigens on the surface effectively stimulated NK cells by enhancing expression of surface activating receptors and boosting anti-tumor function through the secretion of soluble cytotoxic effectors. To conclude, the biomimetic magnetic nanoparticles offer a versatile and powerful tool to present tumor-specific antigens, priming anti-tumor capability, which is promising to enhance NK cell-based adoptive cancer immunotherapy.

Senescent hepatocytes enhance natural killer cell activity via the CXCL-10/CXCR3 axis.

Cellular senescence and natural killer (NK) cells play an important role in liver diseases. Chemokines, a component of the senescence-associated secretory phenotype, can recruit NK cells and are involved in the development of various liver diseases. The effect of the C-X-C motif chemokine ligand (CXCL)-9, −10, −11/C-X-C motif chemokine receptor (CXCR)3 axis in senescent hepatocytes remains unknown. The chemokines secreted by senescent hepatocytes, the contribution of the CXCL-9, −10, −11/CXCR3 axis to the migration of NK cells, and the effect of senescent hepatocytes on the function of NK cells were investigated in the present study. The results demonstrated significantly increased levels of C-C motif chemokine ligand 2 and CXCL-1, −2 and −10 in the supernatant of senescent AML12 cells. Despite increased mRNA expression of CXCL-9, −10, and −11 in these cells, western blotting revealed significantly enhanced expression of only CXCL-10. The expression of CXCR3 on the surface of NK cells stimulated by senescent AML12 cells was upregulated (fold change, >3). Following incubation with the supernatant of senescent hepatocytes, both CD107a and interferon γ expression in NK cells increased by >2.5-fold. The cytotoxic effect of NK cells was notably higher stimulated by senescent AML12 cells. Chemotaxis and blocking assays demonstrated that the senescent hepatocytes enhanced the migration of NK cells via the CXCL-10/CXCR3 axis. The present study suggests that senescent hepatocytes secrete various chemokines, including CXCL-10, resulting in the upregulation and activation of CXCR3 in NK cells and the enhancement of NK cell migration via the CXCL-10/CXCR3 axis.

Role of natural killer cells for immunotherapy in chronic myeloid leukemia (Review).

The majority of natural killer (NK) cells serve an important role in eliminating malignant cells. The cytotoxic effects of NK cells were first identified against leukemia cells, and it is now hypothesized that they may have a critical role in leukemia therapy. The cellular functions of NK cells are mediated by their cell surface receptors, which recognize ligands on cancer cells. The role of NK cells is specifically regulated by the activating or inhibitory killer cell immunoglobulin‑like receptors (KIRs) on their surface, which bind to the human leukocyte antigen (HLA) class I ligands present on the target cells. The association between KIR and HLA is derived from the diversity of KIR/HLA gene profiles present in different individuals, and this determines the cytotoxic effect of NK cells on cancer cells. Chronic myeloid leukemia (CML) is a hematological leukemia characterized by the hyper‑proliferation of myeloid cells, with the majority of patients with CML presenting with abnormal immune cells. Tyrosine kinase inhibitors are the present standard therapy for CML, but are associated with numerous adverse side effects. Various studies have proposed CML therapy by immunotherapeutic approaches targeting the immune cells. This review summarizes the contents of NK cells and the association between KIR/HLA and leukemia, especially CML. This is followed by a discussion on the development of NK cell immunotherapy in hematological malignancies and research into strategies to enhance NK cell function for CML treatment.

Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells.

In this study, in order to investigate the effects of increased macrophage infiltration to radioresistant lung tumors in regulating natural killer (NK) cell-mediated immunity, we examined whether the treatment of radioresistant cells with conditioned medium (CM) from phorbol myristate acetate(PMA)/interleukin(IL)-4 treated THP-1 cells (used as a tumor-associated macrophage source) leads to the development of the additional resistance of tumor cells to NK cell cytotoxicity. We found that the susceptibility of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity was decreased compared to the non-treated cells. In addition, it was found that such a decreased susceptibility was associated with increased programmed death receptor ligand1(PD-L1) and decreased natural killer group2D(NKG2D) ligand levels in tumor cells. We further discovered that the THP-1 cells secreted a high level of IL-6, and that blocking IL-6 action by the addition of a neutralizing antibody(Ab) for IL-6 into the THP-1 CM decreased the resistance of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. Moreover, we discovered that MEK/Erk was the most critical IL-6 downstream signaling pathway in triggering the THP-1 CM effect; thus, the addition of MEK/Erk inhibitor to THP-1 CM enhanced the susceptibility of the THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. On the whole, the findings of this study suggest the existence of a malignant loop characterized by increased macrophage infiltration into radioresistant cells which, in turn, promotes the development of the additional resistance of these cells to NK cell cytotoxicity.

Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia.

Castration-resistant prostate cancer (CRPC) is difficult to treat in current clinical practice. Hypoxia is an important feature of the CRPC microenvironment and is closely associated with the progress of CRPC invasion. However, no research has been performed on the immune escape of CRPC from NK cells. The present study focused on this subject. Firstly, when the CRPC cell lines C4-2 and CWR22Rv1 were induced by hypoxia, the expression of the UL16 binding protein (ULBP) ligand family of natural killer (NK) group 2D (NKG2D; ULBP-1, ULBP-2 and ULBP-3) and MHC class I chain-related proteins A and B (MICA/MICB) decreased. NKG2D is the main activating receptor of NK cells. Tumor cells were then co-cultured with NK cells to conduct NK cell-mediated cytotoxicity experiments, which revealed the decreased immune cytolytic activity of NK cells on hypoxia-induced CRPC cells. In exploring the mechanism behind this observation, an increase in programmed death-ligand 1 (PD-L1) expression in CRPC cells induced by hypoxia was observed, while the addition of PD-L1 antibody effectively reversed the expression of NKG2D ligand and enhanced the cytotoxic effect of NK cells on CRPC cells. In the process of exploring the upstream regulatory factors of PD-L1, inhibition of the Janus kinase (JAK)1,2/signal transducer and activator of transcription 3 (Stat3) signaling pathway decreased the expression of PD-L1 in CRPC cells. Finally, it was observed that combined inhibition of JAK1,2/PD-L1 or Stat3/PD-L1 was more effective than inhibition of a single pathway in enhancing the immune cytolytic activity of NK cells. Taking these results together, it is thought that combined inhibition of the JAK1,2/PD-L1 and Stat3/PD-L1 signaling pathways may enhance the immune cytolytic activity of NK cells toward hypoxia-induced CRPC cells, which is expected to provide novel ideas and targets for the immunotherapy of CRPC.

Abstract LB-268: Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity

Abstract Introduction: Osteosarcoma (OS) is the most common primary malignant tumor of the bone in pediatric patients. Lungs are the most common site of metastasis. Patients with recurrent and metastatic OS are resistant to chemotherapy and have very poor survival rates. Our goal is to find an alternate more effective therapy for OS lung metastasis. Allogeneic natural killer (NK) cells are emerging as a promising treatment strategy for cancers. Although data from our lab and other labs show that OS cells are susceptible to NK cell cytotoxicity, NK cell therapy by itself may not be effective enough to cure OS since tumor cells can escape immunotherapy. One way to augment cytotoxicity of NK cell is using HDAC inhibitors (HDACi). Several studies have shown that HDACis (including entinostat) sensitize tumor cells for cytotoxic effects of NK cells mostly by inducing expression of ligands for NK cells on tumor cells. In this study we sought to investigate whether entinostat would augment the cytotoxic effects NK cells on OS cells. Methods: In our in vitro study we investigated whether entinostat would increase expression of ligands for NK cell receptors on OS cells and as a result would enhance OS cells’ susceptibility to the cytotoxic effects of NK cells. Since the combination of NK cells and entinostat will be used in our in vivo study (a mouse model with OS lung metastasis) we determined the effect of the drug on NK cell viability, receptor expression, and cytotoxicity. NK cells were isolated from healthy donor buffy coats and were expanded ex vivo for 4 weeks in RPMI 1640 media supplemented with artificial antigen-presenting cells (K562) and IL-2. Results: We demonstrated that 2μM entinostat (≤ IC50) for 48hr upregulates expression of NK cell ligands (ULBP1, ULBP2/5/6, and MIC A/B) on OS cell lines (LM7, KRIB, CCHOSD, and CCHOSO). Upregulated ligands on OS cells are stable for at least 24hr after drug removal from the cultured media. Moreover, entinostat treatment increased susceptibility of OS cell lines to cytotoxic killing by NK cells. By using blocking antibodies for NK receptors we confirmed that NK cell cytotoxicity toward OS cells is dependent on receptor and ligand interaction. Treatment of NK cells with up to 2μM entinostat did not affect the viability of NK cells. This drug did not affect the expression of NK cell receptors (NKG2D, NKp30, NKp44, NKp46, and DNAM-1) within the first 24hr; however, after 48hr 1μM and 2μM of the drug decreased expression of receptors except DNAM-1. Pre-treatment of NK cells with entinostat did not decrease cytotoxicity of NK cell to OS cell lines. Conclusion: Our results demonstrate that entinostat immunosensitizes OS cells to NK cell lysis by inducing upregulation of ligands for NK cells on OS cells. We also show that up to 2μM treatment of entinostat for 24 hr does not have any adverse effect on NK cells. These results tell us the time point at which to administer NK cells and entinostat in combination to mice in our in vivo. These results support our hypothesis, saying that combining entinostat with NK cell therapy would augment the cytotoxicity effect of NK cells on osteosarcoma lung metastasis. Citation Format: Simin Kiany, Ling Yu, Michele Guindani, Jimin Wu, John Stewart, Eugenie S Kleinerman. Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2014-LB-268

IL-17A Promotes Immune Cell Recruitment in Human Esophageal Cancers and the Infiltrating Dendritic Cells Represent a Positive Prognostic Marker for Patient Survival

We previously reported that tumor-infiltrating interleukin (IL)-17A-producing cells play a protective role in human esophageal squamous cell carcinoma (ESCC). However, the potential mechanisms involved remain unclear. In the present study, we investigated the effects of IL-17A on immune cell recruitment and function in ESCC. In vitro chemotaxis assays using the ESCC cell lines EC109 and KYSE30 demonstrated that although IL-17A showed no significant direct effects on the migration of T cells, natural killer (NK) cells as well as dendritic cells (DCs), it could induce ESCC tumor cells to produce inflammatory chemokines, for example, CXCL9, CXCL10 and CCL2, CCL20, which are associated with the migration of T cells, NK cells, and DCs, respectively. In addition, IL-17A enhanced the cytotoxic effects of NK cells against tumor cells by augmenting the expression of cytotoxic molecules, for example, tumor necrosis factor-α, interferon-γ, Perforin, and Granzyme B and activation receptors, for example, NKp46, NKp44, NTB-A, and NKG2D on NK cells. Furthermore, immunohistochemical analysis revealed that the density of IL-17A-producing cells was positively and significantly associated with the density of CD1a DCs in tumor tissues. With the analyses of 181 ESCC patients, we found a correlation of higher number of tumor-infiltrating CD1a DCs with significantly improved overall survival of patients with ESCC. This study provides further understanding of the roles of Th17 cells in ESCC, which may contribute to the development of novel cancer immunotherapy strategies.

Hyperthermia increases natural killer cell cytotoxicity against SW-872 liposarcoma cell line.

Although there is convincing data in support of the effectiveness of hyperthermia in tumor therapy, the molecular mechanisms underlying the clinical effects of hyperthermia are still poorly understood. To investigate natural killer (NK) cell cytotoxicity against heat-treated SW-872 and HeLa tumor cell lines. NKG2D ligands and HLA class I transcription were examined using quantitative real-time PCR in treated tumor cell lines at 0, 2, 4, 6 and 12 h following thermal treatment at 39C and 42C for 1 h. The expression of MICA/B, ULBP1 and ULBP2 were also determined by flow cytometry. NK92-MI cytotoxic activity against heat-treated target cell lines was assessed by LDH release as well as annexin-V and 7-AAD assays. Our results showed that heat treatment at 39C improved the cytolytic activity of NK cells against SW-872 cells without increasing NKG2D ligand concentration or decreasing HLA class I levels. The observed increase in the cytotoxicity of NK cells against SW-872 cells after hyperthermia does not coincide with changes in MICA/B, ULBP1 and ULBP2 ligands of NKG2, however, the expression of other ligands in target cells may have made the cells susceptible to the cytotoxic effect of NK cells.

Mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis

The mechanism of natural killer (NK) cell regulatory role in experimental autoimmune encephalomyelitis (EAE) was studied in SJL/J mice. In vivo experiments showed that NK cell depletion by anti-NK1.1 monoclonal antibody treatment enhanced EAE in mice. To investigate the mechanism, we cultured proteolipid protein (PLP) 136–150 peptide-specific, encephalitogenic T cell lines, which were used as the NK cell target. Our results show that NK cells exert a direct cytotoxic effect on autoantigen-specific, encephalitogenic T cells. Furthermore, cytotoxicity to PLP-specific, encephalitogenic T line cells was enhanced by using enriched NK cells as effector cells. However, the cytotoxic effect of NK cells to ovalbumin-specific T line cells and ConA-stimulated T cells could also be detected with a lesser efficiency. Our studies indicate that NK cells play a regulatory role in EAE through killing of syngeneic T cells which include myelin antigen-specific, encephalitogenic T cells, and thus ameliorate EAE.