IL-2 Activated Natural Killer Cells Research Articles

Human decidua basalis mesenchymal stem/stromal cells enhance anticancer properties of human natural killer cells, in vitro.

Mesenchymal stem cells/stromal cells from the Decidua Basalis of the human placenta (DBMSCs) express wide range of effector molecules that modulate the functions of their target cells. These properties make them potential candidate for use in cellular therapy. In this study, we have investigated the consequences of interaction between DBMSCs and natural killer (NK) cells for both cell types. DBMSCs were cultured with IL-2-activated and resting non-activated NK cells isolated from healthy human peripheral blood and various functional assays were performed including, NK cell proliferation and cytolytic activities. Flow cytometry and microscopic studies were performed to examine the expression of NK cell receptors that mediate these cytolytic activities against DBMSCs. Moreover, the mechanism underlying these effects was also investigated. Our findings revealed that, co-culture of DBMSCs and NK cells resulted in inhibition of proliferation of resting NK cells, while proliferation of IL-2 activated NK cells was increased. Contrarily, treatment of DBMSC's with comparatively high numbers of IL-2 activated NK cells, resulted in their lysis, whereas treatment with low numbers resulted in reduction in their proliferation. Cytolytic activity of NK cells against DBMSCs was mediated by several activating NK cell receptors. In spite of the expression of HLA class I molecules by DBMSCs, they were still lysed by NK cells, excluding their involvement in cytolytic activity. In addition, preconditioning NK cells by DBMSCs, enhanced their ability to suppress tumor cell proliferation and in severe cases resulted in their partial lysis. Lysis and decrease of tumor cell proliferation is associated with increased expression of important molecules involved in anticancer activities. We conclude that DBMSCs exhibit dualfunctions on NK cells that enhance their anticancer therapeutic potential.

Activated Natural Killer Cells Withstand the Relatively Low Glucose Concentrations Found in the Bone Marrow of Multiple Myeloma Patients.

Infusion of ex vivo expanded and cytokine-activated natural killer (NK) cells is a promising alternative way to treat multiple myeloma (MM). However, the tumor microenvironment (TME) may suppress their function. While reduced glucose availability is a TME hallmark of many solid tumors, glucose levels within the TME of hematological malignancies residing in the bone marrow (BM) remain unknown. Here, we measured glucose levels in the BM of MM patients and tested the effect of different glucose levels on NK cells. BM glucose levels were measured using a biochemical analyzer. Compared to the normal range of blood glucose, BM glucose levels were lower in 6 of 9 patients (479-1231 mg/L; mean=731.8 mg/L). The effect of different glucose levels on NK cell cytotoxicity was tested in 4-hour cytotoxicity assays with tumor cells. 500 mg/L glucose (representing low range of MM BM) during the 4-hour cytotoxicity assay did not negatively affect cytotoxicity of activated NK cells, while higher glucose concentrations (4000 mg/L) diminished NK cell cytotoxicity. Since clinical application of NK cell therapy might require ex vivo expansion, expanded NK cells were exposed to a range of glucose concentrations from 500-4000 mg/L for a longer period (4 days). This did not reduce cytotoxicity or IFN-γ secretion nor affected their phenotypic profile. In summary, low glucose concentrations, as found in BM of MM patients, by itself did not compromise the anti-tumor potential of IL-2 activated NK cells in vitro. Although follow up studies in models with a more complex TME would be relevant, our data suggest that highly activated NK cells could be used to target tumors with a reduced glucose environment.

Apoptotic Potential of Secretome from Interleukin-Induced Natural Killer Cells toward Breast Cancer Cell Line by Transwell Assay

Breast cancer (BC) is the number one cause of deaths from cancer in women. Metastasis in BC is caused by immunosurveillance deficiency, including impairment of Natural Killer (NK) cell maturation, low NK activity, and decreasing cytotoxicity. This study was performed to improve activating receptors and cytotoxicity of NK cells using interleukin 15 (IL15) against BC cells. Human recombinant IL15 was used to induce NK cells. To evaluate the potential of IL15 in inducing NK cells, we measured the activating and inhibiting receptors (NKG2D, NKG2A), apoptotic potency of NK cells on BC cells (MCF7) using transwell assay. The IL15 inducer on the NK cell were measured NKG2D, NKG2A gene expression with quantitative polymerase chain reaction (qPCR), (GzmB) secretion using ELISA, apoptotic gene expression of MCF7 using qPCR. IL15 increased NKG2D expression 4.01-9.13%, but IL15 could not affect toward NKG2A expression on NK cells. IL15-activated NK cells, inhibited BC cells proliferation, induced apoptotic BC cells 25.89-32.19%, induced apoptotic genes of BC cells bax, p53. IL15 increase NK activating receptor (NKG2D), inhibit BC cells proliferation, induce apoptotic percentage and induce apoptotic gene expression.

Cord Blood IL-15 Activated NK Cells Increase Cord Blood CD34+CD133+CD45lo Cell Function through IFN-y Production and Direct Cell Contact

Delayed engraftment following cord blood (CB) transplantation remains a significant challenge. While cell dose is clearly limiting, CB derived hematopoietic stem cells (HSCs) also exhibit a deficit in homing and engraftment. IL-15 activated natural killer (NK) cells have been shown as potential promoters of homing and engraftment of CB HSCs. However, the role of NK cells and their underlying mechanisms in promoting CB HSCs requires further study. Here, we explore the effect of IL-15 activated CB NK cells on the functional properties of CB CD34+CD133+CD45lo HSCs. In addition, we define the mechanistic interaction between NK cells and HSCs that may increase CB-HSC engraftment and improve patient outcome post-HSC transplantation.We first determined whether IL-15 activated NK cells could improve HSC function in vitro. Purified CD56+CD3- NK cells from CB were stimulated overnight with IL-15 and cultured at a 1:5 cell ratio with autologous purified CD34+CD133+CD45lo cells (CB HSCs). IL-15 activated NK cells increased CXCR4 expression on CB HSCs when compared to cultures with resting NK cells or CB HSCS alone. As HSCs must migrate to the bone marrow in order to engraft and facilitate long-term immune reconstitution, we next assessed whether NK cells also impact on HSC migration, clonogenicity and proliferation. We found that elevated levels of CXCR4 on CB HSCs cultured with activated NK cells also translated into enhanced chemotaxis towards SDF-1α in vitro. IL-15 activated CB NK cells also increased CB HSC clonogenicity as evaluated by short-term in vitro cultures. The effect of activated NK cells on the clonogenic capacity of CB HSCs was cell dose dependent with the highest effect observed at a ratio of 1:10. To study CB HSC proliferation, CFSE stained CD34+CD133+CD45lo cells were cultured either alone, with resting NK cells or IL-15 activated NK cells. Cultures with IL-15 activated NK cells significantly increased CB HSC proliferation when compared to cultures with resting NK cells or CB HSCs alone [median percentage of proliferating CB-HSCs; 38.4% (34%-44.6%) vs. 46.7% (36%-53.4%) vs. 69% (59.6%-78.5%)]. Moreover, following the ability of IL-15 activated NK cells to upregulate CB HSC proliferation, we investigated whether CB HSCs still retained their long-term engraftment potential. We found that proliferating CB HSCs still recalled both their short-term and long term clonogenic capacity as evaluated by CFU assays and cobblestone cultures followed by long-term culture (LTC-IC) respectively. Finally, we demonstrated that IL-15 activated NK cells also possessed the ability to activate pAkt/pErk and pStat3. As pAkt/pErk and pStat3 are key mediators of cell survival proliferation, our findings identify that NK cells may promote the survival and proliferation of CB HSCs through activating pAkt/pErk and pStat3. These data suggest that IL-15 activated NK cells from CB are endowed with properties to promote the functional profile of CB HSCs that contribute to improved engraftment.To further understand the underlying mechanisms through which IL-15 activated NK cells exert their ability to upregulate the functional profile of CB HSCs, we used antibody blockade experiments. We showed that the ability of NK cells to increase CXCR4 expression on CB HSCs was mediated via the provision of IFN-γ, but not TNF-α or TNF-β. Whereas, the effect of NK cells on CB HSC function studied through clonogenicity, proliferation and signalling studies was only partially dependent on IFN-γ production by IL-15 activated NK cells. Using transwell experiments, we further determined that the ability of activated NK cells to upregulate CB HSC function is also partly dependent on direct NK cell/HSC cell contact. Subsequently, we found that the addition of blocking antibody against 2B4 in cultures containing IL-15 activated NK cells and CB HSC partially reversed the ability of NK cells to increase the clonogenic capacity, proliferation and Akt/Erk and Stat3 signalling of CB HSCs. Thus, the ability of IL-15 activated NK cells to increase the functional profile of CB HSCs depends on IFN-γ production and cell-cell contact involving 2B4.Our combined studies demonstrate a novel effect of IL-15 activated CB NK cells and their key factors as potential mediators of stem cell homing and engraftment, which could be utilized to develop strategies that will benefit all patients with haematological malignancies and improve CB transplantation. DisclosuresNo relevant conflicts of interest to declare.

C/Ebpg (CCAAT/Enhancer Binding Protein Gamma) Balances Cytotoxic and Secretory Potential of Natural Killer Cells

C/EBPs (CCAAT/enhance-binding proteins) are a family of transcription factors involved in a variety of hematopoietic processes, regulating both terminal differentiation and cellular proliferation. Among these, it was previously reported that C/EBP gamma (C/EBPg) has a role in the development of Natural Killer (NK) cells. However, the mechanisms of such regulation are unknown. NK cells are lymphocytes with effector functions of cytotoxicity and production of cytokines, both dependent on a dynamic equilibrium between the expression of activating and inhibitory receptors as well as cytokine receptors. The two functions (cytotoxic and secretory) make NK cells important components of hematopoiesis, able to eliminate susceptible targets as well as recruit other cells to amplify inflammatory responses. With the aim of studying the regulation of NK cells by C/EBPg, we isolated NK cells from transgenic Cebpg knockout (KO) mice and controls to analyze their function. To characterize NK cells, we analyzed their frequency (Lineage-/CD3-/NK1.1+ cells) and the expression of the receptors NKG2D, Ly49D and NKG2A by flow cytometry of splenocytes. Both analyses showed no difference between control or Cebpg KO NK cells. Although the numbers of NK cells and their receptors were similar between Cebpg WT and KO animals, a functional assay that measured NK cell degranulation by CD107a expression after co-incubation with YAC-1 target cells showed that the expression of this marker was 5-times lower in Cebpg KO splenocytes than in controls (CT = 12.44 ± 2.50%; KO = 2.255 ± 0.67%, p=0.007), suggesting that Cebpg deficient NK cells are not fully activated after target cell recognition. In addition, a cytotoxicity assay by flow cytometry was performed using a fluorescent probe (Cell Tracker Orange) that was incorporated to YAC-1 cells upon exposure to sorted and IL-2 activated NK cells in culture. In the 10:1 NK:target cells ratio, Cebpg KO cells were significantly less cytotoxic than NK control cells (CT = 23.36 ± 8.67%; KO = 10.60 ± 1.66%, p=0.038). The other NK:target cells ratios of 5:1 and 1:1 showed the same tendency. In addition, the functional subtypes of these cells were characterized according to the expression of CD27 and CD11b, which allowed the identification of NK subpopulations as immature secretory, mature secretory, cytotoxic or tolerant. The KO animals showed higher percentages of secretory cells (CT = 10.77 ± 5.38%; KO = 12.98 ± 13.63%, p=0.0002) and a reduction of cytotoxic cells in comparison to the NK control cells (CT = 12.22 ± 11.08%; KO = 10.65 ± 3.82% p=0.013). Cytokine levels of IL-2, IL-4, IL-6, IL-10, IL-17α, TNFα and IFNγ, obtained from NK culture supernatants, were measured by flow cytometry, after IL-2 activation. Among these cytokines, the production of IFNγ by Cebpg-deficient NK cells was reduced (CT = 37.68 ± 0.51 pg/mL; KO = 22.34 ± 0.14 pg/mL, p=0.023). Together, these experiments indicate that C/EBPg regulates NK cell cytotoxicity. This may be explained, at least in part, by the reduced frequency of the mature cytotoxic NK subpopulation as compared to the secretory subtypes. Moreover, IFNγ may be an important target for the regulation of NK cell function. Finally, C/EBPg seems to be critical to mediate NK cell functions and not only for their development from the ontogenetic point of view. DisclosuresNo relevant conflicts of interest to declare.

Deficient mitochondrial biogenesis in IL-2 activated NK cells correlates with impaired PGC1-α upregulation in elderly humans.

Immunosenescence has been described as age-associated changes in the immune function which are thought to be responsible for the increased morbidity with age. Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in immune defense against tumor and microbial diseases. Interestingly, aging-related NK cell dysfunction is associated with features of aging such as tumor incidence, reduced vaccination efficacy, and short survival due to infection. It is known that NK cell effector functions are critically dependent on cytokines and metabolic activity. Our aim was to determine whether there is a difference in purified human NK cell function in response to high concentration of IL-2 between young and elder donors. Here, we report that the stimulation of human NK cells with IL-2 (2000 U/mL) enhance NK cell cytotoxic activity from both young and elderly donors. However, while NK cells from young people responded to IL-2 signaling by increasing mitochondrial mass and mitochondrial membrane potential, no increase in these mitochondrial functional parameters was seen in purified NK cells from elderly subjects. Moreover, as purified NK cells from the young exhibited an almost three-fold increase in PGC-1α expression after IL-2 (2000 U/mL) stimulation, PGC-1α expression was inhibited in purified NK cells from elders. Furthermore, this response upon PGC-1α expression after IL-2 stimulation promoted an increase in ROS production in NK cells from elderly humans, while no increase in ROS production was observed in NK cells of young donors. Our data show that IL-2 stimulates NK cell effector function through a signaling pathway which involves a PGC-1α-dependent mitochondrial function in young NK cells, however it seems that NK cells from older donors exhibit an altered IL-2 signaling which affects mitochondrial function associated with an increased production of ROS which could represent a feature of NK cell senescence.

Peritumoral IL-15 Promotes Regional NK Activation and DC Licensing to Augment Radiation-Induced Anti-tumor Immunity

Radiotherapy (RT) can elicit anti-tumor T cells by multiple mechanisms but the induced immune response is limited by the immunosuppression in the tumor microenvironment. The common gamma-chain cytokines IL-15 promotes the activation of natural killer (NK) cells and the proliferation of activated CD8+ T cells and is under investigation for its ability to induce T cell response in cancer patients. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate durable tumor-specific immunity. The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic wild type (WT) or batf3-deficient BALB/c mice and when tumors reached 5mm in average diameter, were randomly assigned to one of 4 treatment groups: control, RT, IL-15 or RT+IL-15. Local RT was delivered consecutively in 8 Gy fractions on days 13, 14, and 15 post tumor implantation. IL-15 (2 μg/mouse) was administered s.c. peritumorally daily for 10 days starting on the first day of RT. Mice were followed for tumor growth and surviving mice subsequently re-challenged. Some animals were depleted of CD8 and NK cells (anti-asialo-GM1 Ab) using standard protocols. A parallel experiment was done to characterize tumor-infiltrating lymphocytes (TILs) and measure ex vivo IFNg production by tumor-specific CD8 T-cells in the draining lymph nodes at the end of treatment (day 22). IL-15 as monotherapy had no effect on tumor control but significantly improved the ability of RT to delay tumor growth in WT mice (P < 0.01, RT vs RT+IL-15). Combination treatment led to durable and robust memory responses in 50% of animals compared to 0% of mice treated with RT alone. RT enhanced intratumoral infiltration of DX5+NKp46+ NK cells and was further increased by the addition of IL-15 (22.86% of CD45 TILs in RT vs 32% in RT+IL-15, P<0.05). CD8+ T cells expressing the activation marker CD137 were significantly increased in tumors of RT+IL-15-treated mice while the increase was modest with each monotherapy (35.3% in RT+IL-15 vs 18.8% in RT, 20.7% in IL-15, 5.90% in control, P < 0.05). Systemic depletion of either CD8 T-cells or NK cells abrogated the therapeutic effect of the combination. RT+IL-15 elicited more priming of tumor-specific T-cells in response to tumor epitope AH-1-A5 (1238 pg/mL in RT+IL-15 vs 3.3 pg/mL in control vs 28.7 pg/mL in IL-15 RT vs 2.63 pg/mL in RT; P<0.05). Batf3-/- mice that CD103+ DCs showed reduce response to RT+IL-15 compared to WT mice. Here we show that local IL-15 administration improves priming of anti-tumor CD8+ T cell and leads to long-term memory responses by a mechanism that is dependent on NK cells and Batf3-dependent DCs. Data suggest that IL-15 activated NK cells kill tumor cells and license DCs leading to a long-term T cell-mediated memory response. Overall these results support the testing of IL-15 in combination with tumor-targeted radiotherapy.

P 135 - HO-1 down-regulation increases the efficacy of BRAFV600E inhibition-based therapy in primary melanoma cells

Heme oxygenase 1 (HO-1) plays a key role in maintaining cell redox balance. Under stress conditions, HO-1 is up-regulated and generates bilirubin, ferritin and carbon monoxide, with antioxidant, antiapoptotic and anti-inflammatory properties [Jozkowicz et al., 2007]. HO-1 induction favors cancer progression [Furfaro et al., 2016] and its involvement in tumor resistance to therapy [Was et al., 2006] and immune-escape has been highlighted in melanoma-bearing mice [Di Biase et al., 2016]. In this work, the role of HO-1 in melanoma cell resistance to Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600 has been investigated, as well as HO-1 involvement in natural killer (NK)-mediated killing. BRAFV600E mutant primary melanoma cells isolated in our lab have been treated for 24 h with 1 μM PLX4032. The treatment reduced viability (-23%), increased HO-1 mRNA level of two-fold, and decreased the ability of IL-15 activated NK cells to degranulate in response to PLX4032-treated cells, when compared to untreated melanoma cells. HO-1 silencing increased PLX4032 efficacy further reducing cell viability to 47% in comparison to cells treated with PLX4032 alone and restored the degranulation potential of NK. Thus, we hypothesize that HO-1 inhibition can effectively improve the efficacy of mutant-BRAF inhibitors and favors NK-depending killing of PLX4032-treated melanoma cells.

Interleukin-15 stimulates natural killer cell-mediated killing of both human pancreatic cancer and stellate cells.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

Differentiation by NK cells is a prerequisite for effective targeting of cancer stem cells/poorly differentiated tumors by chemopreventive and chemotherapeutic drugs.

Natural Killer (NK) cells target oral, pancreatic, lung, breast, glioblastoma and melanoma stem-like/poorly differentiated tumors. Differentiation of the abovementioned tumors with supernatants from split-anergized NK cells decreases their susceptibility to NK cells, but increases their sensitivity to cisplatin (CDDP)-mediated cell death. Breast and melanoma tumor cells with CD44 knockdown display enhanced susceptibility to NK cell-mediated lysis, potentially due to decreased differentiation. We also demonstrate that sulindac, a non-steroidal anti-inflammatory drug and a chemopreventive agent, not only limits the growth of oral tumor cells, but also aids in cancer cell elimination by NK cells. Treatment of oral tumors with sulindac, but not adriamycin inversely modulates the expression and function of NFκB and JNK, resulting in a significant down-regulation of IL-6, and VEGF secretion by oral tumor cells. In addition, increased secretion of IL-6 and VEGF is blocked by sulindac during interaction of oral tumors with NK cells. Sulindac treatment prevents synergistic induction of VEGF secretion by the tumor cells after their co-culture with untreated NK cells since non-activated NK cells lack the ability to efficiently kill tumor cells. Moreover, sulindac is able to profoundly reduce VEGF secretion by tumor cells cultured with IL-2 activated NK cells, which are able to significantly lyse the tumor cells. Based on the data presented in this study, we propose the following combinatorial approach for the treatment of stem-like/ poorly differentiated tumors in cancer patients with metastatic disease. Stem-like/ poorly differentiated tumor cells may in part undergo lysis or differentiation after NK cell immunotherapy, followed by treatment of differentiated tumors with chemotherapy and chemopreventive agents to eliminate the bulk of the tumor. This dual approach should limit tumor growth and prevent metastasis.

275 - HO-1 Down-Regulation Increases the Efficacy of BRAFV600E Inhibition-Based Therapy in Primary Melanoma Cells

Heme oxygenase 1 (HO-1) plays a key role in maintaining cell redox balance. Under stress conditions, HO-1 is up-regulated and generates bilirubin, ferritin and carbon monoxide, with antioxidant, antiapoptotic and anti-inflammatory properties [Jozkowicz et al., 2007]. HO-1 induction favors cancer progression [Furfaro et al., 2016] and its involvement in tumor resistance to therapy [Was et al., 2006] and immune-escape has been highlighted in melanoma-bearing mice [Di Biase et al., 2016]. In this work, the role of HO-1 in melanoma cell resistance to Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600 has been investigated, as well as HO-1 involvement in natural killer (NK)-mediated killing. BRAFV600E mutant primary melanoma cells isolated in our lab have been treated for 24 h with 1 μM PLX4032. The treatment reduced viability (-23%), increased HO-1 mRNA level of two-fold, and decreased the ability of IL-15 activated NK cells to degranulate in response to PLX4032-treated cells, when compared to untreated melanoma cells. HO-1 silencing increased PLX4032 efficacy further reducing cell viability to 47% in comparison to cells treated with PLX4032 alone and restored the degranulation potential of NK. Thus, we hypothesize that HO-1 inhibition can effectively improve the efficacy of mutant-BRAF inhibitors and favors NK-depending killing of PLX4032-treated melanoma cells.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor

Background aimsFew studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. MethodsThe expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15–activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription–polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. ResultsThe activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R2 = 0.91; P < 0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. ConclusionsOur data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.

Expanded and Activated Natural Killer Cells for Immunotherapy of Hepatocellular Carcinoma.

Viral infection of the liver is a major risk factor for hepatocellular carcinoma (HCC). Natural killer (NK) cells recognize virally infected and oncogenically transformed cells, suggesting a therapeutic role for NK-cell infusions in HCC. Using the K562-mb15-41BBL cell line as a stimulus, we obtained large numbers of activated NK cells from the peripheral blood of healthy donors. Expanded NK cells exerted remarkably high cytotoxicity against HCC cell lines, which was generally much higher than that of unstimulated or IL2-activated NK cells. In immunodeficient NOD/scid IL2RGnull mice engrafted with Hep3B, treatment with expanded NK cells markedly reduced tumor growth and improved overall survival. HCC cells exposed for 48 hours to 5 μmol/L of sorafenib, a kinase inhibitor currently used for HCC treatment, remained highly sensitive to expanded NK cells. HCC cell reductions of 39.2% to 53.8% caused by sorafenib in three cell lines further increased to 80.5% to 87.6% after 4 hours of culture with NK cells at a 1:1 effector-to-target ratio. NK-cell cytotoxicity persisted even in the presence of sorafenib. We found that NKG2D, an NK-cell-activating receptor, was an important mediator of anti-HCC activity. We therefore enhanced its signaling capacity with a chimeric NKG2D-CD3ζ-DAP10 receptor. This considerably increased the anti-HCC cytotoxicity of expanded NK cells in vitro and in immunodeficient mice. The NK expansion and activation method applied in this study has been adapted to clinical-grade conditions. Hence, these results warrant clinical testing of expanded NK-cell infusions in patients with HCC, possibly after genetic modification with NKG2D-CD3ζ-DAP10. Cancer Immunol Res; 4(7); 574-81. ©2016 AACR.

Haploidentical Natural Killer Cells Infused before Allogeneic Stem Cell Transplantation for Myeloid Malignancies: A Phase I Trial

Allogeneic stem cell transplantation is an effective treatment for high-risk myeloid malignancies, but relapse remains the major post-transplantation cause of treatment failure. Alloreactive natural killer (NK) cells mediate a potent antileukemic effect and may also enhance engraftment and reduce graft-versus-host disease (GVHD). Haploidentical transplantations provide a setting in which NK cell alloreactivity can be manipulated, but they are associated with high rates of GVHD. We performed a phase I study infusing escalating doses of NK cells from an HLA haploidentical–related donor—selected for alloreactivity when possible—as a component of the preparative regimen for allotransplantation from a separate HLA-identical donor. The goal of infusing third-party alloreactive NK cells was to augment the antileukemic effect of the transplantation without worsening GVHD and, thus, improve the overall outcome of hematopoietic transplantation. Twenty-one patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myelogenous leukemia refractory or beyond first remission received a preparative regimen with busulfan and fludarabine followed by infusion of apheresis-derived, antibody-selected, and IL-2–activated NK cells. Doses were initially based on total nucleated cell (TNC) content and later based on CD56+ cells to reduce variability. CD56+ content ranged from .02 to 8.32 × 106/kg. IL-2, .5 × 106 units/m2 subcutaneously was administered daily for 5 days in the final cohort (n = 10). CD3+ cells in the NK cell product were required to be < 105/kg. Median relapse-free, overall, and GVHD-free/relapse-free survival for all patients enrolled was 102, 233, and 89 days, respectively. Five patients are alive, 5 patients died of transplantation-related causes, and 11 patients died of relapse. Despite the small sample size, survival was highly associated with CD56+ cells delivered (P = .022) and development of ≥ grade 3 GVHD (P = .006). There were nonsignificant trends toward higher survival rates in those receiving NK cells from KIR ligand–mismatched donors and KIR-B haplotype donors. There was no association with disease type, remission at time of transplantation, or KIR content. GVHD was not associated with TNC, CD56+, or CD3+ cells infused in the NK cell product or the stem cell product. This trial demonstrates a lack of major toxicity attributable to third-party NK cell infusions delivered in combination with an HLA-compatible allogeneic transplantation. The infusion of haploidentical alloreactive NK cells was well tolerated and did not interfere with engraftment or increase the rate of GVHD after allogeneic hematopoietic transplantation. Durable complete remissions occurred in 5 patients at high risk for disease recurrence. This approach is being further developed in a phase I/II trial with ex vivo–expanded NK cells to increase the NK cell dose with the objective of reducing relapse and improving the outcome of allogeneic hematopoietic transplantation for AML/MDS.

Human cancer cells with stem cell-like phenotype exhibit enhanced sensitivity to the cytotoxicity of IL-2 and IL-15 activated natural killer cells

Tumors harbor a population of cancer stem cells (CSCs) which can drive tumor progression and therapeutical resistance. Nature killer (NK) cells are best known for their ability to directly recognize and kill malignant cells. However, the susceptibility of cancer stem cells to NK cells is not fully understood. Here we demonstrated that human CD44+CD24− breast CSCs were shown enhanced sensitivity to IL-2 and IL-15 activated NK cells. CD44+CD24− CSCs expressed higher levels of NKG2D ligands ULBP1, ULBP2 and MICA. Blockade assay showed that the sensitivity of CSCs to NK cells-mediated lysis was mainly dependent on NKG2D. Furthermore, redox oxygen species (ROS)-low tumor cells were more sensitive to NK cells. The presence of antioxidant enzymes inhibitor L-S,R-buthionine sulfoximine or H2O2 retarded the cytotoxicity of NK cells to CD44+CD24− CSCs. In addition, NK cells could readily target CD133+ colonal CSCs. Our findings provide novel targets for NK cells-based immunotherapy and are of great importance for translational medicine.

Increased sMICA and TGFβ1 levels in HNSCC patients impair NKG2D-dependent functionality of activated NK cells

Disseminated head-and-neck squamous cell carcinoma (HNSCC) escapes immune surveillance and thus frequently manifests as fatal disease. Here, we report on the distribution of distinct immune cell subpopulations, natural killer (NK) cell cytotoxicity and tumor immune escape mechanisms (TIEMs) in 55 HNSCC patients, either at initial diagnosis or present with tumor relapse. Compared to healthy controls, the regulatory NK cells and the ratio of pro/anti-inflammatory cytokines were decreased in HNSCC patients, while soluble major histocompatibility complex Class I chain-related peptide A (sMICA) and transforming growth factor β1 (TGFβ1) plasma levels were markedly elevated. Increased sMICA and TGFβ1 concentrations correlated with tumor progression and staging characteristics in 7 follow-up HNSCC patients, with significantly elevated levels of both soluble factors from the time of initial diagnosis to that of relapse. Patient plasma containing elevated sMICA and TGFβ1 markedly impaired NKG2D-dependent cytotoxicity against HNSCC cells upon incubation with patient-derived and IL-2 activated NK cells vs. those derived from healthy donors. Decreased antitumor recognition was accompanied by reduced NKG2D expression on the NK cell surface and an enhanced caspase-3 activity. In-vitro blocking and neutralization experiments demonstrated a synergistic negative impact of sMICA and TGFβ1 on NK cell functionality. Although we previously showed the feasibility and safety of transfer of allogeneic donor NK cells in a prior clinical study encompassing various leukemia and tumor patients, our present results suggest the need for caution regarding the sole use of adoptive NK cell transfer. The presence of soluble NKG2D ligands in the plasma of HNSCC patients and the decreased NK cell cytotoxicity due to several factors, especially TGFβ1, indicates timely depletion of these immunosuppressing molecules may promote NK cell-based immunotherapy.

N6-isopentenyladenosine affects cytotoxic activity and cytokines production by IL-2 activated NK cells and exerts topical anti-inflammatory activity in mice

N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (<1μM) increased the immune response of natural killer (NK) cells against cancer targets. In the present study, we evaluated the effect of iPA at high concentration (10μM) on IL-2-activated NK cells. iPA, inhibited NK cell proliferation and cytotoxicity against their conventional tumor target, human K562 cells. This inhibition was associated with decreased expression and functionality of NK cell activating receptors NKp44 and NKG2D as well as impaired cyto/chemokines secretion (RANTES, MIP-1α, TNF-α and IFN-γ). ERK/MAPK and STAT5 activation in IL-2-activated NK cells were inhibited by iPA. The results obtained in vitro were validated in vivo in the inflammatory murine model of croton oil-induced ear dermatitis. The topical application of iPA significantly reduced mouse ear oedema, thus suggesting anti-inflammatory properties of this molecule.These results show the ability of iPA to exert anti-inflammatory effects both in vitro and in vivo directly targeting NK cells, providing a novel pharmacological tool in those diseases characterized by a deregulated immune-response, such as cancer or inflammatory conditions.

Cystatin F Regulates Proteinase Activity in IL-2-activated Natural Killer Cells

Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture

Background aimsInterleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture. MethodsIL-2/IL-15–activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry. ResultsCompared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the “first week” group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the “first week” group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of “first week” NK cells that received IL-21 treatment for an additional 2 days compared with the “first week” NK cells (P < 0.05). ConclusionsThese data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.

Upregulation of NKG2D ligands in acute lymphoblastic leukemia and non-Hodgkin lymphoma cells by romidepsin and enhanced in vitro and in vivo natural killer cell cytotoxicity

Background aimsThere is a critical need to prevent and/or treat hematological relapse after allogeneic hematopoietic stem cell transplantation. The activating NKG2D receptor expressed on natural killer (NK) cells, when engaged by its corresponding ligands (MIC A/B), activates NK cells to become cytotoxic against malignant cells. MethodsWe incubated acute lymphoblastic leukemia and non-Hodgkin lymphoma cells for 24 h with 10 ng/mL of romidepsin. Flow cytometry was performed to demonstrate changes in surface expression of NKG2D ligands MIC A/B. In vitro and in vivo cytotoxicity was measured by means of modified Europium assay, and non-obese diabetic/severe combined immunodeficiency mice were xenografted with RS 4:11 cells. ResultsWe demonstrated an approximately 50, 200, 1300 and 180-fold increase in the number of cells positive for the surface expression of MIC A/B in RS 4:11 (P < 0.001), REH (P < 0.001), Ramos (P < 0.001) and Jurkat cells (P < 0.001), respectively. We further demonstrated a significant increase in NK cell–mediated in vitro cytotoxicity against RS 4:11 (P < 0.004), Ramos (P < 0.05), Jurkat (P < 0.001) and REH cells (P < 0.01), respectively. Romidepsin-mediated NK cytotoxicity was blocked by pre-incubating NK cells with anti-NKG2D-Fc in RS 4:11 (P < 0.03) and Ramos cells (P < 0.01), respectively. Finally, non-obese diabetic/severe combined immunodeficiency mice xenografted with RS 4:11 cells had a significant increase in survival (P < 0.02) in mice treated with romidepsin and interleukin-2–activated NK cells compared with each of these other treatment groups. ConclusionsRomidepsin significantly enhanced in vitro and in vivo NK cell cytotoxicity mediated in part by increased MIC A/B expression on malignant cells. This translational approach of the use of romidepsin and interleukin-2–activated NK cells should be considered in patients with relapsed/refractory leukemia or lymphoma.