Cytotoxic Activity Of Natural Killer Cells Research Articles

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

Abstract 3699: Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy

Abstract INTRODUCTION: Taking advantage of aerosol for repeated local applications in the lung, we previously demonstrated that combination of Poly(I:C) and CpG-ODN, TLR3 and 9 ligands capable to activate innate effectors, induced a significant antitumor effect against B16 melanoma lung metastases, which correlated with a strong activation of NK cells. We investigated the sequence of events that lead to NK cells activation by aerosolized TLR agonists in the lung. Although NK cells express certain TLRs, controversies exist regarding their direct responsiveness or dependence on macrophages. Alveolar macrophages have several unique features both promoting host defense mechanisms against invading microorganisms, but also establishing an immunosuppressive microenvironment to avoid inflammatory responses to inhaled particles. Our findings, establishing the sequence of events of lung immune cells activation, might represent the basis for the development of combined aerosol therapies that favor NK cell triggering. EXPERIMENTAL PROCEDURE: Alveolar macrophages and NK cells were recovered by bronchoalveolar lavage and spleen of C57BL/6 mice, respectively. Mice i.v. injected with 5×105 B16 cells were treated starting 7 days later every three days with aerosolized Poly(I:C)/CpG-ODN alone or combined to nebulized myeloid derived suppressive cells (MDSC)-depleting RB6-8C5 antibody. Immune cell populations and expression of M1/M2 markers were analysed by flow cytometry and Real Time PCR, respectively, in digested lungs. Cytotoxic activity of NK cells was assessed by 51Cr release assay. RESULTS: In vitro TLR3/TLR9 stimulation induced IFN-γ secretion by naïve NK cells, but an increase in their cytotoxicity was detected only when NK cells were cocultured with alveolar macrophages pretreated with the two agonists. Alveolar macrophages from melanoma lung metastases-bearing mice treated with aerosolized TLR agonists also promoted NK cell cytotoxicity ex vivo. Moreover, naïve alveolar macrophages incubated with activated NK cells from lungs of melanoma metastases-bearing mice aerosolized with TLR9/TLR3 agonists, up-regulated M1- and downregulated M2-related genes, suggesting a bidirectional crosstalk between NK cells and macrophage. Thus, TLR agonists-activated lung macrophages promote NK cell cytotoxicity and, reciprocally, NK cells have the potential to shape the macrophage programming to M1-like phenotype. Our results also indicated that this positive interplay can be further improved by the depletion of myeloid derived suppressive cells that exert a negative effect on macrophage activation. CONCLUSION: This study points to the promise of combinations of immunotherapeutic agents delivered locally by inhalation to promote macrophages-NK cell activation in the lung as a novel strategy to treat lung cancer patients. Citation Format: Michele Sommariva, Valentino Le Noci, Elda Tagliabue, Andrea Balsari, Lucia Sfondrini. Activation of NK cells cytotoxicity mediated by alveolar macrophages in the lung against murine melanoma metastases by combined aerosol immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2017-3699

Abstract 2997: The tetravalent bispecific antibody AFM13 engages and primes innate immune cells for anti-cancer immunity

Abstract AFM13 is a tetravalent bispecific antibody with bivalent binding to both CD30 and CD16A. It has been shown to engage NK-cells through CD16A with high affinity and specificity, resulting in strong NK-cell cytotoxicity, and is currently being tested in Phase 2 monotherapy and in combination with pembrolizumab in Phase 1b clinical trials. We have previously shown that AFM13-dependent activation of NK-cell cytotoxicity towards CD30+ tumor cells is more pronounced than that of anti-CD30 mAbs. In addition, AFM13 enhances NK-cell sensitivity to low doses of IL-2 and IL-15, leading to an increased NK-cell proliferative potential. Here, we have extended the panel of phenotypic markers on NK-cells that are modulated after exposure to CD30+ tumor cells in the presence of AFM13. Targeting some of these markers may enable the development of novel combination therapies. Moreover, we have analyzed the kinetics of NK-cell responses to AFM13 exposure. Even though short-term exposure to AFM13 significantly enhanced NK-cell cytotoxicity, long-term exposure led to a partial, transient functionally exhausted phenotype in vitro, which could be fully restored by cytokine stimulation for several days in the absence of AFM13. Importantly, these recovered cells displayed high cytotoxicity towards CD30+ target cells in the presence of AFM13. Interestingly, the transient NK-cell exhaustion was not related to the expression of typical exhaustion markers or insufficient levels of perforin and granzyme. These data may warrant the development of novel metronomic application regimens of AFM13. Further studies imply that immune cells other than NK-cells are able to inhibit growth of CD30+ tumor cells in an AFM13-dependent manner. This appears to be strictly dependent on CD16A and a specific cytokine milieu. Taken together, AFM13 specifically enhances the cytotoxic, proliferative and cytokine-producing potential of NK-cells, parameters that can be utilized to monitor NK-cell responses during AFM13 therapy. Moreover, based on our data, engagement of CD16A+ cells to the tumor site might enable several innate immune effector functions within the tumor microenvironment for synergistic anti-tumor activity. Citation Format: Jens Pahl, Joachim Koch, Uwe Reusch, Thorsten Gantke, Adelheid Cerwenka, Martin Treder. The tetravalent bispecific antibody AFM13 engages and primes innate immune cells for anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2997. doi:10.1158/1538-7445.AM2017-2997

A Two-Phase Expansion Protocol Combining Interleukin (IL)-15 and IL-21 Improves Natural Killer Cell Proliferation and Cytotoxicity against Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children. Despite intensive research in recent decades the prognosis for patients with metastatic or relapsed diseases has hardly improved. New therapeutic concepts in anti-tumor therapy aim to modulate the patient’s immune system to increase its aggressiveness or targeted effects toward tumor cells. Besides surgery, radiotherapy and chemotherapy, immune activation by direct application of cytokines, antibodies or adoptive cell therapy are promising approaches. In the last years, adoptive transfer of natural killer (NK) cells came into the focus of translational medicine, because of their high cytotoxic potential against transformed malignant cells. A main challenge of NK cell therapy is that it requires a high amount of functional NK cells. Therefore, ex vivo NK cell expansion protocols are currently being developed. Many culturing strategies are based on the addition of feeder or accessory cells, which need to be removed prior to the clinical application of the final NK cell product. In this study, we addressed feeder cell-free expansion methods using common γ-chain cytokines, especially IL-15 and IL-21. Our results demonstrated high potential of IL-15 for NK cell expansion, while IL-21 triggered NK cell maturation and functionality. Hence, we established a two-phase expansion protocol with IL-15 to induce an early NK cell expansion, followed by short exposure to IL-21 that boosted the cytotoxic activity of NK cells against RMS cells. Further functional analyses revealed enhanced degranulation and secretion of pro-inflammatory cytokines such as interferon-γ and tumor necrosis factor-α. In a proof of concept in vivo study, we also observed a therapeutic effect of adoptively transferred IL-15 expanded and IL-21 boosted NK cells in combination with image guided high precision radiation therapy using a luciferase-transduced RMS xenograft model. In summary, this two-phased feeder cell-free ex vivo culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.

Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease

BackgroundMesenchymal stromal cells (MSC) possess immunomodulatory properties and low immunogenicity, both crucial properties for their development into an effective cellular immunotherapy. They have shown benefit in clinical trials targeting liver diseases; however the efficacy of MSC therapy will benefit from improvement of the immunomodulatory and immunogenic properties of MSC.MethodsMSC derived from human umbilical cords (ucMSC) were treated for 3 days in vitro with various inflammatory factors, interleukins, vitamins and serum deprivation. Their immunogenicity and immunomodulatory capacity were examined by gene-expression analysis, surface-marker expressions, IDO activity, PGE2 secretion and inhibition of T cell proliferation and IFNγ production. Furthermore, their activation of NK cell cytotoxicity was investigated via CD107a expression on NK cells. The immunomodulatory capacity, biodistribution and survival of pre-treated ucMSC were investigated in a CCl4-induced liver disease mouse model. In addition, capacity of pre-treated MSC to ameliorate liver inflammation was examined in an ex vivo liver inflammation co-culture model.ResultsIFN-γ and a multiple cytokine cocktail (MC) consisting of IFN-γ, TGFβ and retinoic acid upregulated the expression of immunomodulatory factor PD-L1 and IDO activity. Subsequently, both treatments enhanced the capacity of ucMSC to inhibit CD4 and CD8 T cell proliferation and IFN-γ production. The susceptibility of ucMSC for NK cell lysis was decreased by IFN-β, TGFβ and MC treatment. In vivo, no immunomodulation was observed by the ucMSC. Four hours after intravenous infusion in mice with CCl4-induced inflammatory liver injury, the majority of ucMSC were trapped in the lungs. Rapid clearance of ucMSC(VitB6), ucMSC(Starv + VitB6) and ucMSC(MC) and altered bio-distribution of ucMSC(TGFβ) compared to untreated ucMSC was observed. In the ex vivo co-culture system with inflammatory liver slices ucMSC(MC) showed significantly enhanced modulatory capacity compared to untreated ucMSC.ConclusionsThe present study demonstrates the responsiveness of ucMSC to in vitro optimisation treatment. The observed improvements in immunomodulatory capacity as well as immunogenicity after MC treatment may improve the efficacy of ucMSC as immunotherapy targeted towards liver inflammation.

The immunological personality of close relatives of SLE patients.

Immunological abnormalities seen in relatives of patients with autoimmune disorders can be useful in understanding the pathogenesis of the disease since, unlike in patients, they cannot result from the disease process or drug treatment. In this article we present a brief overview of our studies of the basic immunological status of close relatives of SLE patients. We looked at blood levels of IgG, IgM and antibodies to double-stranded DNA, as well as at NK cell numbers and cytotoxic activity and the levels of NKT, B and T cells. As many as 60% of relatives showed one or more abnormalities in these assays. Most notably there were increased levels of IgG in male and female relatives and a reduction of IgM in females. IgG correlated inversely with NKT cell numbers adding strength to the concept that the presence of IgG autoantibodies in patients is due to impaired regulation by NKT cells. IgM, on the other hand, correlated inversely with NK cells which may thus have a role in bringing about the reduced IgM seen in some patients. Immunological abnormalities were found to be more often associated with parents and offspring of patients than with their siblings, pointing to the involvement of environmental or epigenetic influences in lupus pathogenesis.

Utilization of protein-peptide conjugates for NK cell activation as a strategy for cancer immunotherapy.

Abstract Cancer-based immunotherapy aims to develop biologics that can stimulate immune responses towards tumor eradication, as is the case of monoclonal antibodies (mAb) and their related analogs. Although the latter have been the most prevalently used biologics in cancer immunotherapy strategies, novel therapeutics may overcome their production, administration, and pharmacological limitations. The synthesis of intermediate size molecules, particularly chemically synthesized protein-peptide bioconjugates (PPB), possessing the targeting and effector functions of antibodies, represent a novel class of immunotherapeutics that may circumvent these limitations. In this study we proposed and executed the rational design of a novel class of cancer-targeting immunostimulatory protein-peptide conjugates (CTIPPC) that may potentially work as a synthetic antibody mimic. In this proof-of-concept, the CTIPPC aimed to target the tumor expressed GRP78, while recruiting and activating NK cells for tumor targeted immunotherapy. The sustained capabilities of the CTIPP conjugates to elicit NK-dependent immunostimmulatory activities were assessed via measuring cytokine secretion, NK cell migration, and the overall anti-tumor cytotoxic activity of NK cells. These studies shed important insights into the effective design, preparation and biological evaluation of a new class of semi-synthetic protein-peptide conjugates.

Cytotoxic activity and calcium signalling in natural killer cells in multiple sclerosis

Objectives Natural killer (NK) cell populations are affected in MS, with reduced circulating numbers and cytotoxic activity, altered cytokine production, and profile between CD56Dim and CD56Bright NK cell populations. Calcium permeable cation channels, transient receptor potential melastatin3 (TRPM3), are expressed on NK cells. However, the role of receptor/calcium interaction in NK cells has not been investigated in MS patients. Furthermore, the role of calcium mobilisation mediating NK lysis through receptors such as TRPM3, has not been studied in MS. Methods We examined NK cell cytotoxic activity and intracellular calcium mobilisation in untreated and alemtuzumab treated MS patients. We examined TRPM3 surface expression as a mechanism for altered NK function in MS. 22 MS (10 untreated, 12 treated alemtuzumab) and 22 healthy controls (HC) were examined. NK cytotoxic activity and calcium signalling were examined under stimulation with ionomycin, pregenelone sulphate (PregS), 2-Aminoethoxydiphenyl borate (2-ABP) and thapsigargin (TG). Receptor expression (TRPM3, CD69, CD107a) was determined on CD56Dim and CD56Bright NK cells. Results There was no difference in NK cytotoxic activity. CD69 expression was significantly lower on CD56Dimcells of untreated MS group when compared to HC (p-value 0.031) and treated MS group (p=0.036) in unstimulated conditions. This effect was abrogated in treated MS group in unstimulated and stimulated conditions. Calcium signalling was increased in CD56Bright cells of treated MS group compared to untreated MS group under stimulation with PregS, 2-ABP and TG. No difference in calcium signalling was observed in CD56Dimcells. TRPM3 expression was increased in untreated MS group compared to HC with PregS alone. Conclusions Altered NK cell profiling may be implicated in MS. Furthermore, alemtuzumab may mediate therapeutic effect via altered expression of CD56Dim and CD56Bright cells. Alemtuzumab alters calcium signalling in CD56Brightcells, however intracellular calcium signalling alone appears insufficient to explain all changes in NK cell profiling in alemtuzumab treated MS patients.

Stimulatory effect of desipramine on lung metastases of adenocarcinoma MADB 106 in stress highly-sensitive and stress non-reactive rats

The effect of antidepressant drugs on tumor progress is very poorly recognized. The aim of the present study was to examine the effect of individual reactivity to stress and 24-day desipramine (DES) administration on the metastatic colonization of adenocarcinoma MADB 106 cells in the lungs of Wistar rats. Wistar rats were subjected to stress procedure according to the chronic mild stress (CMS) model of depression for two weeks and stress highly-sensitive (SHS) and stress non-reactive (SNR) rats were selected. SHS rats were more prone to cancer metastasis than SNR ones and chronic DES treatment further increased the number of lung metastases by 59% and 50% in comparison to vehicle-treated appropriate control rats. The increase in lung metastases was connected with DES-induced skew macrophage activity towards M2 functional phenotype in SHS and SNR rats. Moreover, during 24h after DES injection in healthy rats, the decreased number of TCD8+ and B cells in SHS and SNR rats as well as NK cell cytotoxic activity in SNR rats could be attributed to the lowered capacity to defend against cancer metastasis observed in chronic DES treated and tumor injected rats.

Enriching the Housing Environment for Mice Enhances Their NK Cell Antitumor Immunity via Sympathetic Nerve-Dependent Regulation of NKG2D and CCR5.

Mice housed in an enriched environment display a tumor-resistant phenotype due to eustress stimulation. However, the mechanisms underlying enriched environment-induced protection against cancers remain largely unexplained. In this study, we observed a significant antitumor effect induced by enriched environment in murine pancreatic cancer and lung cancer models. This effect remained intact in T/B lymphocyte-deficient Rag1-/- mice, but was nearly eliminated in natural killer (NK) cell-deficient Beige mice or in antibody-mediated NK-cell-depleted mice, suggesting a predominant role of NK cells in enriched environment-induced tumor inhibition. Exposure to enriched environment enhanced NK-cell activity against tumors and promoted tumoral infiltration of NK cells. Enriched environment increased the expression levels of CCR5 and NKG2D (KLRK1) in NK cells; blocking their function effectively blunted the enriched environment-induced enhancement of tumoral infiltration and cytotoxic activity of NK cells. Moreover, blockade of β-adrenergic signaling or chemical sympathectomy abolished the effects of enriched environment on NK cells and attenuated the antitumor effect of enriched environment. Taken together, our results provide new insight into the mechanism by which eustress exerts a beneficial effect against cancer. Cancer Res; 77(7); 1611-22. ©2017 AACR.

Influence of Carbohydrate Ingestion from Bananas or Sugar Beverage on Natural Killer Cells During Recovery from 75‐km Cycling

Natural killer cells are part of the first line of defense against infection. They are also the most responsive of all immune cell types to acute exercise bouts, and their depletion from the bloodstream and potential cytotoxic capabilities loss during the recovery phase open a window for opportunistic infections to develop. Carbohydrate intake has multiple effects for the exercising athlete, including enhanced glucose availability to the working muscle, resulting in reduced post‐exercise cortisol and epinephrine levels, decreased leukocytosis and phagocytosis, and lower inflammatory cytokines. The effect of carbohydrate intake on post‐exercise NK bioactivity has never been directly assessed, and the purpose of this study was to compare ingestion of Cavendish bananas with an equicaloric, sugar‐only beverage or water alone on NK cell release and cytotoxic activity during recovery from an intensive 75‐km cycling bout. Participants included 16 male and female cyclists (age 39.9±2.4 years) who regularly competed in road races and were capable of cycling 75‐km at race pace. This study utilized a randomized crossover approach, and participants engaged in three 75‐km cycling time trials while ingesting water only, bananas and water, and a 6% sugar beverage in an overnight fasted state, with data analyzed using a repeated measures two‐way analysis of variance (ANOVA), or paired two‐tailed student t‐test. Before the exercise bout, participants ingested 5 ml/kg water alone, 6% sugar beverage, Cavendish (with water) adjusted to quantities providing 0.4 g carbohydrate per kilogram of body weight (g carb/kg). During the cycling bout, water was ingested at a rate of 3 ml/kg every 15 minutes, with carbohydrate intake from the 6% sugar beverage and banana adjusted to 0.2 g carb/kg every 15 minutes. Blood samples were collected pre‐ and post‐exercise, and then at 1.5 h and 21 h post‐exercise. Isolation and count of NK cells from whole blood was done using a Magnetic Assisted Cell Sorting technology (Miltenyi, Germany) using a CD56 positive selection. NK cell cytotoxicity was assessed using an Amnis Imagestream flow cytometer (Millipore, MA). NK cell count profile was significantly different in the water group compared to all the other groups (group interaction p=0.0051 by ANOVA), with post‐exercise NK cell counts about 71% higher in the water‐treated group vs all other groups (p<0.05 by Student t‐test). NK cell cytotoxicity was significantly decreased about 70% 1.5 h post‐exercise for all treatments, with no trial differences (time effect, p=0.0057 by ANOVA). In conclusion, these data indicate that carbohydrate ingestion from bananas with water or a 6% sugar beverage compared to water alone attenuates the sharp post‐exercise release of NK cells in the blood compartment, but has little effect on countering the post‐exercise decrement in NK cell cytotoxic potential.Support or Funding InformationSupport provided by Dole Foods, Inc., Westlake Village, CA

Immunoregulatory Role of NK Cells in Tissue Inflammation and Regeneration.

NK cells represent an important first line of defense against viral infection and cancer and are also involved in tissue homeostasis. Studies of NK cell activation in the last decade have revealed that they are able to respond to the inflammatory stimuli evoked by tissue damage and contribute to both progression and resolution of diseases. Exacerbation of the inflammatory response through interactions between immune effector cells facilitates the progression of non-alcoholic fatty liver disease (NAFLD) into steatosis, cirrhosis, and hepatocellular carcinoma (HCC). When hepatic damage is incurred, macrophage activation is crucial for initiating cross talk with neighboring cells present in the liver, including hepatocytes and NK cells, and the importance of this interaction in shaping the immune response in liver disease is increasingly recognized. Inflicted structural damage can be in part regenerated via the process of self-limiting fibrosis, though persistent hepatic damage will lead to chronic fibrosis and loss of tissue organization and function. The cytotoxic activity of NK cells plays an important role in inducing hepatic stellate cell apoptosis and thus curtailing the progression of fibrosis. Alternatively, in some diseases, such as HCC, NK cells may become dysregulated, promoting an immunosuppressive state where tumors are able to escape immune surveillance. This review describes the current understanding of the contributions of NK cells to tissue inflammation and metabolic liver diseases and the ongoing effort to develop therapeutics that target the immunoregulatory function of NK cells.

Chitosan promotes immune responses, ameliorating total mature white blood cell numbers, but increases glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, and ameliorates lactate dehydrogenase levels in leukemia mice in vivo.

The aim of the present study was to investigate the effect of chitosan (a naturally derived polymer) on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) levels in WEHI-3 cell-generated leukemia mice. Mice were divided into control, WEHI-3 control, acetic acid (vehicle)-treated, and 5 and 20 mg/kg chitosan-treated groups. Mice were subsequently weighed, blood was collected, and liver and spleen samples were isolated and weighed. Blood samples were measured for cell markers, the spleen underwent phagocytosis and natural killer (NK) cell activity examination, and cell proliferation was analyzed by flow cytometry. Chitosan did not significantly affect the weights of body, liver and spleen at 5 and 20 mg/kg treatment. Chitosan increased the percentage of CD3 (T cells marker), decreased the levels of CD19 (B-cell marker) and CD11b at 5 mg/kg treatment, and decreased the levels of Mac-3 at 5 and 20 mg/kg treatment. Chitosan significantly increased macrophage phagocytosis of PBMCs, but did not significantly affect macrophage phagocytosis in the peritoneal cavity. Chitosan treatment did not significantly affect the cytotoxic activity of NK cells, and also did not affect T- and B-cell proliferation. Chitosan significantly increased total white blood cell numbers, and GOT and GPT activities were both significantly increased. However, chitosan did not significantly affect LDH activity in leukemia mice. Chitosan may aid in future studies on improving immune responses in the treatment of leukemia.

Abstract B80: Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer

Abstract Background: While the majority of Triple Negative Breast Cancer (TNBC) overexpress epidermal growth factor receptor (EGFR), EGFR inhibition as a monotherapy with Cetuximab is not effective. The study of its mechanism of action is important to find out potentially synergistic combinations. Previously, we demonstrated that Cetuximab can trigger antibody-dependent NK cell-mediated cytotoxicity against TNBC cells. The aim of this study was to investigate the effect of NK cell activation by Cetuximab-coated TNBC cells on Dendritic Cell (DC) function, and the result of its combination with IL-15. Methods: Human monocyte-derived DCs were co-cultured with autologous isolated NK cells, TNBC cells, Cetuximab and/or IL-15 for 24hs. Costimulatory molecule and activation marker expression in DC and NK cells were analyzed by flow cytometry, and cytokine concentration in culture supernatants was assessed by ELISA. DCs obtained after co-cultures were incubated for 24hs with CD40 ligand transfected fibroblasts to mimic the interaction with CD40L expressing Th cells, and IL-12 was analyzed in culture supernatants by ELISA. Results: Cetuximab treatment of TNBC cells enhanced DC maturation only when NK cells were present in the co-culture (%CD83+: 6.6±0.7 vs 61.0±17, p<0.001; CD86 MIF: 121±36 vs 1868±833, p<0.01). In concert, NK cells were activated by Cetuximab showing increased CD25 expression and IFN-γ production, a cytokine known to induce DC activation. After subsequent CD40 ligand stimulation, DCs that where co-cultured with Cetuximab activated NK cells had an increased ability to produce IL-12p70. Furthermore, IL-15 combination with Cetuximab not only promoted NK cell cytotoxic activity against TNBC cells, but also induced IFN-γ production and DC maturation. Conclusions: Our results suggest that besides its anti-proliferative mechanism of action, Cetuximab has immunological effects in an in vitro model of TNBC, promoting NK cell activation and DC maturation, which could be potentiated by the combination with IL-15. This could ultimately result in the priming of T cell-based immunity, which will be evaluated in further studies. Citation Format: Estefanía P. Juliá, María Betina Pampena, Yamila S. Rocca, José Mordoh, Estrella M. Levy. Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B80.

Melanoma-associated fibroblasts decrease tumor cell susceptibility to NK cell-mediated killing through matrix-metalloproteinases secretion.

Cancer-associated fibroblasts (CAFs) play a central role in the complex process of tumor-stroma interaction and promote tumor growth. Emerging evidences also suggest that these fibroblasts are involved in the alteration of the anti-tumor immune response by impacting several immune cell populations, especially through their secretion of pro-inflammatory and immunosuppressive factors in the tumor microenvironment. However, the underlying immuno-modulating mechanisms triggered by these fibroblasts are still only partially defined. In this study, we provide evidence that melanoma-associated fibroblasts decrease the susceptibility of melanoma tumor cells to NK-mediated lysis through the secretion of active matrix metalloproteinases. This secretion reduces the expression of the two NKG2D ligands, MICA/B, at the surface of tumor cells and consequently decreases the NKG2D-dependent cytotoxic activity of NK cells against melanoma tumor cells. Together, our data demonstrate that the modification of tumor cell susceptibility to killer cells is an important determinant of the anti-tumor immune response alteration triggered by CAFs.

Biallelic IRF8 Mutations Causing NK Cell Deficiency

Human primary immunodeficiencies result in an exacerbated susceptibility to contracting infectious diseases. Recent work by Mace et al., published in the Journal of Clinical Investigation, unveils a novel genetic cause for the development of familial natural killer (NK) cell deficiency: a biallelic compound heterozygous mutation in IRF8, which leads to impaired NK cell development and cytotoxic activity.

Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

Decreased expression of the NKG2D ligand ULBP4 may be an indicator of poor prognosis in patients with nasopharyngeal carcinoma.

U16-binding protein 4 (ULBP4), a human ligand for natural killer group 2, member D (NKG2D) receptor on NK cells and subsets of T cells, is thought to activate anticancer immune responses. However, the expression pattern and prognostic effect of ULBP4 in nasopharyngeal carcinoma (NPC) has not been investigated. We first compared ULBP4 expression between archival 15 NPC tissues and 8 normal nasopharynx (NP) tissues using qPCR. Then ULBP4 expression among 111 NPC specimens was validated on immunohistochemical examination. In addition, the association of ULBP4 expression with clinical characteristics and survival outcomes was analyzed. Furthermore, the impact of ULBP4 expression in NPC cells on the cytotoxic activity of NK cells was investigated. Both mRNA and protein ULBP4 expressions of NPC tissues were significantly lower than those in normal NP tissues. However, no association of ULBP4 expression with clinical characteristics was observed. Patients with NPC having decreased expression of UBLP4 had significantly poorer overall survival (OS), progression-free survival (PFS), and distant metastasis-free survival (DMFS) than those with preserved levels of ULBP4. On multivariate analyses, low expression of ULBP4 was of borderline significance for OS, PFS, and DMFS (P = 0.060, 0.053, and 0.076, respectively). Further, LDH analysis demonstrated that the cytotoxic activitity of NK cells against C666-1 or 5-8F NPC cells with lenti-ULBP4 was considerably increased as compared to those with lenti-vector at various E/T ratios. Hence, restoration of ULBP4 expression may be a novel therapeutic strategy for treatment of NPC. However, further study is required to confirm these findings.

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis

ABSTRACTExosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70+ exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.

Activation of NK cell cytotoxicity by aerosolized CpG-ODN/poly(I:C) against lung melanoma metastases is mediated by alveolar macrophages

Controversies remain about NK cells direct responsiveness to Toll-like receptor (TLR) agonists or dependence on macrophages. In a melanoma lung metastasis model, aerosolized TLR9 and TLR3 agonists have been reported to induce antitumor immunity through NK cells activation. In the current study, we demonstrated that in vitro TLR9/TLR3 stimulation induced IFN-γ secretion by NK cells, but an increase in their cytotoxicity was detected only after NK cells co-culture with in vitro TLR9/TLR3 agonists pretreated alveolar macrophages. Alveolar macrophages from melanoma lung metastases-bearing mice, treated with aerosolized TLR agonists, also promoted NK cell cytotoxicity. Activated NK cells from lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists were able to polarize naive alveolar macrophages toward a M1-like phenotype. Our results demonstrate that activation of NK cells in the lung after TLR engagement is mediated by alveolar macrophages and that activated NK cells shape macrophage behavior.