Science.gov

Sample records for nk cells mediated

  1. Altered NK Cell Development and Enhanced NK Cell-Mediated Resistance to MCMV in NKG2D-Deficient Mice

    PubMed Central

    Zafirova, Biljana; Mandarić, Sanja; Antulov, Ronald; Krmpotić, Astrid; Jonsson, Helena; Yokoyama, Wayne M.; Jonjić, Stipan; Polić, Bojan

    2009-01-01

    Summary NKG2D is a potent activating receptor on NK cells which acts as a molecular sensor for stressed cells expressing NKG2D ligands such as infected or tumor transformed cells. Although NKG2D is expressed on NK cell precursors, its role in NK cell development is still not known. We have generated NKG2D-deficient mice by targeting the Klrk1 locus. Here we provide evidence for an important regulatory role of NKG2D in the development of NK cells. The absence of NKG2D causes faster division of NK cells, perturbation in size of some NK cell subpopulations and their augmented sensitivity to apoptosis. As expected, NKG2D−/− NK cells are less responsive to tumor targets expressing NKG2D ligands. NKG2D−/− mice, however, show an enhanced NK cell-mediated resistance to MCMV infection as a consequence of NK cell dysregulation. Altogether, these findings provide evidence for yet unknown regulatory function of NKG2D in NK cell physiology. PMID:19631564

  2. Effects of reactive nitrogen scavengers on NK-cell-mediated killing of K562 cells.

    PubMed

    Zeng, Yili; Huang, Qinmiao; Zheng, Meizhu; Guo, Jianxin; Pan, Jingxin

    2012-01-01

    This study explored the effects of reactive nitrogen metabolites (RNMS) on natural-killer- (NK-) cell-mediated killing of K562 cells and the influence of RNM scavengers, such as tiopronin (TIP), glutamylcysteinylglycine (GSH), and histamine dihydrochloride (DHT), on reversing the suppressing effect of RNM. We administered exogenous and endogenous RNM in the NK + K562 culture system and then added RNM scavengers. The concentrations of RNM, TNF-β and IFN-γ, and NK-cell cytotoxicity (NCC) and the percentage of living NK cells were then examined. We found that both exogenous and endogenous RNM caused the KIR to decrease (P < 0.01); however, RNM scavengers such as TIP and GSH rescued this phenomenon dose dependently. In conclusion, our data suggests that RNM scavengers such as TIP and GSH enhance the antineoplasmic activity of NK cells.

  3. IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA.

    PubMed

    Kloess, Stephan; Huenecke, Sabine; Piechulek, Daniel; Esser, Ruth; Koch, Joachim; Brehm, Claudia; Soerensen, Jan; Gardlowski, Tanja; Brinkmann, Andrea; Bader, Peter; Passweg, Jakob; Klingebiel, Thomas; Schwabe, Dirk; Koehl, Ulrike

    2010-11-01

    NK group 2D (NKG2D)-expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain-related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D-dependent NK-cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D-MICA interaction have been elucidated, its impact for donor NK (dNK) cell-based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL-2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high-risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK-cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK-cell-mediated cytotoxicity. This effect could be reversed by high-dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non-occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.

  4. Role of complement and NK cells in antibody mediated rejection.

    PubMed

    Akiyoshi, Takurin; Hirohashi, Tsutomu; Alessandrini, Alessandro; Chase, Catherine M; Farkash, Evan A; Neal Smith, R; Madsen, Joren C; Russell, Paul S; Colvin, Robert B

    2012-12-01

    Despite extensive research on T cells and potent immunosuppressive regimens that target cellular mediated rejection, few regimens have been proved to be effective on antibody-mediated rejection (AMR), particularly in the chronic setting. C4d deposition in the graft has been proved to be a useful marker for AMR; however, there is an imperfect association between C4d and AMR. While complement has been considered as the main player in acute AMR, the effector mechanisms in chronic AMR are still debated. Recent studies support the role of NK cells and direct effects of antibody on endothelium cells in a mechanism suggesting the presence of a complement-independent pathway. Here, we review the history, currently available systems and progress in experimental animal research. Although there are consistent findings from human and animal research, transposing the experimental results from rodent to human has been hampered by the differences in endothelial functions between species. We briefly describe the findings from patients and compare them with results from animals, to propose a combined perspective.

  5. Langerhans Cells Suppress CD49a+ NK Cell-Mediated Skin Inflammation.

    PubMed

    Scholz, Felix; Naik, Shruti; Sutterwala, Fayyaz S; Kaplan, Daniel H

    2015-09-01

    Recruitment of innate immune effector cells into sites of infection is a critical component of resistance to pathogen infection. Using a model of intradermal footpad injection of Candida albicans, we observed that inflammation as measured by footpad thickness and neutrophil recruitment occurred independent of adoptive immunity but was significantly reduced in MyD88(-/-) and IL-6(-/-) mice. Unexpectedly, huLangerin-DTA mice (ΔLC) that lack Langerhans cells (LC) developed increased skin inflammation and expressed higher amounts of IL-6, suggesting a suppressive role for LC. Increased inflammation also occurred in Rag1(-/-) ΔLC mice but was reversed by Ab-mediated ablation of NK cells. CXCR6(+)CD49a(+) NK cells are a liver-resident subset that can mediate inflammatory skin responses. We found that exaggerated skin inflammation was absent in ΔLC × CXCR6(-/-) mice. Moreover, the exaggerated response in ΔLC mice could be adoptively transferred with liver CD49a(+) NK cells. Finally, CD49a(+) NK cells in ΔLC but not control mice were recruited to the skin, and inhibition of their recruitment prevented the exaggerated response. Thus, in the absence of LC, CD49a(+) liver NK cells display an inappropriately proinflammatory phenotype that results in increased local skin inflammation. These data reveal a novel function for LC in the regulation of this recently described subset of skin tropic NK cells. PMID:26209621

  6. NK Cells and γδ T Cells Mediate Resistance to Polyomavirus–Induced Tumors

    PubMed Central

    Mishra, Rabinarayan; Chen, Alex T.; Welsh, Raymond M.; Szomolanyi-Tsuda, Eva

    2010-01-01

    NK and γδ T cells can eliminate tumor cells in many experimental models, but their effect on the development of tumors caused by virus infections in vivo is not known. Polyomavirus (PyV) induces tumors in neonatally infected mice of susceptible strains and in adult mice with certain immune deficiencies, and CD8+ αβ T cells are regarded as the main effectors in anti-tumor immunity. Here we report that adult TCRβ knockout (KO) mice that lack αβ but have γδ T cells remain tumor-free after PyV infection, whereas TCRβ×δ KO mice that lack all T cells develop tumors. In addition, E26 mice, which lack NK and T cells, develop the tumors earlier than TCRβ×δ KO mice. These observations implicate γδ T and NK cells in the resistance to PyV-induced tumors. Cell lines established from PyV-induced tumors activate NK and γδ T cells both in culture and in vivo and express Rae-1, an NKG2D ligand. Moreover, these PyV tumor cells are killed by NK cells in vitro, and this cytotoxicity is prevented by treatment with NKG2D-blocking antibodies. Our findings demonstrate a protective role for NK and γδ T cells against naturally occurring virus-induced tumors and suggest the involvement of NKG2D-mediated mechanisms. PMID:20523894

  7. FoxO1-mediated autophagy is required for NK cell development and innate immunity

    PubMed Central

    Wang, Shuo; Xia, Pengyan; Huang, Guanling; Zhu, Pingping; Liu, Jing; Ye, Buqing; Du, Ying; Fan, Zusen

    2016-01-01

    Natural killer (NK) cells exert a crucial role in early immune responses as a major innate effector component. However, the underlying mechanisms of NK cell development remain largely elusive. Here we show that robust autophagy appears in the stage of immature NK cells (iNKs), which is required for NK cell development. Autophagy defects result in damaged mitochondria and accumulation of reactive oxygen species (ROS) that leads to apoptosis of NK cells. Autophagy protects NK cell viability during development through removal of damaged mitochondria and intracellular ROS. Phosphorylated Forkhead box O (FoxO)1 is located to the cytoplasm of iNKs and interacts with Atg7, leading to induction of autophagy. FoxO1 deficiency or an inactive FoxO1AAA mutant abrogates autophagy initiation in iNKs and impairs NK cell development and viral clearance. Therefore we conclude that FoxO1-mediated autophagy is required for NK cell development and NK cell-induced innate immunity. PMID:27010363

  8. Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV/SHIV Infection

    PubMed Central

    He, Xuan; Li, Dan; Luo, Zhenwu; Liang, Hua; Peng, Hong; Zhao, Yangyang; Wang, Nidan; Liu, Donghua; Qin, Chuan; Wei, Qiang; Yan, Huimin; Shao, Yiming

    2013-01-01

    Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies. PMID:23424655

  9. RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

    PubMed

    Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

    2015-01-01

    Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

  10. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    PubMed Central

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  11. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy.

    PubMed

    Wang, Wei; Erbe, Amy K; Hank, Jacquelyn A; Morris, Zachary S; Sondel, Paul M

    2015-01-01

    Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be "specifically activated" through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies.

  12. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma.

    PubMed

    Xu, Liyun; Huang, Yanyan; Tan, Linlin; Yu, Wei; Chen, Dongdong; Lu, ChangChang; He, Jianying; Wu, Guoqing; Liu, Xiaoguang; Zhang, Yongkui

    2015-12-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56+ cells (P<0.05) and CD3-CD56(dim) cells (P<0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56+ NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P<0.05) or with tumor stage T3-T4 (P<0.05). Tim-3 expression in CD56(dim) NK-cell subset was higher in patients with tumor size ≥3cm (P<0.05), or LNM (P<0.05) or with tumor stage T3-T4 (P<0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56+ NK cells and CD56(dim) NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P=0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.

  13. Sleep-deprivation reduces NK cell number and function mediated by β-adrenergic signalling.

    PubMed

    De Lorenzo, Beatriz H P; de Oliveira Marchioro, Laís; Greco, Carollina Ribeiro; Suchecki, Deborah

    2015-07-01

    Reduction of sleep time triggers a stress response, leading to augmented levels of glucocorticoids and adrenaline. These hormones regulate components of the innate immune system such as natural killer (NK) and NKT cells. In the present study, we sought to investigate whether and how stress hormones could alter the population and function of NK and NKT cells of mice submitted to different lengths of paradoxical sleep deprivation (PSD, from 24 to 72 h). Results showed that 72h of PSD decreased not only NK and NKT cell counts, but also their cytotoxic activity against B16F10 melanoma cells in vitro. Propranolol treatment during PSD reversed these effects, indicating a major inhibitory role of beta-adrenergic receptors (β-AR) on NK cells function. Moreover, both corticosterone plasma levels and expression of beta 2-adrenergic receptors (β2-AR) in NK cells increased by 48 h of PSD. In vitro incubation of NK cells with dexamethasone augmented the level of β2-AR in the cell surface, suggesting that glucocorticoids could induce β2-AR expression. In summary, we propose that reduction of NK and NKT cell number and cytotoxic activity appears to be mediated by glucocorticoids-induced increased expression of β2-AR in these cells. PMID:25929826

  14. Phenotypically distinct helper NK cells are required for gp96-mediated anti-tumor immunity

    PubMed Central

    Sedlacek, Abigail L.; Kinner-Bibeau, Lauren B.; Binder, Robert J.

    2016-01-01

    A number of Heat Shock Proteins (HSPs), in the extracellular environment, are immunogenic. Following cross-presentation of HSP-chaperoned peptides by CD91+ antigen presenting cells (APCs), T cells are primed with specificity for the derivative antigen-bearing cell. Accordingly, tumor-derived HSPs are in clinical trials for cancer immunotherapy. We investigate the role of NK cells in gp96-mediated anti-tumor immune responses given their propensity to lyse tumor cells. We show that gp96-mediated rejection of tumors requires a unique and necessary helper role in NK cells. This helper role occurs during the effector phase of the anti-tumor immune response and is required for T cell and APC function. Gp96 activates NK cells indirectly via APCs to a phenotype distinct from NK cells activated by other mechanisms such as IL-2. While NK cells have both lytic and cytokine producing properties, we show that gp96 selectively activates cytokine production in NK cells, which is important in the HSP anti-tumor immune response, and leaves their cytotoxic capacity unchanged. PMID:27431727

  15. The skin test antigen stimulated killer (STAK) cell mediating NK like CMC is OKM1 positive and OKT3 negative.

    PubMed Central

    Tartof, D; Curran, J J; Levitt, D; Loken, M R

    1983-01-01

    Recently we demonstrated that candida antigen stimulated natural killer cell like cell-mediated cytolysis (NK like CMC) in peripheral blood mononuclear cells (PBMNC) isolated from normal individuals (Tartof et al., 1980). Utilizing monoclonal antibodies directed against human mononuclear cell subpopulations in conjunction with a fluorescence activated cell sorter (FACS) we determined that, similar to the previously described NK cell, the skin test antigen stimulated killer (STAK) cell is a larger OKM1 positive, OKT3 negative cell. We obtained similar results using two different skin test antigens. Thus, stimulation of NK like CMC in PBMNC by skin test antigens probably represents activation of NK or NK like cells. PMID:6360439

  16. CAM and NK Cells

    PubMed Central

    2004-01-01

    It is believed that tumor development, outgrowth and metastasis are under the surveillance of the immune system. Although both innate and acquired immune systems play roles, innate immunity is the spearhead against tumors. Recent studies have revealed the critical role of natural killer (NK) cells in immune surveillance and that NK cell activity is considerably influenced by various agents, such as environmental factors, stress, foods and drugs. Some of these NK cell stimulants have been used in complementary and alternative medicine (CAM) since ancient times. Therefore, the value of CAM should be re-evaluated from this point of view. In this review, we overview the intimate correlation between NK cell functions and CAM agents, and discuss possible underlying mechanisms mediating this. In particular, neuro-immune crosstalk and receptors for CAM agents are the most important and interesting candidates for such mechanisms. PMID:15257322

  17. Cytolysis of oligodendrocytes is mediated by killer (K) cells but not by natural killer (NK) cells.

    PubMed

    Satoh, J; Kim, S U; Kastrukoff, L F

    1991-03-01

    The cytotoxic activity of killer (K) cells against enriched cultures of bovine oligodendrocytes (BOL) was investigated in multiple sclerosis (MS) and controls. Human K cells mediated cytotoxicity to primary cultures of BOL in the presence of anti-BOL antiserum in all study groups, while BOL were resistant to human natural killer (NK) cells. Cytotoxic activity was significantly reduced in MS when compared to age-matched normal controls but not when compared to other neurologic disease (OND) patients. K cell-mediated lysis of BOL could also be induced with anti-galactocerebroside antibody but not with other antibodies including those specific for OL antigens (myelin basic protein, proteolipid apoprotein, and 2',3'-cyclic nucleotide 3'-phosphodiesterase). Enrichment of the effector population indicated that antibody-dependent cell-mediated cytotoxicity (ADCC) to BOL was mediated by large granular lymphocytes, and the effector population was further characterized by flow cytometry. The effector cells mediating ADCC could be inhibited by protein A of Staphylococcus aureus, and by K562 cells in cold competition assay. These observations indicate that oligodendrocytes are resistant to NK cells but are susceptible to cytolysis mediated by K cells. This may represent a potentially important immune mechanism in the pathogenesis of MS.

  18. Escape of HIV-1-infected dendritic cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk--a pivotal role of HMGB1.

    PubMed

    Melki, Marie-Thérèse; Saïdi, Héla; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Gougeon, Marie-Lise

    2010-04-01

    Early stages of Human Immunodeficiency Virus-1 (HIV-1) infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK) cells and dendritic cells (DCs). Immature DCs (iDCs) capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them ("editing process") at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL)-Death Receptor 4 (DR4) pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC(HIV) become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC(HIV). The escape of DC(HIV) from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP) and the cellular inhibitor of apoptosis 2 (c-IAP2), induced by NK-DC(HIV) cognate interaction. High-mobility group box 1 (HMGB1), an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC(HIV). Finally, we demonstrate that restoration of DC(HIV) susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific si

  19. WASH has a critical role in NK cell cytotoxicity through Lck-mediated phosphorylation

    PubMed Central

    Huang, L; Zhu, P; Xia, P; Fan, Z

    2016-01-01

    Natural killer (NK) cells are important effector cells of the innate immune system to kill certain virus-infected and transformed cells. Wiskott–Aldrich Syndrome protein (WASP) and SCAR homolog (WASH) has been identified as a member of WASP family proteins implicated in regulating the cytoskeletal reorganization, yet little is known about its function in lymphocytes. Here we demonstrate that WASH is crucial for NK cell cytotoxicity. WASH was found to colocalize with lytic granules upon NK cell activation. Knockdown of WASH expression substantially inhibited polarization and release of lytic granules to the immune synapse, resulting in the impairment of NK cell cytotoxicity. More importantly, our data also define a previously unappreciated mechanism for WASH function, in which Src family kinase Lck can interact with WASH and induce WASH phosphorylation. Mutation of tyrosine residue Y141, identified here as the major site of WASH phosphorylation, partially blocked WASH tyrosine phosphorylation and NK cell cytotoxicity. Taken together, these observations suggest that WASH has a pivotal role for regulation of NK cell cytotoxicity through Lck-mediated Y141 tyrosine phosphorylation. PMID:27441653

  20. Inflammatory cytokine-mediated evasion of virus-induced tumors from NK cell control

    PubMed Central

    Mishra, Rabinarayan; Polic, Bojan; Welsh, Raymond M.; Szomolanyi-Tsuda, Eva

    2013-01-01

    Infections with DNA tumor viruses, including members of the polyomavirus family, often result in tumor formation in immune-deficient hosts. The complex control involved in antiviral and antitumor immune responses during these infections can be studied in murine polyomavirus (PyV)-infected mice as a model. We found that NK cells efficiently kill cells derived from PyV-induced salivary gland tumors in vitro in an NKG2D (effector cell) -RAE-1 (target cell) - dependent manner, but in T cell-deficient mice NK cells only delay but do not prevent the development of PyV-induced tumors. Here we show that the PyV-induced tumors have infiltrating functional NK cells. The freshly removed tumors, however, lack surface RAE-1 expression, and the tumor tissues produce soluble factors that down-regulate RAE-1. These factors include the pro-inflammatory cytokines IL-1α, IL-1β, IL-33, and TNF. Each of these cytokines down-regulate RAE-1 expression and susceptibility to NK cell mediated cytotoxicity. CD11b+F4/80+ macrophages infiltrating the PyV-induced tumors produce high amounts of IL-1β and TNF. Thus, our data suggest a new mechanism whereby inflammatory cytokines generated in the tumor environment lead to evasion of NK cell-mediated control of virus-induced tumors. PMID:23772039

  1. NLRC5 shields T lymphocytes from NK-cell-mediated elimination under inflammatory conditions

    PubMed Central

    Ludigs, Kristina; Jandus, Camilla; Utzschneider, Daniel T.; Staehli, Francesco; Bessoles, Stéphanie; Dang, Anh Thu; Rota, Giorgia; Castro, Wilson; Zehn, Dietmar; Vivier, Eric; Held, Werner; Romero, Pedro; Guarda, Greta

    2016-01-01

    NLRC5 is a transcriptional regulator of MHC class I (MHCI), which maintains high MHCI expression particularly in T cells. Recent evidence highlights an important NK–T-cell crosstalk, raising the question on whether NLRC5 specifically modulates this interaction. Here we show that NK cells from Nlrc5-deficient mice exhibit moderate alterations in inhibitory receptor expression and responsiveness. Interestingly, NLRC5 expression in T cells is required to protect them from NK-cell-mediated elimination upon inflammation. Using T-cell-specific Nlrc5-deficient mice, we show that NK cells surprisingly break tolerance even towards ‘self' Nlrc5-deficient T cells under inflammatory conditions. Furthermore, during chronic LCMV infection, the total CD8+ T-cell population is severely decreased in these mice, a phenotype reverted by NK-cell depletion. These findings strongly suggest that endogenous T cells with low MHCI expression become NK-cell targets, having thus important implications for T-cell responses in naturally or therapeutically induced inflammatory conditions. PMID:26861112

  2. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

    PubMed

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A; Varadarajan, Navin

    2014-11-20

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

  3. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

    PubMed Central

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A.

    2014-01-01

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

  4. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

    PubMed

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A; Varadarajan, Navin

    2014-11-20

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

  5. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Dixon, Amera; Orange, Jordan S.

    2016-01-01

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation. PMID:27435370

  6. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation.

    PubMed

    Brennan, Todd V; Lin, Liwen; Brandstadter, Joshua D; Rendell, Victoria R; Dredge, Keith; Huang, Xiaopei; Yang, Yiping

    2016-01-01

    Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell-based antitumor therapies. PMID:26649979

  7. Chip-based platform for dynamic analysis of NK cell cytolysis mediated by a triplebody.

    PubMed

    Chatzopoulou, Elisavet I; Roskopf, Claudia C; Sekhavati, Farzad; Braciak, Todd A; Fenn, Nadja C; Hopfner, Karl-Peter; Oduncu, Fuat S; Fey, Georg H; Rädler, Joachim O

    2016-04-01

    Cancer therapy via redirected lysis mediated by antibodies and antibody-derived agents relies on the availability of substantial numbers of sufficiently active immune effector cells. To monitor antitumor responses before and during therapy, sensitive methods are needed, capable of quantitating specific lysis of target cells. Here we present a chip-based single-cell cytometric assay, which uses adherent human target cells arrayed in structured micro-fields. Using a fluorescent indicator of cell death and time-lapse microscopy in an automated high-throughput mode, we measured specific target cell lysis by activated human NK cells, mediated by the therapeutic single chain triplebody SPM-2 (33-16-123). This antibody-derived tri-specific fusion protein carries binding sites for the myeloid antigens CD33 and CD123 and recruits NK cells via a binding site for the Fc-receptor CD16. Specific lysis increased with increasing triplebody concentration, and the single-cell assay was validated by direct comparison with a standard calcein-release assay. The chip-based approach allowed measurement of lysis events over 16 hours (compared to 4 hours for the calcein assay) and required far smaller numbers of primary cells. In addition, dynamic properties inaccessible to conventional methods provide new details about the activation of cytolytic effector cells by antibody-derived agents. Thus, the killing rate exhibited a dose-dependent maximum during the reaction interval. In clinical applications ex vivo monitoring of NK activity of patient's endogenous cells will likely help to choose appropriate therapy, to detect impaired or recovered NK function, and possibly to identify rare subsets of cancer cells with particular sensitivity to effector-cell mediated lysis. PMID:26958659

  8. IL-15 Superagonist–Mediated Immunotoxicity: Role of NK Cells and IFN-γ

    PubMed Central

    Guo, Yin; Luan, Liming; Rabacal, Whitney; Bohannon, Julia K.; Fensterheim, Benjamin A.; Hernandez, Antonio

    2015-01-01

    IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8+ T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8+ T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA–induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA–mediated immunotoxicity. PMID:26216888

  9. Drug-induced hyperploidy stimulates an antitumor NK cell response mediated by NKG2D and DNAM-1 receptors

    PubMed Central

    Acebes-Huerta, Andrea; Lorenzo-Herrero, Seila; Folgueras, Alicia R.; Huergo-Zapico, Leticia; Lopez-Larrea, Carlos; López-Soto, Alejandro; Gonzalez, Segundo

    2016-01-01

    ABSTRACT Formation of polyploid or aneuploid cells is a pathological hallmark of malignant tumors. Cell cycle checkpoint mechanisms play a crucial role in ensuring genomic integrity during mitosis, avoiding the generation of aneuploid cells. Additionally, cancer cell DNA ploidy is subjected to extrinsic controls operated by activation of adaptive immune responses mediated by T cells. NK cells exert a central role in the innate anticancer immunity; however, the mechanisms involved in the recognition of tumor cells by NK cells have not been fully elucidated. Herein, we report that drug-induced polyploidy in cancer cells activates antitumor responses mediated by NK cells. Thus, hyperploidy-inducing chemotherapeutic agents strongly upregulate the tumor expression of ligands for the NK cell activating receptors NKG2D and DNAM-1. Drug-induced hyperploidy modulated the repertoire of activating receptors and the cytokine profile of NK cells, rendering tumor cells more susceptible to NK cell-mediated lysis through the activation of NKG2D and DNAM-1 receptors. In addition, hyperploidization stimulated the production of IL-2 by CD4 T cells, which induced NK cell proliferation and activity. The stimulation of MICA, a key NKG2D ligand, in hyperploid cells was mainly mediated by ATM protein kinase. Likewise, pharmacological inhibition of key regulators of endoplasmic reticulum stress in certain cell models supports a role for this pathway in NKG2D ligand upregulation. Overall, our findings indicate that, besides the cytotoxic effect on tumor cells, the therapeutic activity of anti-mitotic drugs may be mediated by the induction of a coordinated antitumor immune response involving NK and T cells. PMID:27057443

  10. The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses

    PubMed Central

    Russier, Marion; Reynard, Stéphanie; Carnec, Xavier

    2014-01-01

    ABSTRACT Lassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a

  11. Delta-short consensus repeat 4-decay accelerating factor (DAF: CD55) inhibits complement-mediated cytolysis but not NK cell-mediated cytolysis.

    PubMed

    Miyagawa, Shuji; Kubo, Tomoko; Matsunami, Katsuyoshi; Kusama, Tamiko; Beppu, Keiko; Nozaki, Hiroshi; Moritan, Toshiyuki; Ahn, Curie; Kim, Jae Young; Fukuta, Daisuke; Shirakura, Ryota

    2004-09-15

    NK cells play a critical role in the rejection of xenografts. In this study, we report on an investigation of the effect of complement regulatory protein, a decay accelerating factor (DAF: CD55), in particular, on NK cell-mediated cytolysis. Amelioration of human NK cell-mediated pig endothelial cell (PEC) and pig fibroblast cell lyses by various deletion mutants and point substitutions of DAF was tested, and compared with their complement regulatory function. Although wild-type DAF and the delta-short consensus repeat (SCR) 1-DAF showed clear inhibition of both complement-mediated and NK-mediated PEC lyses, delta-SCR2-DAF and delta-SCR3-DAF failed to suppress either process. However, delta-SCR4-DAF showed a clear complement regulatory effect, but had no effect on NK cells. Conversely, the point substitution of DAF (L147 x F148 to SS and KKK(125-127) to TTT) was half down-regulated in complement inhibitory function, but the inhibition of NK-mediated PEC lysis remained unchanged. Other complement regulatory proteins, such as the cell membrane-bound form factor H, fH-PI, and C1-inactivator, C1-INH-PI, and CD59 were also assessed, but no suppressive effect on NK cell-mediated PEC lysis was found. These data suggest, for DAF to function on NK cells, SCR2-4 is required but no relation to its complement regulatory function exists.

  12. STING in tumor and host cells cooperatively work for NK cell-mediated tumor growth retardation.

    PubMed

    Takashima, Ken; Takeda, Yohei; Oshiumi, Hiroyuki; Shime, Hiroaki; Okabe, Masaru; Ikawa, Masahito; Matsumoto, Misako; Seya, Tsukasa

    2016-09-30

    An interferon-inducing DNA sensor STING participates in tumor rejection in mouse models. Here we examined what mechanisms contribute to STING-dependent growth retardation of B16 melanoma sublines by NK cells in vivo. The studies were designed using WT and STING KO black mice, and B16D8 (an NK-sensitive melanoma line having STING) and STING KO B16D8 sublines established for this study. The results from tumor-implant studies suggested that STING in host immune cells and tumor cells induced distinct profiles of chemokines including CXCL10, CCL5 and IL-33, and both participated in NK cell infiltration and activation in B16D8 tumor. Spontaneous activation of STING occurs in host-immune and tumor cells of this NK-sensitive tumor, thereby B16D8 tumor growth being suppressed in this model. Our data show that STING induces tumor cytotoxicity by NK cells through tumor and host immune cell network to contribute to innate surveillance and suppression of tumors in vivo. PMID:27608599

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

    PubMed

    Castriconi, Roberta; Dondero, Alessandra; Negri, Francesca; Bellora, Francesca; Nozza, Paolo; Carnemolla, Barbara; Raso, Alessandro; Moretta, Lorenzo; Moretta, Alessandro; Bottino, Cristina

    2007-11-01

    Adoptive cellular immunotherapy has been proposed as an additional treatment of medulloblastoma, an intracranial tumor characterized by a particularly poor prognosis. However, little is known on the ability of the immune system to effectively attack this tumor. In this study, we show that activated human NK cells efficiently kill medulloblastoma cell lines in vitro. NK-mediated killing involved different activating receptors (including NKp46, NKp30, DNAM-1 and NKG2D) and correlated with the presence of their specific ligands on tumor cells. In contrast, the absence of major adhesion interactions, such as LFA-1/ICAM did not impair the NK-mediated cytotoxicity. Medulloblastoma expressed a number of tumor-associated molecules including CD146 and CD133, considered a marker for cancer stem cells. Remarkably, both CD133-positive and CD133-negative cell lines were susceptible to lysis. Tumor cells also expressed molecules that are currently used as diagnostic tools for neuroblastoma cell identification. In particular, B7 homolog 3 (B7-H3) was expressed by all the medulloblastoma cell lines analyzed, while the presence of GD(2) and NB84 was restricted to given cell lines and/or marked a defined tumor cell subset.

  14. Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function

    PubMed Central

    Goodier, Martin R.; Lusa, Chiara; Sherratt, Sam; Rodriguez-Galan, Ana; Behrens, Ron; Riley, Eleanor M.

    2016-01-01

    Cross-linking of FcγRIII (CD16) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, downregulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal) influenza vaccination. CD16 downregulation persisted for at least 12 weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal) vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long-term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16high CD57+ NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression). CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25 (IL-2Rα) expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on trivalent influenza vaccine-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 downregulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways. PMID:27725819

  15. NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens

    PubMed Central

    Habib, Samar; El Andaloussi, Abdeljabar; Hisham, Ahmed; Ismail, Nahed

    2016-01-01

    Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia. PMID:27092553

  16. Human Induced Pluripotent Stem Cells Are Targets for Allogeneic and Autologous Natural Killer (NK) Cells and Killing Is Partly Mediated by the Activating NK Receptor DNAM-1

    PubMed Central

    Monecke, Sebastian; Cyganek, Lukas; Elsner, Leslie; Hübscher, Daniela; Walter, Lutz; Streckfuss-Bömeke, Katrin; Guan, Kaomei; Dressel, Ralf

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P=1.69x10-39). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P=1.72x10-6) and ranged between 46 % and 64 % in 51Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63x10-6). Killing was partly dependent on the activating NK receptor DNAM-1 (P=8.22x10-7). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells. PMID:25950680

  17. NK Cells Promote Th-17 Mediated Corneal Barrier Disruption in Dry Eye

    PubMed Central

    Zhang, Xiaobo; Volpe, Eugene A.; Gandhi, Niral B.; Schaumburg, Chris S.; Siemasko, Karyn F.; Pangelinan, Solherny B.; Kelly, Scott D.; Hayday, Adrian C.; Li, De-Quan; Stern, Michael E.; Niederkorn, Jerry Y.; Pflugfelder, Stephen C.; De Paiva, Cintia S.

    2012-01-01

    Background The conjunctiva contains a specialized population of lymphocytes that reside in the epithelium, named intraepithelial lymphocytes (IEL). Methodology/Principal Findings Here we characterized the IEL population prior to and after experimental desiccating stress (DS) for 5 or 10 days (DS5, DS10) and evaluated the effect of NK depletion on DS. The frequency of IELs in normal murine conjunctiva was CD3+CD103+ (∼22%), CD3+γδ+ (∼9.6%), CD3+NK+ (2%), CD3−NK+ (∼4.4%), CD3+CD8α (∼0.9%), and CD4 (∼0.6%). Systemic depletion of NK cells prior and during DS led to a decrease in the frequency of total and activated DCs, a decrease in T helper-17+ cells in the cervical lymph nodes and generation of less pathogenic CD4+T cells. B6.nude recipient mice of adoptively transferred CD4+T cells isolated from NK-depleted DS5 donor mice showed significantly less corneal barrier disruption, lower levels of IL-17A, CCL20 and MMP-3 in the cornea epithelia compared to recipients of control CD4+T cells. Conclusions/Significance Taken together, these results show that the NK IELs are involved in the acute immune response to desiccation-induced dry eye by activating DC, which in turn coordinate generation of the pathogenic Th-17 response. PMID:22590618

  18. NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

    PubMed Central

    Lakshmikanth, Tadepally; Burke, Shannon; Ali, Talib Hassan; Kimpfler, Silvia; Ursini, Francesco; Ruggeri, Loredana; Capanni, Marusca; Umansky, Viktor; Paschen, Annette; Sucker, Antje; Pende, Daniela; Groh, Veronika; Biassoni, Roberto; Höglund, Petter; Kato, Masashi; Shibuya, Kazuko; Schadendorf, Dirk; Anichini, Andrea; Ferrone, Soldano; Velardi, Andrea; Kärre, Klas; Shibuya, Akira; Carbone, Ennio; Colucci, Francesco

    2009-01-01

    NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell–mediated innate immunity to melanoma cells and provide a background to design NK cell–based immunotherapeutic strategies against melanoma and possibly other tumors. PMID:19349689

  19. Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1–dependent NK cell-mediated lysis of infected cells

    PubMed Central

    Grauwet, Korneel; Cantoni, Claudia; Parodi, Monica; De Maria, Andrea; Devriendt, Bert; Pende, Daniela; Moretta, Lorenzo; Vitale, Massimo; Favoreel, Herman W.

    2014-01-01

    Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2–infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry. PMID:25352670

  20. Education of hyporesponsive NK cells by cytokines.

    PubMed

    Juelke, Kerstin; Killig, Monica; Thiel, Andreas; Dong, Jun; Romagnani, Chiara

    2009-09-01

    NK-cell tolerance to self is mediated via engagement of inhibitory receptors by cognate MHC molecules. This event is critical for NK-cell education to achieve functional competence. Thus, NK cells expressing self-MHC-specific inhibitory receptors are responsive to activating stimuli while those lacking such receptors are hyporesponsive. Nevertheless, the mechanisms underlying NK-cell education are still poorly understood. Here, we show that after stimulation with cytokines, hyporesponsive NK cells acquire stable expression of killer Ig-like receptors (KIR) as reflected by DNA hypomethylation of their KIR locus. Remarkably, only hyporesponsive NK cells that acquire KIR in the presence of their cognate MHC molecule gain functional competence and this process can occur in the absence of any accessory cells. Acquisition of competence does not result in autoreactivity, since acquired KIR are functional and therefore able to inhibit NK-cell cytotoxicity. Our data demonstrate that competent NK cells can be generated by cytokine stimulation, suggesting that NK-cell education might not only be an early event which takes place during NK-cell development but might also occur in the periphery during an immune response.

  1. A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells.

    PubMed

    Schilling, Daniela; Tetzlaff, Fabian; Konrad, Sarah; Li, Wei; Multhoff, Gabriele

    2015-01-01

    Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.

  2. Induction of the Lytic Cycle Sensitizes Epstein-Barr Virus-Infected B Cells to NK Cell Killing That Is Counteracted by Virus-Mediated NK Cell Evasion Mechanisms in the Late Lytic Cycle

    PubMed Central

    Williams, Luke R.; Quinn, Laura L.; Rowe, Martin

    2015-01-01

    ABSTRACT Epstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replication in vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses. IMPORTANCE This report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2

  3. Critical role of the NKG2D receptor for NK cell-mediated control and immune escape of B-cell lymphoma.

    PubMed

    Belting, Lena; Hömberg, Nadine; Przewoznik, Margarethe; Brenner, Christoph; Riedel, Tanja; Flatley, Andrew; Polić, Bojan; Busch, Dirk H; Röcken, Martin; Mocikat, Ralph

    2015-09-01

    Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ-myc-transgenic model of endogenously arising lymphoma and NKG2D-deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN-γ expression, which was effected by MHCI(low) tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D-deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D-deficient λ-myc-transgenic mice was significantly accelerated compared to NKG2D-competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D-L) in late disease stages, this did not occur in NKG2D-deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D-L loss mutants provides a mechanism of tumor escape. PMID:26151313

  4. Critical role of the NKG2D receptor for NK cell-mediated control and immune escape of B-cell lymphoma.

    PubMed

    Belting, Lena; Hömberg, Nadine; Przewoznik, Margarethe; Brenner, Christoph; Riedel, Tanja; Flatley, Andrew; Polić, Bojan; Busch, Dirk H; Röcken, Martin; Mocikat, Ralph

    2015-09-01

    Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ-myc-transgenic model of endogenously arising lymphoma and NKG2D-deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN-γ expression, which was effected by MHCI(low) tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D-deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D-deficient λ-myc-transgenic mice was significantly accelerated compared to NKG2D-competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D-L) in late disease stages, this did not occur in NKG2D-deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D-L loss mutants provides a mechanism of tumor escape.

  5. Soluble HLA-G generated by proteolytic shedding inhibits NK-mediated cell lysis.

    PubMed

    Park, Gyu Man; Lee, Sunray; Park, Boyoun; Kim, Eunkyung; Shin, Jinwook; Cho, Kwangmin; Ahn, Kwangseog

    2004-01-16

    In contrast to the classical HLA class Ia molecules, the nonclassical HLA-G primary transcript is alternatively spliced to generate several mRNAs that encode four membrane-bound and three soluble isoforms. This study demonstrated that the soluble form of HLA-G can also be generated by metalloproteinase-dependent shedding at post-translational level. These soluble HLA-G1 molecules generated by the cleavage of membrane-bound HLA-G1 associate with beta2-microglobulin and contain bound peptides that are stable at physiological conditions. This report further showed that the soluble HLA-G1 is able to protect HLA class I-negative K562 cells from NK lysis, suggesting that soluble HLA-G could act as an immunoregulator in NK cell recognition and possibly in other immune responses.

  6. Modulation of MICA on the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing

    PubMed Central

    Ibana, Joyce Altamarino; Aiyar, Ashok; Quayle, Alison Jane; Schust, Danny Joseph

    2012-01-01

    Chlamydia trachomatis serovars D-K are obligate intracellular bacteria that have tropism for the columnar epithelial cells of the genital tract. Chlamydia trachomatis infection has been reported to induce modifications in immune cell ligand expression on epithelial host cells. In this study, we used an in vitro infection model that resulted in a partial infection of C. trachomatis-exposed primary-like immortalized endocervical epithelial cells (A2EN). Using this model, we demonstrated that expression of the natural killer (NK) cell activating ligand, MHC class I-related protein A (MICA), was upregulated on C. trachomatis-infected, but not on noninfected bystander cells. MICA upregulation was concomitant with MHC class I downregulation and impacted the susceptibility of C. trachomatis-infected cells to NK cell activity. The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU) when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability of the host to counter C. trachomatis infection. PMID:22251247

  7. NK Cells and Type 1 Diabetes

    PubMed Central

    Rodacki, Melanie; Milech, Adolpho; de Oliveira, José Egídio Paulo

    2006-01-01

    Type 1 diabetes (T1D) is characterized by an immuno-mediated progressive destruction of the pancreatic β cells. Due to the ability of NK cells to kill target cells as well as to interact with antigen-presenting and T cells, it has been suggested that they could be involved in one or multiple steps of the immune-mediated attack that leads to T1D. Abnormalities in the frequency and activity of NK cells have been described both in animal models and patients with T1D. Some of these alterations are linked to its onset while others seem to be a consequence of the disease. Here, we discuss the main characteristics of NK cells and review the studies that investigated the role of NK cells in T1D, both in mouse models and humans. PMID:17162353

  8. Ab-IL2 fusion proteins mediate NK cell immune synapse formation by polarizing CD25 to the target cell-effector cell interface.

    PubMed

    Gubbels, Jennifer A A; Gadbaw, Brian; Buhtoiarov, Ilia N; Horibata, Sachi; Kapur, Arvinder K; Patel, Dhara; Hank, Jacquelyn A; Gillies, Stephen D; Sondel, Paul M; Patankar, Manish S; Connor, Joseph

    2011-12-01

    The huKS-IL2 immunocytokine (IC) consists of IL2 fused to a mAb against EpCAM, while the hu14.18-IL2 IC recognizes the GD2 disialoganglioside. They are under evaluation for treatment of EpCAM(+) (ovarian) and GD2(+) (neuroblastoma and melanoma) malignancies because of their proven ability to enhance tumor cell killing by antibody-dependent cell-mediated cytotoxicity (ADCC) and by antitumor cytotoxic T cells. Here, we demonstrate that huKS-IL2 and hu14.18-IL2 bind to tumor cells via their antibody components and increase adhesion and activating immune synapse (AIS) formation with NK cells by engaging the immune cells' IL-2 receptors (IL2R). The NK leukemia cell line, NKL (which expresses high affinity IL2Rs), shows fivefold increase in binding to tumor targets when treated with IC compared to matching controls. This increase in binding is effectively inhibited by blocking antibodies against CD25, the α-chain of the IL2R. NK cells isolated from the peritoneal environment of ovarian cancer patients, known to be impaired in mediating ADCC, bind to huKS-IL2 via CD25. The increased binding between tumor and effector cells via ICs is due to the formation of AIS that are characterized by the simultaneous polarization of LFA-1, CD2 and F-actin at the cellular interface. AIS formation of peritoneal NK and NKL cells is inhibited by anti-CD25 blocking antibody and is 50-200% higher with IC versus the parent antibody. These findings demonstrate that the IL-2 component of the IC allows IL2Rs to function not only as receptors for this cytokine but also as facilitators of peritoneal NK cell binding to IC-coated tumor cells.

  9. NK Cells and Trophoblasts

    PubMed Central

    Parham, Peter

    2004-01-01

    In placental mammals, viviparity—the production of living young within the mother's body—evolved under the auspices of the immune system. Elements of immunity were incorporated, giving pregnancy a mildly inflammatory character. Formation of the placenta, the organ that feeds the fetus, involves a cooperation between maternal natural killer (NK) cells and fetal trophoblast cells that remodels the blood supply. Recent research reveals that this process and human reproductive success are influenced by polymorphic HLA-C ligands and their killer cell immunoglobulin-like receptors (KIR). PMID:15492121

  10. Valproic acid cooperates with hydralazine to augment the susceptibility of human osteosarcoma cells to Fas- and NK cell-mediated cell death.

    PubMed

    Yamanegi, Koji; Yamane, Junko; Kobayashi, Kenta; Kato-Kogoe, Nahoko; Ohyama, Hideki; Nakasho, Keiji; Yamada, Naoko; Hata, Masaki; Fukunaga, Satoru; Futani, Hiroyuki; Okamura, Haruki; Terada, Nobuyuki

    2012-07-01

    We investigated the effects of valproic acid (VPA), a histone deacetylase inhibitor, in combination with hydralazine, a DNA methylation inhibitor, on the expression of cell-surface Fas and MHC-class I-related chain molecules A and B (MICA and B), the ligands of NKG2D which is an activating receptor of NK cells, and on production of their soluble forms in HOS, U-2 OS and SaOS-2 human osteosarcoma cell lines. We also examined the susceptibility of these cells to Fas- and NK cell-mediated cell death. VPA did not increase the expression of Fas on the surface of osteosarcoma cells, while hydralazine did, and the combination of VPA with hydralazine increased the expression of cell-surface Fas. In contrast, the combination of VPA with hydralazine did not increase the production of soluble Fas by osteosarcoma cells. Both VPA and hydralazine increased the expression of cell-surface MICA and B in osteosarcoma cells, and their combination induced a greater increase in their expression. VPA inhibited the production of both soluble MICA and MICB by osteosarcoma cells while hydralazine produced no effect. Both VPA and hydralazine enhanced the susceptibility of osteosarcoma cells to Fas- and NK cell-mediated cell death and the combination of VPA with hydralazine further enhanced the effects. The present results suggest that combined administration of VPA and hydrazine is valuable for enhancing the therapeutic effects of immunotherapy for osteosarcomas.

  11. "Natural Regulators": NK Cells as Modulators of T Cell Immunity.

    PubMed

    Schuster, Iona S; Coudert, Jerome D; Andoniou, Christopher E; Degli-Esposti, Mariapia A

    2016-01-01

    Natural killer (NK) cells are known as frontline responders capable of rapidly mediating a response upon encountering transformed or infected cells. Recent findings indicate that NK cells, in addition to acting as innate effectors, can also regulate adaptive immune responses. Here, we review recent studies on the immunoregulatory function of NK cells with a specific focus on their ability to affect the generation of early, as well as long-term antiviral T cell responses, and their role in modulating immune pathology and disease. In addition, we summarize the current knowledge of the factors governing regulatory NK cell responses and discuss origin, tissue specificity, and open questions about the classification of regulatory NK cells as classical NK cells versus group 1 innate lymphoid cells. PMID:27379097

  12. Characterization of Murine Cytomegalovirus m157 from Infected Cells and Identification of Critical Residues Mediating Recognition by the NK Cell Receptor, Ly49H

    PubMed Central

    Davis, Aja H.; Guseva, Natalya V.; Ball, Brianne L.; Heusel, Jonathan W.

    2008-01-01

    Activated natural killer (NK) cells mediate potent cytolytic and secretory effector functions, and are vital components of the early antiviral immune response. NK cell activities are regulated by the assortment of inhibitory receptors that recognize major histocompatibility class I ligands expressed on healthy cells and activating receptors that recognize inducible host ligands or ligands that are not well characterized. The activating Ly49H receptor of mouse NK cells is unique in that it specifically recognizes a virally encoded ligand, the m157 glycoprotein of murine cytomegalovirus (MCMV). The Ly49H-m157 interaction underlies a potent resistance mechanism (Cmv1) in C57BL/6 mice, and serves as an excellent model in which to understand how NK cells are specifically activated in vivo, as similar receptor systems are operative for human NK cells. For transduced cells expressing m157 in isolation and for MCMV-infected cells, we show that m157 is expressed in multiple isoforms with marked differences in abundance between infected fibroblasts (high) and macrophages (low). At the cell surface m157 is exclusively a glycosylphosphatidylinositol-associated protein in MCMV-infected cells. Through random and site-directed mutagenesis of m157 we identify unique residues that provide for efficient cell surface expression of m157, but fail to activate Ly49H-expressing reporter cells. These m157 mutations are predicted to alter the conformation of a putative m157 interface with Ly49H, one that relies on the position of a critical α0-helix of m157. These findings support an emerging model for a novel interaction between this important NK cell receptor and its viral ligand. PMID:18566392

  13. Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

    PubMed

    Lu, Chia-Chen; Hsu, Ya-Jing; Chang, Chih-Jung; Lin, Chuan-Sheng; Martel, Jan; Ojcius, David M; Ko, Yun-Fei; Lai, Hsin-Chih; Young, John D

    2016-10-01

    Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases. PMID:27469258

  14. The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis.

    PubMed

    Matsunami, K; Miyagawa, S; Nakai, R; Murase, A; Shirakura, R

    2001-10-01

    The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human beta2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human beta2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human beta2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.

  15. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

    PubMed

    Tseng, Han-Ching; Kanayama, Keiichi; Kaur, Kawaljit; Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E; Nishimura, Ichiro; Jewett, Anahid

    2015-08-21

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer. PMID:26343372

  16. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

    PubMed Central

    Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E.; Nishimura, Ichiro; Jewett, Anahid

    2015-01-01

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing due to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer. PMID:26343372

  17. Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

    PubMed

    Tseng, Han-Ching; Kanayama, Keiichi; Kaur, Kawaljit; Park, So-Hyun; Park, Sil; Kozlowska, Anna; Sun, Shuting; McKenna, Charles E; Nishimura, Ichiro; Jewett, Anahid

    2015-08-21

    The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1β, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer.

  18. NK cell cytotoxicity mediated by 2B4 and NTB-A is dependent on SAP acting downstream of receptor phosphorylation.

    PubMed

    Meinke, Stephan; Watzl, Carsten

    2013-01-01

    2B4 (CD244) and NK-T-B-antigen (NTB-A, CD352) are activating receptors on human natural killer (NK) cells and belong to the family of signaling lymphocyte activation molecule (SLAM)-related receptors (SRR). Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SLAM-associated protein (SAP) and Ewing's sarcoma-activated transcript-2 (EAT-2). X-linked lymphoproliferative syndrome (XLP) is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.

  19. Differential requirement for Nfil3 during NK cell development.

    PubMed

    Seillet, Cyril; Huntington, Nicholas D; Gangatirkar, Pradnya; Axelsson, Elin; Minnich, Martina; Brady, Hugh J M; Busslinger, Meinrad; Smyth, Mark J; Belz, Gabrielle T; Carotta, Sebastian

    2014-03-15

    NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow-derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL(+) Eomes(-) NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3(-/-) progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3. PMID:24532575

  20. Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A.

    PubMed

    Nielsen, Natasja; Ødum, Niels; Ursø, Birgitte; Lanier, Lewis L; Spee, Pieter

    2012-01-01

    In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation. PMID:22384114

  1. IL-27 stimulates human NK-cell effector functions and primes NK cells for IL-18 responsiveness.

    PubMed

    Ziblat, Andrea; Domaica, Carolina I; Spallanzani, Raúl G; Iraolagoitia, Ximena L Raffo; Rossi, Lucas E; Avila, Damián E; Torres, Nicolás I; Fuertes, Mercedes B; Zwirner, Norberto W

    2015-01-01

    IL-27, a member of the IL-12 family of cytokines, is produced by APCs, and displays pro- and anti-inflammatory effects. How IL-27 affects human NK cells still remains unknown. In this study, we observed that mature DCs secreted IL-27 and that blockade of IL-27R (CD130) reduced the amount of IFN-γ produced by NK cells during their coculture, showing the importance of IL-27 during DC-NK-cell crosstalk. Accordingly, human rIL-27 stimulated IFN-γ secretion by NK cells in a STAT1-dependent manner, induced upregulation of CD25 and CD69 on NK cells, and displayed a synergistic effect with IL-18. Preincubation experiments demonstrated that IL-27 primed NK cells for IL-18-induced IFN-γ secretion, which was associated with an IL-27-driven upregulation of T-bet expression. Also, IL-27 triggered NKp46-dependent NK-cell-mediated cytotoxicity against Raji, T-47D, and HCT116 cells, and IL-18 enhanced this cytotoxic response. Such NK-cell-mediated cytotoxicity involved upregulation of perforin, granule exocytosis, and TRAIL-mediated cytotoxicity but not Fas-FasL interaction. Moreover, IL-27 also potentiated Ab-dependent cell-mediated cytotoxicity against mAb-coated target cells. Taken together, IL-27 stimulates NK-cell effector functions, which might be relevant in different physiological and pathological situations. PMID:25308526

  2. The CC chemokine CK beta-11/MIP-3 beta/ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through NK cells.

    PubMed

    Braun, S E; Chen, K; Foster, R G; Kim, C H; Hromas, R; Kaplan, M H; Broxmeyer, H E; Cornetta, K

    2000-04-15

    CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.

  3. B7-H6-mediated downregulation of NKp30 in NK cells contributes to ovarian carcinoma immune escape

    PubMed Central

    Pesce, Silvia; Tabellini, Giovanna; Cantoni, Claudia; Patrizi, Ornella; Coltrini, Daniela; Rampinelli, Fabio; Matta, Jessica; Vivier, Eric; Moretta, Alessandro; Parolini, Silvia; Marcenaro, Emanuela

    2015-01-01

    In this study the phenotype and function of tumor-associated NK cells from peritoneal fluids of a selected cohort of patients with seropapillary ovarian carcinoma were analyzed. In > 50% of these patients, the expression of the activating receptor NKp30 in tumor-associated NK cells was substantially reduced as compared to autologous peripheral blood (PB) NK cells. The impaired expression of this receptor was associated with the presence of one of its cellular ligands (B7-H6), which was detectable as a surface/cytosolic molecule in tumor cells and as a soluble molecule in the peritoneal fluid. NK cells from patients expressing this NKp30low phenotype displayed an impaired interferon-gamma (IFNγ) production and cytolytic function when tested against target cells expressing surface B7-H6. Our data also suggest that in these patients, the defective expression and function of NKp30 may be induced by the chronic engagement of this receptor by soluble B7-H6 or by tumor cells expressing this ligand. The impairment of NK cell functions described herein could represent a novel mechanism by which the tumor microenvironment may contribute to the escape from immune surveillance. PMID:26137398

  4. Recombinant TLR5 agonist CBLB502 promotes NK cell-mediated anti-CMV immunity in mice.

    PubMed

    Hossain, Mohammad S; Ramachandiran, Sampath; Gewirtz, Andrew T; Waller, Edmund K

    2014-01-01

    Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

  5. The Human NK Cell Response to Yellow Fever Virus 17D Is Primarily Governed by NK Cell Differentiation Independently of NK Cell Education.

    PubMed

    Marquardt, Nicole; Ivarsson, Martin A; Blom, Kim; Gonzalez, Veronica D; Braun, Monika; Falconer, Karolin; Gustafsson, Rasmus; Fogdell-Hahn, Anna; Sandberg, Johan K; Michaëlsson, Jakob

    2015-10-01

    NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57(-) NK cells. In contrast, NK cells expressing self- and nonself-HLA class I-binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I-mediated inhibition or education.

  6. Shaping of NK cell subsets by aging.

    PubMed

    Solana, Rafael; Campos, Carmen; Pera, Alejandra; Tarazona, Raquel

    2014-08-01

    NK cells are key players in the innate immune response against virus infection and tumors. Here we describe the current knowledge on age-associated changes in NK cells and the role of persistent CMV infection in configuring NK cell compartment in the elderly. Aging but not CMV causes a redistribution of NK cell subsets as shown by a decrease of CD56bright cells and an increase of CD56-CD16+ NK cells. On the contrary the changes in CD56dimCD16+ NK cells are compatible with the accumulation of CD57+ long-lived NK cells that can also be observed in young CMV-seropositive individuals. NK cell function and dynamics in the elderly will be related not only with age but also with exposure to pathogens, especially CMV.

  7. Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans.

    PubMed

    Xiang, Richard F; Stack, Danuta; Huston, Shaunna M; Li, Shu Shun; Ogbomo, Henry; Kyei, Stephen K; Mody, Christopher H

    2016-03-25

    The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans. PMID:26867574

  8. Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans.

    PubMed

    Xiang, Richard F; Stack, Danuta; Huston, Shaunna M; Li, Shu Shun; Ogbomo, Henry; Kyei, Stephen K; Mody, Christopher H

    2016-03-25

    The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.

  9. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

    PubMed

    Morandi, Fabio; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Chiesa, Sabrina; Imperatori, Andrea; Zanellato, Silvia; Mortara, Lorenzo; Gattorno, Marco; Pistoia, Vito; Malavasi, Fabio

    2015-08-01

    Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases. PMID:26091716

  10. NAP-2 Secreted by Human NK Cells Can Stimulate Mesenchymal Stem/Stromal Cell Recruitment

    PubMed Central

    Almeida, Catarina R.; Caires, Hugo R.; Vasconcelos, Daniela P.; Barbosa, Mário A.

    2016-01-01

    Summary Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. PMID:27052313

  11. Contrasting effects of anti-Ly49A due to MHC class I cis-binding on NK cell mediated allogeneic bone marrow cell resistance

    PubMed Central

    Alvarez, Maite; Sungur, Can M.; Ames, Erik; Anderson, Stephen K.; Pomeroy, Claire; Murphy, William J.

    2013-01-01

    NK subsets have activating and inhibitory receptors that bind MHC-I. Ly49A is a mouse inhibitory receptor that binds with high affinity to H2d in both a cis- and trans-manner. Ly49A cis-associations limit trans-interactions with H2d-expressing targets as well as monoclonal antibody (mAb)-binding. We demonstrate that cis-interactions affect mAb effector functions. In vivo administration of anti-Ly49A depleted NK cells in H2b but not H2d mice. Despite lack of depletion, in vivo treatment with anti-Ly49A reduced NK killing capabilities and inhibited activation, partially due to its agonistic effect. These data explain the previously described in vivo effects on bone marrow allograft rejection observed with anti-Ly49A treatment in H2d-haplotype mice. However, prior treatment of mice with poly(I:C) or MCMV infection resulted in increased Ly49A expression and Ly49A+ NK cell depletion in H2d mice. These data indicate that although Ly49 mAbs can exert similar in vivo effects in mice with different MHC-haplotypes, these effects are mediated via different mechanisms of action correlating with Ly49A expression levels and can be altered within the same strain contingent on stimuli. This illustrates the marked diversity of mAb effector functions due to the regulation of the level of expression of target antigens and responses by stimulatory incidents such as infection. PMID:23752612

  12. NK cell-mediated lysis of autologous antigen-presenting cells is triggered by the engagement of the phosphatidylinositol 3-kinase upon ligation of the natural cytotoxicity receptors NKp30 and NKp46.

    PubMed

    Spaggiari, G M; Carosio, R; Pende, D; Marcenaro, S; Rivera, P; Zocchi, M R; Moretta, L; Poggi, A

    2001-06-01

    Interleukin-2 (IL-2)-activated polyclonal or clonal NK cells lysed autologous antigen presenting cells (APC) through the engagement of the natural cytotoxicity receptors (NCR) NKp30 and NKp46. NK cell-mediated cytolysis of APC correlated with the surface density of these NCR. Indeed, NK cell clones bearing low amounts of NKp30 and NKp46 did not lyse autologous APC, whereas NK cell clones with bright expression of these NCR efficiently killed autologous APC. Upon masking of NKp30 or NKp46 by specific monoclonal antibodies a strong reduction (by 50%) of APC lysis could be detected and the complete inhibition was achieved by the simultaneous masking of these NCR. Interestingly, NK cell-mediated APC lysis was impaired by the phosphatidylinositol 3-kinase (PI-3 K) inhibitors LY294002 or wortmannin. Similarly, these drugs strongly reduced NK cell activation triggered by NKp30 or NKp46 in a re-directed killing assay as well as the activation of Akt/PKB, substrate of PI-3 K, induced by the engagement of these receptors. Altogether, these findings strongly suggest that NCR are responsible for the killing of autologous APC through the activation of PI-3 K.

  13. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

    PubMed

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

  14. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

    PubMed Central

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART. PMID:26696395

  15. NK cell development and function--plasticity and redundancy unleashed.

    PubMed

    Cichocki, Frank; Sitnicka, Ewa; Bryceson, Yenan T

    2014-04-01

    Bone marrow-derived natural killer (NK) cells constitute the major subset of cytotoxic lymphocytes in peripheral blood. They provide innate defense against intracellular infection or malignancy and contribute to immune homeostasis. Large numbers of NK cells are also present in tissues, including the liver and uterus, where they can mediate immunosurveillance but also play important roles in tissue remodeling and vascularization. Here, we review the pathways involved in NK cell lineage commitment and differentiation, discussing relationships to other lymphocyte populations and highlighting genetic determinants. Characterizing NK cells from distinct tissues and during infections have revealed subset specializations, reflecting inherent cellular plasticity. In this context, we discuss how different environmental and inflammatory stimuli may shape NK cells. Particular emphasis is placed on genes identified as being critical for NK cell development, differentiation, and function from studies of model organisms or associations with disease. Such studies are also revealing important cellular redundancies. Here, we provide a view of the genetic framework constraining NK cell development and function, pinpointing molecules required for these processes but also underscoring plasticity and redundancy that may underlie robust immunological function. With this view, built in redundancy may highlight the importance of NK cells to immunity.

  16. NK Cell Subtypes as Regulators of Autoimmune Liver Disease

    PubMed Central

    2016-01-01

    As major components of innate immunity, NK cells not only exert cell-mediated cytotoxicity to destroy tumors or infected cells, but also act to regulate the functions of other cells in the immune system by secreting cytokines and chemokines. Thus, NK cells provide surveillance in the early defense against viruses, intracellular bacteria, and cancer cells. However, the effecter function of NK cells must be exquisitely controlled to prevent inadvertent attack against normal “self” cells. In an organ such as the liver, where the distinction between immunotolerance and immune defense against routinely processed pathogens is critical, the plethora of NK cells has a unique role in the maintenance of homeostasis. Once self-tolerance is broken, autoimmune liver disease resulted. NK cells act as a “two-edged weapon” and even play opposite roles with both regulatory and inducer activities in the hepatic environment. That is, NK cells act not only to produce inflammatory cytokines and chemokines, but also to alter the proliferation and activation of associated lymphocytes. However, the precise regulatory mechanisms at work in autoimmune liver diseases remain to be identified. In this review, we focus on recent research with NK cells and their potential role in the development of autoimmune liver disease. PMID:27462349

  17. NK Cell Subtypes as Regulators of Autoimmune Liver Disease.

    PubMed

    Jiao, Guohui; Wang, Bangmao

    2016-01-01

    As major components of innate immunity, NK cells not only exert cell-mediated cytotoxicity to destroy tumors or infected cells, but also act to regulate the functions of other cells in the immune system by secreting cytokines and chemokines. Thus, NK cells provide surveillance in the early defense against viruses, intracellular bacteria, and cancer cells. However, the effecter function of NK cells must be exquisitely controlled to prevent inadvertent attack against normal "self" cells. In an organ such as the liver, where the distinction between immunotolerance and immune defense against routinely processed pathogens is critical, the plethora of NK cells has a unique role in the maintenance of homeostasis. Once self-tolerance is broken, autoimmune liver disease resulted. NK cells act as a "two-edged weapon" and even play opposite roles with both regulatory and inducer activities in the hepatic environment. That is, NK cells act not only to produce inflammatory cytokines and chemokines, but also to alter the proliferation and activation of associated lymphocytes. However, the precise regulatory mechanisms at work in autoimmune liver diseases remain to be identified. In this review, we focus on recent research with NK cells and their potential role in the development of autoimmune liver disease. PMID:27462349

  18. NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection

    PubMed Central

    Cheekatla, Satyanarayana Swamy; Tripathi, Deepak; Venkatasubramanian, Sambasivan; Nathella, Pavan Kumar; Paidipally, Padmaja; Ishibashi, Munenori; Welch, Elwyn; Tvinnereim, Amy R.; Ikebe, Mitsuo; Valluri, Vijaya Lakshmi; Babu, Subash; Kornfeld, Hardy; Vankayalapati, Ramakrishna

    2016-01-01

    In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice. PMID:27783671

  19. Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

    PubMed Central

    Okita, Riki; Wolf, Diana; Yasuda, Koichiro; Maeda, Ai; Yukawa, Takuro; Saisho, Shinsuke; Shimizu, Katsuhiko; Yamaguchi, Yoshiyuki; Oka, Mikio; Nakayama, Eiichi; Lundqvist, Andreas; Kiessling, Rolf; Seliger, Barbara; Nakata, Masao

    2015-01-01

    Introduction Several cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown. Methods This study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis. Results We demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a. Conclusion In keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells. PMID:26439264

  20. Histone Deacetylase Inhibitors Enhance CD4 T Cell Susceptibility to NK Cell Killing but Reduce NK Cell Function

    PubMed Central

    Pace, Matthew; Williams, James; Kurioka, Ayako; Gerry, Andrew B.; Jakobsen, Bent; Klenerman, Paul; Nwokolo, Nneka; Fox, Julie

    2016-01-01

    In the search for a cure for HIV-1 infection, histone deacetylase inhibitors (HDACi) are being investigated as activators of latently infected CD4 T cells to promote their targeting by cytotoxic T-lymphocytes (CTL). However, HDACi may also inhibit CTL function, suggesting different immunotherapy approaches may need to be explored. Here, we study the impact of different HDACi on both Natural Killer (NK) and CTL targeting of HIV-1 infected cells. We found HDACi down-regulated HLA class I expression independently of HIV-1 Nef which, without significantly compromising CTL function, led to enhanced targeting by NK cells. HDACi-treated HIV-1-infected CD4 T cells were also more effectively cleared than untreated controls during NK co-culture. However, HDACi impaired NK function, reducing degranulation and killing capacity. Depending on the HDACi and dose, this impairment could counteract the benefit gained by treating infected target cells. These data suggest that following HDACi-induced HLA class I down-regulation NK cells kill HIV-1-infected cells, although HDACi-mediated NK cell inhibition may negate this effect. Our data emphasize the importance of studying the effects of potential interventions on both targets and effectors. PMID:27529554

  1. NK Cells and Their Ability to Modulate T Cells during Virus Infections

    PubMed Central

    Cook, Kevin D.; Waggoner, Stephen N.; Whitmire, Jason K.

    2014-01-01

    Natural killer (NK) cells are important in protection against virus infections, and many viruses have evolved mechanisms to thwart NK cell activity. NK cells respond to inflammatory signals at an early stage of virus infection, resulting in proliferation, cytokine production, and cytolytic activity that can reduce virus loads. Moreover, the rapid kinetics of the NK cell response enables NK cells to influence other populations of innate immune cells, affect the inflammatory milieu, and guide adaptive immune responses to infection. Early NK cell interactions with other leukocytes can have long-lasting effects on the number and quality of memory T cells, as well as impact the exhaustion of T cells during chronic infections. The ability of NK cells to modulate T cell responses can be mediated through direct T-NK interactions, cytokine production, or indirectly through dendritic cells and other cell types. Herein, we summarize our current understanding of how NK cells interact with T cells, dendritic cells, B cells, and other cell types involved in adaptive immune responses to virus infection. We outline several mechanisms by which NK cells enhance or suppress adaptive immune response and long-lived immunological memory. PMID:25404045

  2. Primary B-CLL resistance to NK cell cytotoxicity can be overcome in vitro and in vivo by priming NK cells and monoclonal antibody therapy.

    PubMed

    Veuillen, Caroline; Aurran-Schleinitz, Thérèse; Castellano, Rémy; Rey, Jérôme; Mallet, Françoise; Orlanducci, Florence; Pouyet, Laurent; Just-Landi, Sylvaine; Coso, Diane; Ivanov, Vadim; Carcopino, Xavier; Bouabdallah, Réda; Collette, Yves; Fauriat, Cyril; Olive, Daniel

    2012-06-01

    Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy.

  3. Mouse host unlicensed NK cells promote donor allogeneic bone marrow engraftment

    PubMed Central

    Alvarez, Maite; Sun, Kai

    2016-01-01

    Natural killer (NK) cells exist as subsets based on expression of inhibitory receptors that recognize major histocompatibility complex I (MHCI) molecules. NK cell subsets bearing MHCI binding receptors for self-MHCI have been termed as “licensed” and exhibit a higher ability to respond to stimuli. In the context of bone marrow transplantation (BMT), host licensed-NK (L-NK) cells have also been demonstrated to be responsible for the acute rejection of allogeneic and MHCI-deficient BM cells (BMCs) in mice after lethal irradiation. However, the role of recipient unlicensed-NK (U-NK) cells has not been well established with regard to allogeneic BMC resistance. After NK cell stimulation, the prior depletion of host L-NK cells resulted in a marked increase of donor engraftment compared with the untreated group. Surprisingly, this increased donor engraftment was reduced after total host NK cell depletion, indicating that U-NK cells can actually promote donor allogeneic BMC engraftment. Furthermore, direct coculture of U-NK cells with allogeneic but not syngeneic BMCs resulted in increased colony-forming unit cell growth in vitro, which was at least partially mediated by granulocyte macrophage colony-stimulating factor (GM-CSF) production. These data demonstrate that host NK cell subsets exert markedly different roles in allogeneic BMC engraftment where host L- and U-NK cells reject or promote donor allogeneic BMC engraftment, respectively. PMID:26738538

  4. The unconventional expression of IL-15 and its role in NK cell homeostasis.

    PubMed

    Huntington, Nicholas D

    2014-03-01

    Natural killer (NK) cells are the founding members of the innate lymphoid cell family and contribute to the rapid production of inflammatory mediators upon pathogen detection. The evolution of receptors for self major histocompatibility complex-I and stress-induced ligands also bestows upon NK cells an important effector role in the clearance of virus-infected and transformed cells. NK cells are dependent on the pleiotropic cytokine interleukin (IL)-15 for their development, differentiation and optimal function. Here I review the regulation of IL-15 in vivo, its role in driving NK cell differentiation and discuss the function of NK cell diversification with regard to innate immunity.

  5. Different natural killer (NK) receptor expression and immunoglobulin E (IgE) regulation by NK1 and NK2 cells

    PubMed Central

    Aktas, E; Akdis, M; Bilgic, S; Disch, R; Falk, C S; Blaser, K; Akdis, C; Deniz, G

    2005-01-01

    Many studies concerning the role of T cells and cytokines in allergy have been performed, but little is known about the role of natural killer (NK) cells. Accordingly, the expression of co-stimulatory, inhibitory and apoptosis receptors, cytokine profiles and their effect on immunoglobulin isotypes were investigated in polyallergic atopic dermatitis (AD) patients with hyper immunoglobulin E (IgE) and healthy individuals. AD patients showed significantly decreased peripheral blood NK cells compared to healthy individuals. Freshly isolated NK cells of polyallergic patients spontaneously released higher amounts of interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-γ compared to healthy individuals. NK cells were differentiated to NK1 cells by IL-12 and neutralizing anti-IL-4 monoclonal antibodies (mAb), and to NK2 cells by IL-4 and neutralizing anti-IL-12 mAb. Following IL-12 stimulation, NK cells produced increased levels of IFN-γ and decreased IL-4. In contrast, stimulation of NK cells with IL-4 inhibited IFN-γ, but increased IL-13, production. The effect of NK cell subsets on IgE regulation was examined in co-cultures of in vitro differentiated NK cells with peripheral blood mononuclear cells (PBMC) or B cells. NK1 cells significantly inhibited IL-4- and soluble CD40-ligand-stimulated IgE production; however, NK2 cells did not have any effect. The inhibitory effect of NK1 cells on IgE production was blocked by neutralization of IFN-γ. Except for CD40, NK cell subsets showed different expression of killer-inhibitory receptors and co-stimulatory molecules between the polyallergic and healthy subjects. These results indicate that human NK cells show differences in numbers, surface receptor and cytokine phenotypes and functional properties in AD. PMID:15807855

  6. Anti-neuroblastoma effect of ch14.18 antibody produced in CHO cells is mediated by NK-cells in mice.

    PubMed

    Zeng, Yan; Fest, Stefan; Kunert, Renate; Katinger, Hermann; Pistoia, Vito; Michon, Jean; Lewis, Gillan; Ladenstein, Ruth; Lode, Holger N

    2005-07-01

    Successful treatment of stage 4 neuroblastoma remains a major challenge in pediatric oncology. In order to improve the outcome, passive immunotherapy using human-mouse chimeric monoclonal anti-disialoganglioside GD2 antibody ch14.18 has been evaluated in early phase clinical trials with promising results in progressing stage 4 neuroblastoma patients. In preparation of European phase III clinical trial (HR-NBL-1/ESIOP), the cell line used for production of ch14.18 was changed. Specifically, the plasmid encoding for ch14.18 antibody was recloned into CHO cells. Here, we report the in vitro and in vivo anti-neuroblastoma activity of antibody ch14.18 produced in CHO cells (ch14.18/CHO) compared to that of ch14.18 manufactured from SP2/0 (ch14.18/SP2/0) and NS0 cells (ch14.18/NS0). First, we demonstrate identical binding of ch14.18/CHO to the nominal antigen disialoganglioside GD2 in vitro compared to ch14.18/SP2/0 and ch14.18/NS0. Binding was GD2-specific, since all precursor- and metabolite-gangliosides of GD2 tested were not recognized by ch14.18/CHO. Second, the functional properties of ch14.18/CHO were determined in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) reactions against GD2 positive neuroectodermal tumor cell lines in vitro. There was no difference in CDC mediated specific tumor cell lysis among the three different ch14.18 antibody preparations. Interestingly, ch14.18/CHO showed superior ADCC activity at low antibody concentrations. Third, the efficacy of ch14.18/CHO was evaluated in the NXS2 neuroblastoma model in vivo. Importantly, the ch14.18/CHO preparation was effective in suppression of experimental liver metastasis in this model. In vivo depletion of NK-cells completely abrogated this effect, suggesting that the mechanism involved in the ch14.18/CHO induced anti-neuroblastoma effect is mediated by NK-dependent ADCC.

  7. “Natural Regulators”: NK Cells as Modulators of T Cell Immunity

    PubMed Central

    Schuster, Iona S.; Coudert, Jerome D.; Andoniou, Christopher E.; Degli-Esposti, Mariapia A.

    2016-01-01

    Natural killer (NK) cells are known as frontline responders capable of rapidly mediating a response upon encountering transformed or infected cells. Recent findings indicate that NK cells, in addition to acting as innate effectors, can also regulate adaptive immune responses. Here, we review recent studies on the immunoregulatory function of NK cells with a specific focus on their ability to affect the generation of early, as well as long-term antiviral T cell responses, and their role in modulating immune pathology and disease. In addition, we summarize the current knowledge of the factors governing regulatory NK cell responses and discuss origin, tissue specificity, and open questions about the classification of regulatory NK cells as classical NK cells versus group 1 innate lymphoid cells. PMID:27379097

  8. Dendritic cells for NK/LAK activation: rationale for multicellular immunotherapy in neuroblastoma patients.

    PubMed

    Valteau-Couanet, Dominique; Leboulaire, Christophe; Maincent, Kim; Tournier, Muriel; Hartmann, Olivier; Bénard, Jean; Beaujean, Françoise; Boccaccio, Catherine; Zitvogel, Laurence; Angevin, Eric

    2002-10-01

    Natural killer (NK)/lymphokine-activated killer (LAK) cell-based immunotherapy could be beneficial against major histocompatibility complex class I-negative tumor residual disease such as neuroblastoma (NB), provided that interleukin 2 (IL-2) or surrogate nontoxic NK cell stimulatory factors could sustain NK cell activation and survival in vivo. Here we show that human monocyte-derived dendritic cells (MD-DCs) promote potent NK/LAK effector functions and long-term survival, circumventing the need for IL-2. This study demonstrates (1) the feasibility of differentiating granulocyte colony-stimulating factor-mobilized hematopoietic peripheral blood stem cells (PBSCs) into high numbers of functional MD-DCs and NK/LAK cells in a series of 12 children with stage 4 neuroblastoma (NB); (2) potent DC-mediated NK cell activation in autologous settings; (3) the reciprocal capacity of NK/LAK cells to turn immature DCs into maturing cells electively capable of triggering NK cell functions; and (4) the unique capacity of maturing DCs to sustain NK cell survival, superior to that achieved in IL-2. These data show a reciprocal interaction between DCs and NK/LAK cells, leading to the amplification of NK cell effector functions, and support the implementation of DC/NK cell-based immunotherapy for purging the graft and/or controlling minimal residual disease after autologous stem cell transplantation.

  9. L-plastin is involved in NKG2D recruitment into lipid rafts and NKG2D-mediated NK cell migration.

    PubMed

    Serrano-Pertierra, Esther; Cernuda-Morollón, Eva; Brdička, Tomáš; Hoøejši, Václav; López-Larrea, Carlos

    2014-09-01

    Membrane rafts are microdomains of the plasma membrane that have multiple biological functions. The involvement of these structures in the biology of T cells, namely in signal transduction by the TCR, has been widely studied. However, the role of membrane rafts in immunoreceptor signaling in NK cells is less well known. We studied the distribution of the activating NKG2D receptor in lipid rafts by isolating DRMs in a sucrose density gradient or by raft fractionation by β-OG-selective solubility in the NKL cell line. We found that the NKG2D-DAP10 complex and pVav are recruited into rafts upon receptor stimulation. Qualitative proteomic analysis of these fractions showed that the actin cytoskeleton is involved in this process. In particular, we found that the actin-bundling protein L-plastin plays an important role in the clustering of NKG2D into lipid rafts. Moreover, coengagement of the inhibitory receptor NKG2A partially disrupted NKG2D recruitment into rafts. Furthermore, we demonstrated that L-plastin participates in NKG2D-mediated inhibition of NK cell chemotaxis.

  10. Mobilization of NK cells by exercise: downmodulation of adhesion molecules on NK cells by catecholamines.

    PubMed

    Nagao, F; Suzui, M; Takeda, K; Yagita, H; Okumura, K

    2000-10-01

    The change of plasma catecholamine concentration correlates with the change of natural killer (NK) activity and NK cell number in peripheral blood mononuclear cells (PBMC) during and after moderate exercise. We studied the causal relation between exercise-induced catecholamine and expression of adhesion molecules on NK cells during and after exercise. The expression of CD44 and CD18 on CD3(-)CD56(+) NK cells was significantly reduced during exercise (P < 0.01). When PBMC were stimulated with 10(-8)M norepinephrine in vitro, the expression of these adhesion molecules on CD3(-)CD56(+) NK cells was downmodulated within 30 min. The binding capacity of NK cells to a CD44 ligand, hyaluronate, was reduced by the stimulation with norepinephrine (P < 0.01). The intravenous injection of norepinephrine in mice decreased the expression of CD44 and CD18 on CD3(-)NK1.1(+) cells (P < 0.01) and increased the number of CD3(-)NK1.1(+) cells in PBMC (P < 0.01). These findings suggest that exercise-induced catecholamines modulate the expression of adhesion molecules on NK cells, resulting in the mobilization of NK cells into the circulation. PMID:11003990

  11. Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway.

    PubMed

    Sun, Jintang; Yang, Meixiang; Ban, Yanli; Gao, Wenjuan; Song, Bingfeng; Wang, Yang; Zhang, Yun; Shao, Qianqian; Kong, Beihua; Qu, Xun

    2016-01-01

    NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3(+) dNK expressed more levels of mature markers CD94 and CD69 than Tim-3- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.

  12. Homotypic NK cell-to-cell communication controls cytokine responsiveness of innate immune NK cells

    PubMed Central

    Kim, Tae-Jin; Kim, Miju; Kim, Hye Mi; Lim, Seon Ah; Kim, Eun-Ok; Kim, Kwanghee; Song, Kwang Hoon; Kim, Jiyoung; Kumar, Vinay; Yee, Cassian; Doh, Junsang; Lee, Kyung-Mi

    2014-01-01

    While stationary organ cells are in continuous contact with neighboring cells, immune cells circulate throughout the body without an apparent requirement for cell-cell contact to persist in vivo. This study challenges current convention by demonstrating, both in vitro and in vivo, that innate immune NK cells can engage in homotypic NK-to-NK cell interactions for optimal survival, activation, and proliferation. Using a specialized cell-laden microwell approach, we discover that NK cells experiencing constant NK-to-NK contact exhibit a synergistic increase in activation status, cell proliferation, and anti-tumor function in response to IL-2 or IL-15. This effect is dependent on 2B4/CD48 ligation and an active cytoskeleton, resulting in amplification of IL-2 receptor signaling, enhanced CD122/CD132 colocalization, CD25 upregulation, and Stat3 activation. Conversely, ‘orphan' NK cells demonstrate no such synergy and fail to persist. Therefore, our data uncover the existence of homotypic cell-to-cell communication among mobile innate lymphocytes, which promotes functional synergy within the cytokine-rich microenvironment. PMID:25475707

  13. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

    PubMed Central

    Shabrish, Snehal; Gupta, Maya; Madkaikar, Manisha

    2016-01-01

    Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p < 0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells. PMID:27413758

  14. Epigenetic Regulation of Adaptive NK Cell Diversification.

    PubMed

    Tesi, Bianca; Schlums, Heinrich; Cichocki, Frank; Bryceson, Yenan T

    2016-07-01

    Natural killer (NK) cells were previously considered to represent short-lived, innate lymphocytes. However, mouse models have revealed expansion and persistence of differentiated NK cell subsets in response to cytomegalovirus (CMV) infection, paralleling antigen-specific T cell differentiation. Congruently, analyses of humans have uncovered CMV-associated NK cell subsets characterized by epigenetic diversification processes that lead to altered target cell specificities and functional capacities. Here, focusing on responses to viruses, we review similarities and differences between mouse and human adaptive NK cells, identifying molecular analogies that may be key to transcriptional reprogramming and functional alterations. We discuss possible molecular mechanisms underlying epigenetic diversification and hypothesize that processes driving epigenetic diversification may represent a more widespread mechanism for fine-tuning and optimization of cellular immunity.

  15. Ionomycin Treatment Renders NK Cells Hyporesponsive

    PubMed Central

    Romera-Cárdenas, Gema; Thomas, L. Michael; Lopez-Cobo, Sheila; García-Cuesta, Eva M.; Long, Eric O.; Reyburn, Hugh T.

    2016-01-01

    Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes. PMID:27007115

  16. Ionomycin Treatment Renders NK Cells Hyporesponsive.

    PubMed

    Romera-Cárdenas, Gema; Thomas, L Michael; Lopez-Cobo, Sheila; García-Cuesta, Eva M; Long, Eric O; Reyburn, Hugh T

    2016-01-01

    Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes. PMID:27007115

  17. NK cell-based cancer immunotherapy: from basic biology to clinical application.

    PubMed

    Li, Yang; Yin, Jie; Li, Ting; Huang, Shan; Yan, Han; Leavenworth, JianMei; Wang, Xi

    2015-12-01

    Natural killer (NK) cells, which recognize and kill target cells independent of antigen specificity and major histocompatibility complex (MHC) matching, play pivotal roles in immune defence against tumors. However, tumor cells often acquire the ability to escape NK cell-mediated immune surveillance. Thus, understanding mechanisms underlying regulation of NK cell phenotype and function within the tumor environment is instrumental for designing new approaches to improve the current cell-based immunotherapy. In this review, we elaborate the main biological features and molecular mechanisms of NK cells that pertain to regulation of NK cell-mediated anti-tumor activity. We further overview current clinical approaches regarding NK cell-based cancer therapy, including cytokine infusion, adoptive transfer of autologous or allogeneic NK cells, applications of chimeric antigen receptor (CAR)-expressing NK cells and adoptive transfer of memory-like NK cells. With these promising clinical outcomes and fuller understanding the basic questions raised in this review, we foresee that NK cell-based approaches may hold great potential for future cancer immunotherapy.

  18. The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT).

    PubMed

    Foley, Bree; Felices, Martin; Cichocki, Frank; Cooley, Sarah; Verneris, Michael R; Miller, Jeffrey S

    2014-03-01

    Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed 'the missing self hypothesis' to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation.

  19. Novel immune modulators used in hematology: impact on NK cells.

    PubMed

    Krieg, Stephanie; Ullrich, Evelyn

    2012-01-01

    There is a wide range of important pharmaceuticals used in treatment of cancer. Besides their known effects on tumor cells, there is growing evidence for modulation of the immune system. Immunomodulatory drugs (IMiDs(®)) play an important role in the treatment of patients with multiple myeloma or myelodysplastic syndrome and have already demonstrated antitumor, anti-angiogenic, and immunostimulating effects, in particular on natural killer (NK) cells. Tyrosine kinase inhibitors are directly targeting different kinases and are known to regulate effector NK cells and expression of NKG2D ligands (NKG2DLs) on tumor cells. Demethylating agents, histone deacetylases, and proteasome inhibitors interfere with the epigenetic regulation and protein degradation of malignant cells. There are first hints that these drugs also sensitize tumor cells to chemotherapy, radiation, and NK cell-mediated cytotoxicity by enhanced expression of TRAIL and NKG2DLs. However, these pharmaceuticals may also impair NK cell function in a dose- and time-dependent manner. In summary, this review provides an update on the effects of different novel molecules on the immune system focusing NK cells. PMID:23316191

  20. Novel immune modulators used in hematology: impact on NK cells

    PubMed Central

    Krieg, Stephanie; Ullrich, Evelyn

    2013-01-01

    There is a wide range of important pharmaceuticals used in treatment of cancer. Besides their known effects on tumor cells, there is growing evidence for modulation of the immune system. Immunomodulatory drugs (IMiDs®) play an important role in the treatment of patients with multiple myeloma or myelodysplastic syndrome and have already demonstrated antitumor, anti-angiogenic, and immunostimulating effects, in particular on natural killer (NK) cells. Tyrosine kinase inhibitors are directly targeting different kinases and are known to regulate effector NK cells and expression of NKG2D ligands (NKG2DLs) on tumor cells. Demethylating agents, histone deacetylases, and proteasome inhibitors interfere with the epigenetic regulation and protein degradation of malignant cells. There are first hints that these drugs also sensitize tumor cells to chemotherapy, radiation, and NK cell-mediated cytotoxicity by enhanced expression of TRAIL and NKG2DLs. However, these pharmaceuticals may also impair NK cell function in a dose- and time-dependent manner. In summary, this review provides an update on the effects of different novel molecules on the immune system focusing NK cells. PMID:23316191

  1. A peptide antagonist disrupts NK cell inhibitory synapse formation.

    PubMed

    Borhis, Gwenoline; Ahmed, Parvin S; Mbiribindi, Bérénice; Naiyer, Mohammed M; Davis, Daniel M; Purbhoo, Marco A; Khakoo, Salim I

    2013-03-15

    Productive engagement of MHC class I by inhibitory NK cell receptors depends on the peptide bound by the MHC class I molecule. Peptide:MHC complexes that bind weakly to killer cell Ig-like receptors (KIRs) can antagonize the inhibition mediated by high-affinity peptide:MHC complexes and cause NK cell activation. We show that low-affinity peptide:MHC complexes stall inhibitory signaling at the step of Src homology protein tyrosine phosphatase 1 recruitment and do not go on to form the KIR microclusters induced by high-affinity peptide:MHC, which are associated with Vav dephosphorylation and downstream signaling. Furthermore, the low-affinity peptide:MHC complexes prevented the formation of KIR microclusters by high-affinity peptide:MHC. Thus, peptide antagonism of NK cells is an active phenomenon of inhibitory synapse disruption.

  2. Evaluation of Functional NK Cell Responses in Vaccinated and SIV-Infected Rhesus Macaques.

    PubMed

    Vargas-Inchaustegui, Diego A; Ying, Olivia; Demberg, Thorsten; Robert-Guroff, Marjorie

    2016-01-01

    NK cells are crucial components of the innate immune system due to their capacity to exert rapid cytotoxic and immunomodulatory function in the absence of prior sensitization. NK cells can become activated by exposure to target cells and/or by cytokines produced by antigen-presenting cells. In this study, we examined the effects of a simian immunodeficiency virus (SIV) vaccine regimen and subsequent SIV infection on the cytotoxic and immunomodulatory functions of circulatory NK cells. While vaccination did not significantly impact the capacity of NK cells to kill MHC-devoid 721.221 target cells, SIV-infection led to a significant decrease in target cell killing. NK cells from uninfected macaques were responsive to a low dose (5 ng/ml) of IL-15 pre-activation, leading to significant increases in their cytotoxic potential, however, NK cells from SIV-infected macaques required a higher dose (50 ng/ml) of IL-15 pre-activation in order to significantly increase their cytotoxic potential. By contrast, no differences were observed in the capacity of NK cells from vaccinated and SIV-infected macaques to respond to IL-12 and IL-18. Similarly, NK cells both before and after infection exhibited equivalent responses to Fc-mediated activation. Collectively, our results show that early SIV-infection impairs the natural cytotoxic capacity of circulatory NK cells without affecting Fc-mediated or cytokine-producing function. PMID:27630641

  3. Evaluation of Functional NK Cell Responses in Vaccinated and SIV-Infected Rhesus Macaques

    PubMed Central

    Vargas-Inchaustegui, Diego A.; Ying, Olivia; Demberg, Thorsten; Robert-Guroff, Marjorie

    2016-01-01

    NK cells are crucial components of the innate immune system due to their capacity to exert rapid cytotoxic and immunomodulatory function in the absence of prior sensitization. NK cells can become activated by exposure to target cells and/or by cytokines produced by antigen-presenting cells. In this study, we examined the effects of a simian immunodeficiency virus (SIV) vaccine regimen and subsequent SIV infection on the cytotoxic and immunomodulatory functions of circulatory NK cells. While vaccination did not significantly impact the capacity of NK cells to kill MHC-devoid 721.221 target cells, SIV-infection led to a significant decrease in target cell killing. NK cells from uninfected macaques were responsive to a low dose (5 ng/ml) of IL-15 pre-activation, leading to significant increases in their cytotoxic potential, however, NK cells from SIV-infected macaques required a higher dose (50 ng/ml) of IL-15 pre-activation in order to significantly increase their cytotoxic potential. By contrast, no differences were observed in the capacity of NK cells from vaccinated and SIV-infected macaques to respond to IL-12 and IL-18. Similarly, NK cells both before and after infection exhibited equivalent responses to Fc-mediated activation. Collectively, our results show that early SIV-infection impairs the natural cytotoxic capacity of circulatory NK cells without affecting Fc-mediated or cytokine-producing function.

  4. Evaluation of Functional NK Cell Responses in Vaccinated and SIV-Infected Rhesus Macaques

    PubMed Central

    Vargas-Inchaustegui, Diego A.; Ying, Olivia; Demberg, Thorsten; Robert-Guroff, Marjorie

    2016-01-01

    NK cells are crucial components of the innate immune system due to their capacity to exert rapid cytotoxic and immunomodulatory function in the absence of prior sensitization. NK cells can become activated by exposure to target cells and/or by cytokines produced by antigen-presenting cells. In this study, we examined the effects of a simian immunodeficiency virus (SIV) vaccine regimen and subsequent SIV infection on the cytotoxic and immunomodulatory functions of circulatory NK cells. While vaccination did not significantly impact the capacity of NK cells to kill MHC-devoid 721.221 target cells, SIV-infection led to a significant decrease in target cell killing. NK cells from uninfected macaques were responsive to a low dose (5 ng/ml) of IL-15 pre-activation, leading to significant increases in their cytotoxic potential, however, NK cells from SIV-infected macaques required a higher dose (50 ng/ml) of IL-15 pre-activation in order to significantly increase their cytotoxic potential. By contrast, no differences were observed in the capacity of NK cells from vaccinated and SIV-infected macaques to respond to IL-12 and IL-18. Similarly, NK cells both before and after infection exhibited equivalent responses to Fc-mediated activation. Collectively, our results show that early SIV-infection impairs the natural cytotoxic capacity of circulatory NK cells without affecting Fc-mediated or cytokine-producing function. PMID:27630641

  5. Diacylglycerol Kinase ζ Is a Target To Enhance NK Cell Function.

    PubMed

    Yang, Enjun; Singh, Brenal K; Paustian, Amanda M Schmidt; Kambayashi, Taku

    2016-08-01

    Enhancement of NK cell function could be beneficial in treatment of a variety of tumors and infections. However, efforts to improve NK cell function by disrupting negative regulators that target proximal signaling pathways paradoxically results in hyporesponsive rather than hyperresponsive NK cells. In this study, we demonstrate that genetic deletion of diacylglycerol kinase (DGK)ζ, a negative regulator of diacylglycerol-mediated signaling, has the desired effect of enhancing NK cell function due to its distal position in the activating receptor-mediated signaling cascade. Upon stimulation through multiple activating receptors, NK cells from mice lacking DGKζ display increased cytokine production and degranulation in an ERK-dependent manner. Additionally, they have improved cytotoxic functions against tumor cell lines. The enhancement of NK cell function by DGKζ deficiency is NK cell-intrinsic and developmentally independent. Importantly, DGKζ deficiency does not affect inhibitory NK cell receptor expression or function. Thus, DGKζ knockout mice display improved missing self recognition, as evidenced by enhanced rejection of a TAP-deficient tumor in vivo. We propose that enzymes that negatively regulate distal activating receptor signaling pathways such as DGKζ represent novel targets for augmenting the therapeutic potential of NK cells.

  6. NK cell depletion diminish tumour-specific B cell responses.

    PubMed

    Jensen, Markus; Tawadros, Samir; Sedlacek, Hans-Harald; Schultze, Joachim L; Berthold, Frank

    2004-05-15

    Natural killer (NK) cells can exercise immediate cytotoxicity against malignant cells and thus far modulate the development of tumour directed T cell immunity. To investigate the impact of NK cells on the development of tumour directed B cell immunity mice were immunised with IMR5-75 human neuroblastoma cells with or without prior in vivo NK cell depletion. Flow cytometry analyses gave evidence for an impaired IgG response against the cells immunised with. Dissection of Th1 (IgG2a) and Th2 (IgG1) oriented B cell responses revealed Th1 responses as primarily affected, while Th2 oriented B cell responses as measured by flow cytometry and GD2 ganglioside-specific ELISA were enforced. The data reveal an unexpected impact of NK cells on the development of tumour directed B cell responses. Consequently, NK cell function has also to be taken into account when developing B cell-based cancer immunotherapy.

  7. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  8. CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies.

    PubMed

    Romanski, Annette; Uherek, Christoph; Bug, Gesine; Seifried, Erhard; Klingemann, Hans; Wels, Winfried S; Ottmann, Oliver G; Tonn, Torsten

    2016-07-01

    Many B-cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK-92 cells, a continuously growing NK cell line, showing that anti-CD19-CAR (αCD19-CAR) engineered NK-92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK-92 cells. The 'first generation' CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc-tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK-92 and transduced αCD19-CAR NK-92 cells towards CD19 negative targets was found. However, αCD19-CAR NK-92 cells specifically and efficiently lysed CD19 expressing B-precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK-92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK-92 should be considered a treatment option for patients with lymphoid malignancies.

  9. Combination Immune Therapies to Enhance Anti-Tumor Responses by NK Cells

    PubMed Central

    Mentlik James, Ashley; Cohen, Adam D.; Campbell, Kerry S.

    2013-01-01

    Natural killer (NK) cells are critical innate immune lymphocytes capable of destroying virally infected or cancerous cells through targeted cytotoxicity and further assisting in the immune response by releasing inflammatory cytokines. NK cells are thought to contribute to the process of tumor killing by certain therapeutic monoclonal antibodies (mAb) by directing antibody-dependent cellular cytotoxicity (ADCC) through FcγRIIIA (CD16). Numerous therapeutic mAb have been developed that target distinct cancer-specific cell markers and may direct NK cell-mediated ADCC. Recent therapeutic approaches have combined some of these cancer-specific mAb with additional strategies to optimize NK cell cytotoxicity. These include agonistic mAb targeting NK cell activating receptors and mAbs blocking NK cell inhibitory receptors to enhance NK cell functions. Furthermore, several drugs that can potentiate NK cell cytotoxicity through other mechanisms are being used in combination with therapeutic mAb. In this review, we examine the mechanisms employed by several promising agents used in combination therapies that enhance natural or Ab-dependent cytotoxicity of cancer cells by NK cells, with a focus on treatments for leukemia and multiple myeloma. PMID:24391651

  10. A hematopoietic cell-driven mechanism involving SLAMF6 receptor, SAP adaptors and SHP-1 phosphatase regulates NK cell education.

    PubMed

    Wu, Ning; Zhong, Ming-Chao; Roncagalli, Romain; Pérez-Quintero, Luis-Alberto; Guo, Huaijian; Zhang, Zhanguang; Lenoir, Christelle; Dong, Zhongjun; Latour, Sylvain; Veillette, André

    2016-04-01

    Activation of natural killer (NK) cells by hematopoietic target cells is controlled by the SLAM family of receptors and by the associated SAP family of adaptors. Here we found that SLAM receptors also enhanced NK cell activation by nonhematopoietic target cells, which lack ligands for SLAM receptors. This function was mediated by SLAMF6, a homotypic SLAM receptor found on NK cells and other hematopoietic cells, and was regulated by SAP adaptors, which uncoupled SLAM receptors from phosphatase SHP-1 and diminished the effect of SLAMF6 on NK cell responsiveness toward nonhematopoietic cells. Thus, in addition to their role in NK cell activation by hematopoietic cells, the SLAM-SAP pathways influence responsiveness toward nonhematopoietic targets by a process akin to NK cell 'education'.

  11. Stromal Cells from Human Decidua Exert a Strong Inhibitory Effect on NK Cell Function and Dendritic Cell Differentiation

    PubMed Central

    Canegallo, Francesca; Conte, Romana; Venturini, Pier Luigi; Moretta, Lorenzo; Mingari, Maria Cristina

    2014-01-01

    Stromal cells (SC) are an important component of decidual tissues where they are in strict proximity with both NK and CD14+ myelomonocytic cells that play a role in the maintenance of pregnancy. In this study we analyzed whether decidual SC (DSC) could exert a regulatory role on NK and CD14+ cells that migrate from peripheral blood (PB) to decidua during pregnancy. We show that DSCs inhibit the IL15-mediated up-regulation of major activating NK receptors in PB-derived NK cells. In addition, the IL15-induced NK cell proliferation, cytolytic activity and IFN-γ production were severely impaired. DSCs sharply inhibited dendritic cells differentiation and their ability to induce allogeneic T cell proliferation. Indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) mediated the inhibitory effect of DSCs. Our results strongly suggest an important role of DSCs in preventing potentially dangerous immune response, thus contributing to maintenance of pregnancy. PMID:24586479

  12. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

    PubMed

    Wild, J; Schmiedel, B J; Maurer, A; Raab, S; Prokop, L; Stevanović, S; Dörfel, D; Schneider, P; Salih, H R

    2015-08-01

    Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

  13. Lenalidomide augments actin remodeling and lowers NK-cell activation thresholds

    PubMed Central

    Lagrue, Kathryn; Carisey, Alex; Morgan, David J.; Chopra, Rajesh

    2015-01-01

    As multiple myeloma (MM) progresses, natural killer (NK)-cell responses decline against malignant plasma cells. The immunomodulatory drug lenalidomide is widely used for treatment of MM but its influence on NK-cell biology is unclear. Here, we report that lenalidomide lowers the threshold for NK-cell activation, causing a 66% decrease in the 50% effective concentration (EC50) for activation through CD16, and a 38% decrease in EC50 for NK group 2 member D (NKG2D)–mediated activation, allowing NK cells to respond to lower doses of ligand. In addition, lenalidomide augments NK-cell responses, causing a twofold increase in the proportion of primary NK cells producing interferon-γ (IFN-γ), and a 20-fold increase in the amount of IFN-γ produced per cell. Importantly, lenalidomide did not trigger IFN-γ production in unstimulated NK cells. Thus, lenalidomide enhances the NK-cell arm of the immune response, without activating NK cells inappropriately. Of particular clinical importance, lenalidomide also allowed NK cells to be activated by lower doses of rituximab, an anti-CD20 monoclonal antibody (mAb) widely used to treat B-cell malignancies. This supports combined use of lenalidomide and rituximab in a clinical setting. Finally, superresolution microscopy revealed that lenalidomide increased the periodicity of cortical actin at immune synapses, resulting in an increase in the area of the actin mesh predicted to be penetrable to vesicles containing IFN-γ. NK cells from MM patients also responded to lenalidomide in this way. This indicates that nanometer-scale rearrangements in cortical actin, a recently discovered step in immune synapse assembly, are a potential new target for therapeutic compounds. PMID:26002964

  14. Repression of GSK3 restores NK cell cytotoxicity in AML patients.

    PubMed

    Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A; Gupta, Kalpana; deLima, Marcos; Beck, Rose C; Wald, David N

    2016-04-04

    Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.

  15. Repression of GSK3 restores NK cell cytotoxicity in AML patients

    PubMed Central

    Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A.; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A.; Gupta, Kalpana; deLima, Marcos; Beck, Rose C.; Wald, David N.

    2016-01-01

    Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy. PMID:27040177

  16. Perturbation of NK cell peripheral homeostasis accelerates prostate carcinoma metastasis.

    PubMed

    Liu, Gang; Lu, Shengjun; Wang, Xuanjun; Page, Stephanie T; Higano, Celestia S; Plymate, Stephen R; Greenberg, Norman M; Sun, Shaoli; Li, Zihai; Wu, Jennifer D

    2013-10-01

    The activating receptor NK cell group 2 member D (NKG2D) mediates antitumor immunity in experimental animal models. However, whether NKG2D ligands contribute to tumor suppression or progression clinically remains controversial. Here, we have described 2 novel lines of "humanized" bi-transgenic (bi-Tg) mice in which native human NKG2D ligand MHC class I polypeptide-related sequence B (MICB) or the engineered membrane-restricted MICB (MICB.A2) was expressed in the prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous carcinogenesis. Bi-Tg TRAMP/MICB mice exhibited a markedly increased incidence of progressed carcinomas and metastasis, whereas TRAMP/MICB.A2 mice enjoyed long-term tumor-free survival conferred by sustained NKG2D-mediated antitumor immunity. Mechanistically, we found that cancer progression in TRAMP/MICB mice was associated with loss of the peripheral NK cell pool owing to high serum levels of tumor-derived soluble MICB (sMICB). Prostate cancer patients also displayed reduction of peripheral NK cells and high sMIC levels. Our study has not only provided direct evidence in "humanized" mouse models that soluble and membrane-restricted NKG2D ligands pose opposite impacts on cancer progression, but also uncovered a mechanism of sMIC-induced impairment of NK cell antitumor immunity. Our findings suggest that the impact of soluble NKG2D ligands should be considered in NK cell-based cancer immunotherapy and that our unique mouse models should be valuable for therapy optimization. PMID:24018560

  17. Parasite-Mediated Upregulation of NK Cell-Derived Gamma Interferon Protects against Severe Highly Pathogenic H5N1 Influenza Virus Infection▿

    PubMed Central

    O'Brien, Kevin B.; Schultz-Cherry, Stacey; Knoll, Laura J.

    2011-01-01

    Outbreaks of influenza A viruses are associated with significant human morbidity worldwide. Given the increasing resistance to the available influenza drugs, new therapies for the treatment of influenza virus infection are needed. An alternative approach is to identify products that enhance a protective immune response. In these studies, we demonstrate that infecting mice with the Th1-inducing parasite Toxoplasma gondii prior to highly pathogenic avian H5N1 influenza virus infection led to decreased lung viral titers and enhanced survival. A noninfectious fraction of T. gondii soluble antigens (STAg) elicited an immune response similar to that elicited by live parasites, and administration of STAg 2 days after H5N1 influenza virus infection enhanced survival, lowered viral titers, and reduced clinical disease. STAg administration protected H5N1 virus-infected mice lacking lymphocytes, suggesting that while the adaptive immune response was not required for enhanced survival, it was necessary for STAg-mediated viral clearance. Mechanistically, we found that administration of STAg led to increased production of gamma interferon (IFN-γ) from natural killer (NK) cells, which were both necessary and sufficient for survival. Further, administration of exogenous IFN-γ alone enhanced survival from H5N1 influenza virus infection, although not to the same level as STAg treatment. These studies demonstrate that a noninfectious T. gondii extract enhances the protective immune response against severe H5N1 influenza virus infections even when a single dose is administered 2 days postinfection. PMID:21734055

  18. Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality.

    PubMed

    Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W; Böttcher, Sebastian; van Dongen, Jacques J M; Orfao, Alberto; Almeida, Julia

    2015-12-15

    Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

  19. Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality

    PubMed Central

    Bárcena, Paloma; Jara-Acevedo, María; Tabernero, María Dolores; López, Antonio; Sánchez, María Luz; García-Montero, Andrés C.; Muñoz-García, Noemí; Vidriales, María Belén; Paiva, Artur; Lecrevisse, Quentin; Lima, Margarida; Langerak, Anton W.; Böttcher, Sebastian; van Dongen, Jacques J.M.

    2015-01-01

    Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56low NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56low NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94hi/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality. PMID:26556869

  20. NK cell-extrinsic IL-18 signaling is required for efficient NK-cell activation by vaccinia virus.

    PubMed

    Brandstadter, Joshua D; Huang, Xiaopei; Yang, Yiping

    2014-09-01

    NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study using C57BL/6 mice, we first showed that IL-18 is critical for NK-cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK-cell activation upon VV infection in vitro. We further showed in vivo that efficient NK-cell activation in response to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK-cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK-cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK-cell-based therapies for viral infections and cancer.

  1. Flagellin-induced expression of CXCL10 mediates direct fungal killing and recruitment of NK cells to the cornea in response to Candida albicans infection.

    PubMed

    Liu, Xiaowei; Gao, Nan; Dong, Chen; Zhou, Li; Mi, Qing-Sheng; Standiford, Theodore J; Yu, Fu-Shin X

    2014-09-01

    We previously showed that topical flagellin induces profound mucosal innate protection in the cornea against microbial infection, a response involving multiple genes and cell types. In this study, we used a Candida albicans (CA)-C57BL/6 mouse keratitis model to delineate the contribution of CXCL10- and CXCR3-expressing cells in flagellin-induced protection. Flagellin pretreatment markedly enhanced CXCL10 expression at 6 h post CA infection (hpi), but significantly dampened CXCL10 expression at 24 hpi. At the cellular level, CXCL10 was expressed in the epithelia at 6 hpi in flagellin-pretreated corneas, and concentrated at lesion sites 24 hpi. CXCR3-expressing cells were detected in great numbers at 24 hpi, organized within clusters at the lesion sites in CA-infected corneas. CXCL10 or CXCR3 neutralization increased keratitis severity and dampened flagellin-induced protection. CXCR3-positive cells were identified as NK cells, the depletion of which resulted in severe CA keratitis. Contributions from NK T-cells were excluded by finding no change in flagellin-induced protection in Rag1 KO mice. Recombinant CXCL10 inhibited CA growth in vitro and accelerated fungal clearance and inflammation resolution in vivo. Taken together, our data indicate that epithelium-expressed CXCL10 plays a critical role in fungal clearance and that CXCR3-expressing NK cells contribute to CA eradication in mouse corneas.

  2. IL-17 regulates systemic fungal immunity by controlling the functional competence of NK cells.

    PubMed

    Bär, Eva; Whitney, Paul G; Moor, Kathrin; Reis e Sousa, Caetano; LeibundGut-Landmann, Salomé

    2014-01-16

    Interleukin 17 (IL-17)-mediated immunity plays a key role in protection from fungal infections in mice and man. Here, we confirmed that mice deficient in the IL-17 receptor or lacking the ability to secrete IL-17 are highly susceptible to systemic candidiasis, but we found that temporary blockade of the IL-17 pathway during infection in wild-type mice did not impact fungal control. Rather, mice lacking IL-17 receptor signaling had a cell-intrinsic impairment in the development of functional NK cells, which accounted for the susceptibility of these mice to systemic fungal infection. NK cells promoted antifungal immunity by secreting GM-CSF, necessary for the fungicidal activity of neutrophils. These data reveal that NK cells are crucial for antifungal defense and indicate a role for IL-17 family cytokines in NK cell development. The IL-17-NK cell axis may impact immunity against not only fungi but also bacteria, viruses, and tumors.

  3. Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model

    PubMed Central

    Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

    2016-01-01

    Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression.

  4. Cetuximab intensifies the ADCC activity of adoptive NK cells in a nude mouse colorectal cancer xenograft model

    PubMed Central

    Chen, Shanshan; Li, Xuechun; Chen, Rongming; Yin, Mingang; Zheng, Qiuhong

    2016-01-01

    Natural killer (NK) cells, discovered ~40 years ago, are believed to be the most effective cytotoxic lymphocytes to counteract cancer; however, adoptive NK cell therapy in vivo has encountered certain limitations, including a lack of specificity. The drug cetuximab can mediate antibody dependent cell mediated cytotoxicity (ADCC) activity through NK cells in vivo, and has been approved for the first-line treatment of epidermal growth factor receptor (EGFR)-positive metastatic colorectal cancer (CRC). However, the ADCC activity of adoptive NK cells, induced by cetuximab in a nude mouse CRC xenograft model, has not been previously reported. The aim of the present study was to explore the ADCC activity of cetuximab combined with adoptive NK cells in CRC xenograft models with various EGFR expressions. The nude mouse xenograft models were established by subcutaneously injecting LOVO or SW620 cells. The mice were then randomly divided into 6 groups: Phosphate-buffered saline, cetuximab, human immunoglobulin G (hIgG), NK cells, hIgG plus NK cells and cetuximab plus NK cells. The ADCC antitumor activity was evaluated in these CRC models. The results indicated that the cetuximab plus NK cells group showed the greatest tumor inhibition effect compared with the NK cells group in LOVO xenograft tumor models with positive EGFR expression. However, the combination of cetuximab and NK cells did not show a stronger tumor inhibitory effect against the SW620 xenograft tumor models compared with the efficiency of NK cells. In conclusion, cetuximab could intensify the ADCC antitumor activity of adoptive NK cells towards CRC with an increased EGFR expression. The combination of cetuximab and NK cells may be a potential immunotherapy for metastatic CRC patients with positive EGFR expression. PMID:27602116

  5. Multidirectional interactions are bridging human NK cells with plasmacytoid and monocyte-derived dendritic cells during innate immune responses.

    PubMed

    Della Chiesa, Mariella; Romagnani, Chiara; Thiel, Andreas; Moretta, Lorenzo; Moretta, Alessandro

    2006-12-01

    During innate immune responses, natural killer (NK) cells may interact with both plasmacytoid dendritic cells (pDCs) and monocyte-derived dendritic cells (MDDCs). We show that freshly isolated NK cells promote the release by pDCs of IFN-alpha, in a CpG-dependent manner, whereas they induce IL-6 production in a CpG-independent manner. In turn pDC-derived IFN-alpha up-regulates NK-mediated killing, whereas IL-6 could promote B-cell differentiation. We also show that exposure to exogenous IL-12 or coculture with maturing MDDCs up-regulates the NK-cell-dependent IFN-alpha production by pDCs. On the other hand, NK cells cocultured with pDCs acquire the ability to kill immature MDDCs, thus favoring their editing process. Finally, we show that activated NK cells are unable to lyse pDCs because these cells display an intrinsic resistance to lysis. The exposure of pDCs to IL-3 increased their susceptibility to NK-cell cytotoxicity resulting from a de novo expression of ligands for activating NK-cell receptors, such as the DNAM-1 ligand nectin-2. Thus, different cell-to-cell interactions and various cytokines appear to control a multidirectional network between NK cells, MDDCs, and pDCs that is likely to play an important role during the early phase of innate immune responses to viral infections and to tumors. PMID:16873676

  6. Ly49C Impairs NK Cell Memory in Mouse Cytomegalovirus Infection.

    PubMed

    Forbes, Catherine A; Scalzo, Anthony A; Degli-Esposti, Mariapia A; Coudert, Jerome D

    2016-07-01

    NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function, accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed "licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I-dependent manner. The presence of Ly49C impacted the NK cell repertoire in a β2m-dependent manner, with reduced Ly49A(+), Ly49G2(+), and Ly49D(+) subsets, an increased DNAM-1(+) subset, and higher NKG2D expression. Licensed NK cells displayed a skewed distribution of the maturation stages, which was characterized by differential CD27 and CD11b expression, toward the mature phenotypes. We found that Ly49C-mediated licensing induced a split effect on NK cell functions, with increased cytokine-production capabilities following engagement of various activating receptors while cytotoxicity remained unchanged. Analysis of licensed NK cell functions in vivo, in a system of mouse CMV infection, indicated that licensing did not play a major role in the NK cell antiviral response during acute infection, but it strongly impaired the generation and/or persistence of memory NK cells. This study unravels multifaceted effects of licensing on NK cell populations and their functions. PMID:27233959

  7. Recognition and Regulation of T Cells by NK Cells

    PubMed Central

    Pallmer, Katharina; Oxenius, Annette

    2016-01-01

    Regulation of T cell responses by innate lymphoid cells (ILCs) is increasingly documented and studied. Direct or indirect crosstalk between ILCs and T cells early during and after T cell activation can affect their differentiation, polarization, and survival. Natural killer (NK) cells that belong to the ILC1 group were initially described for their function in recognizing and eliminating “altered self” and as source of early inflammatory cytokines, most notably type II interferon. Using signals conveyed by various germ-line encoded activating and inhibitory receptors, NK cells are geared to sense sudden cellular changes that can be caused by infection events, malignant transformation, or cellular stress responses. T cells, when activated by TCR engagement (signal 1), costimulation (signal 2), and cytokines (signal 3), commit to a number of cellular alterations, including entry into rapid cell cycling, metabolic changes, and acquisition of effector functions. These abrupt changes may alert NK cells, and T cells might thereby expose themselves as NK cell targets. Here, we review how activated T cells can be recognized and regulated by NK cells and what consequences such regulation bears for T cell immunity in the context of vaccination, infection, or autoimmunity. Conversely, we will discuss mechanisms by which activated T cells protect themselves against NK cell attack and outline the significance of this safeguard mechanism. PMID:27446081

  8. Salivary gland NK cells are phenotypically and functionally unique.

    PubMed

    Tessmer, Marlowe S; Reilly, Emma C; Brossay, Laurent

    2011-01-13

    Natural killer (NK) cells and CD8(+) T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or T(reg) cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

  9. NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

    PubMed Central

    Grandclément, Camille; Pick, Horst; Vogel, Horst; Held, Werner

    2016-01-01

    Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca2+ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca2+-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS. PMID:26963818

  10. Teach Your NK Cells Well.

    PubMed

    Cooper, Megan A

    2016-08-16

    Natural killer cells readily kill target cells, and education ensures tolerance to self. In this issue of Immunity, Boudreau et al. (2016) and Chen et al. (2016) report new mechanisms of human and mouse natural killer cell education by inhibitory and activating receptors. PMID:27533007

  11. Teach Your NK Cells Well.

    PubMed

    Cooper, Megan A

    2016-08-16

    Natural killer cells readily kill target cells, and education ensures tolerance to self. In this issue of Immunity, Boudreau et al. (2016) and Chen et al. (2016) report new mechanisms of human and mouse natural killer cell education by inhibitory and activating receptors.

  12. Models to study NK cell biology and possible clinical application

    PubMed Central

    Zamora, Anthony E.; Grossenbacher, Steven K.; Aguilar, Ethan G.; Murphy, William J.

    2016-01-01

    Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes resulting from the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been described as natural killers that provide a first line of defense prior to the induction of adaptive immunity, a more complex view of NK cells is beginning to emerge indicating they may also function in various immunoregulatory roles and have the capacity to shape adaptive immune responses. With the growing appreciation for the diverse functions of NK cells and recent technological advancements that allow for a more in-depth understanding of NK cell biology, we can now begin to explore new ways to manipulate NK cells to increase their clinical utility. In this overview unit, we introduce the reader to various aspects of NK cell biology by reviewing topics ranging from NK cell diversity and function, mouse models and the roles of NK cells in health and disease, to potential clinical applications. PMID:26237009

  13. Models to Study NK Cell Biology and Possible Clinical Application.

    PubMed

    Zamora, Anthony E; Grossenbacher, Steven K; Aguilar, Ethan G; Murphy, William J

    2015-08-03

    Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes that is due to the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been described as natural killers that provide a first line of defense prior to the induction of adaptive immunity, a more complex view of NK cells is beginning to emerge, indicating they may also function in various immunoregulatory roles and have the capacity to shape adaptive immune responses. With the growing appreciation for the diverse functions of NK cells, and recent technological advancements that allow for a more in-depth understanding of NK cell biology, we can now begin to explore new ways to manipulate NK cells to increase their clinical utility. In this overview unit, we introduce the reader to various aspects of NK cell biology by reviewing topics ranging from NK cell diversity and function, mouse models, and the roles of NK cells in health and disease, to potential clinical applications. © 2015 by John Wiley & Sons, Inc.

  14. NK Cell-based Immunotherapies in Pediatric Oncology

    PubMed Central

    McDowell, Kimberly A.; Hank, Jacquelyn A.; DeSantes, Kenneth B.; Capitini, Christian M.; Otto, Mario; Sondel, Paul M.

    2015-01-01

    The past decade has seen several anti-cancer immunotherapeutic strategies transition from “promising preclinical models” to treatments with proven clinical activity or benefit. In 2013, the journal Science selected the field of Cancer Immunotherapy as the overall number-1 breakthrough for the year in all of scientific research. In the setting of cancer immunotherapy for adult malignancies, many of these immunotherapy strategies have relied on the cancer patient’s endogenous anti-tumor T cell response. While much promising research in pediatric oncology is similarly focused on T cell reactivity, several pediatric malignancies themselves, or the chemo-radiotherapy used to achieve initial responses, can be associated with profound immune suppression, particularly of the T cell system. A separate component of the immune system, also able to mediate anti-tumor effects and less suppressed by conventional cancer treatment, is the NK cell system. In recent years, several distinct immunotherapeutic approaches that rely on the activity of NK cells have moved from preclinical development into clinical testing, and some have shown clear antitumor benefit. This review provides an overview of NK cell-based immunotherapy efforts that are directed towards childhood malignancies, with an emphasis on protocols that are already in clinical testing. PMID:25590232

  15. Altered effector functions of NK cells in chronic hepatitis C are associated with IFNL3 polymorphism.

    PubMed

    Rogalska-Taranta, Magdalena; Markova, Antoaneta A; Taranta, Andrzej; Lunemann, Sebastian; Schlaphoff, Verena; Flisiak, Robert; Manns, Michael P; Cornberg, Markus; Kraft, Anke R M; Wedemeyer, Heiner

    2015-08-01

    Interferon α-mediated effector functions of NK cells may contribute to the control of HCV replication and the pathogenesis of liver disease. The single-nucleotide polymorphism rs12979860 near IFNL3 (previously known as IL28B) is important in response to IFN-α treatment and in spontaneous resolution of acute hepatitis C. The role of the IFNL3 polymorphism in NK cell function is unclear. Thus, we investigated the role of IFNL3 polymorphism in type I IFN-dependent regulation of NK cell functions in patients with cHC and healthy control subjects. We demonstrated a marked polarization of NK cells toward cytotoxicity in response to IFN-α stimulation in patients with hepatitis C. That TRAIL up-regulation was present, particularly in patients with the IFNL3-TT allele, was supported by a shift in the pSTAT-1:pSTAT-4 ratios toward pSTAT-1. In patients bearing the IFNL3-TT allele, NK cell effector function correlated with liver disease activity. In contrast, higher cytokine production of NK cells was observed in healthy individuals with the IFNL3-CC genotype, which may support spontaneous HCV clearance in acute infection. Overall, these findings show that the role of NK cells may differ in chronic infection vs. early antiviral defense and that the IFNL3 genotype differentially influences NK cell function. PMID:26034208

  16. The Protozoan Neospora caninum Directly Triggers Bovine NK Cells To Produce Gamma Interferon and To Kill Infected Fibroblasts

    PubMed Central

    Boysen, Preben; Klevar, Siv; Olsen, Ingrid; Storset, Anne K.

    2006-01-01

    Natural killer (NK) cells are considered to be key players in the early innate responses to protozoan infections, primarily indirectly by producing gamma interferon (IFN-γ) in response to cytokines, like interleukin 12 (IL-12). We demonstrate that live, as well as heat-inactivated, tachyzoites of Neospora caninum, a Toxoplasma-like protozoan, directly trigger production of IFN-γ from purified, IL-2-activated bovine NK cells. This response occurred independently of IL-12 but was increased by the addition of the cytokine. A similar IFN-γ response was measured in cocultures of NK cells and N. caninum-infected autologous fibroblasts. However, no NK cell-derived IFN-γ response was detected when cells were cultured with soluble antigens from the organism, indicating that intact tachyzoites or nonsoluble components are necessary for NK cell triggering. Furthermore, N. caninum-infected autologous fibroblasts had increased susceptibility to NK cell cytotoxicity compared to uninfected fibroblasts. This cytotoxicity was largely mediated by a perforin-mediated mechanism. The activating receptor NKp46 was involved in cytotoxicity against fibroblasts but could not explain the increased cytotoxicity against infected targets. Interestingly, N. caninum tachyzoites were able to infect cultured NK cells, in which tachyzoites proliferated inside parasitophorous vacuoles. Together, these findings underscore the role of NK cells as primary responders during a protozoan infection, describe intracellular protozoan infection of NK cells in vitro for the first time, and represent the first functional study of purified bovine NK cells in response to infection. PMID:16428740

  17. Metastatic consequences of immune escape from NK cell cytotoxicity by human breast cancer stem cells.

    PubMed

    Wang, Bin; Wang, Qiang; Wang, Zhe; Jiang, Jun; Yu, Shi-Cang; Ping, Yi-Fang; Yang, Jing; Xu, Sen-Lin; Ye, Xian-Zong; Xu, Chuan; Yang, Lang; Qian, Cheng; Wang, Ji Ming; Cui, You-Hong; Zhang, Xia; Bian, Xiu-Wu

    2014-10-15

    Breast cancer stem-like cells (BCSC) are crucial for metastasis but the underlying mechanisms remain elusive. Here, we report that tumor-infiltrating natural killer (NK) cells failed to limit metastasis and were not associated with improved therapeutic outcome of BCSC-rich breast cancer. Primary BCSCs were resistant to cytotoxicity mediated by autologous/allogeneic NK cells due to reduced expression of MICA and MICB, two ligands for the stimulatory NK cell receptor NKG2D. Furthermore, the downregulation of MICA/MICB in BCSCs was mediated by aberrantly expressed oncogenic miR20a, which promoted the resistance of BCSC to NK cell cytotoxicity and resultant lung metastasis. The breast cancer cell differentiation-inducing agent, all-trans retinoic acid, restored the miR20a-MICA/MICB axis and sensitized BCSC to NK cell-mediated killing, thereby reducing immune escape-associated BCSC metastasis. Together, our findings reveal a novel mechanism for immune escape of human BCSC and identify the miR20a-MICA/MICB signaling axis as a therapeutic target to limit metastatic breast cancer.

  18. Bacterial lipopolysaccharide activates CD57-negative human NK cells.

    PubMed

    Kanevskiy, L M; Erokhina, S A; Streltsova, M A; Telford, W G; Sapozhnikov, A M; Kovalenko, E I

    2014-12-01

    NK cells play an important regulatory role in sepsis by induction and augmentation of proinflammatory reactions in early stages of the septic process and by suppression of immune response in later stages of inflammation. The present work was aimed at the effect of bacterial lipopolysaccharide (LPS), the main pathogenic factor of sepsis development, on human NK cells ex vivo. We show that LPS activates immature CD57-negative NK cells, which typically constitute less than half of the normal NK cell population in human peripheral blood. Under conditions of NK cell stimulation with IL-2, addition of LPS provokes an increase in IFN-γ production. However, LPS both increased and inhibited NK cell cytotoxic activity. It is important to note that the activation of NK cells on LPS addition was observed in the absence of TLR4 on the NK cell surface. These results confirm our previous data arguing for a direct interaction of LPS with NK cells and evidence an atypical mechanism of LPS-induced NK cell activation without the involvement of surface TLR4.

  19. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

    PubMed

    Frenkel, Deborah; Zhang, Fengqiu; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Radwanska, Magdalena; Magez, Stefan; Black, Samuel J

    2016-07-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  20. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells

    PubMed Central

    Frenkel, Deborah; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Magez, Stefan; Black, Samuel J.

    2016-01-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  1. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

    PubMed

    Frenkel, Deborah; Zhang, Fengqiu; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Radwanska, Magdalena; Magez, Stefan; Black, Samuel J

    2016-07-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  2. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  3. Glycocalyx Engineering Reveals a Siglec-Based Mechanism for NK Cell Immunoevasion

    PubMed Central

    Hudak, Jason E.; Canham, Stephen M.; Bertozzi, Carolyn R.

    2013-01-01

    The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human NK cell activation through the recruitment of Siglec-7. Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy. PMID:24292068

  4. In vivo tumor surveillance by NK cells requires TYK2 but not TYK2 kinase activity

    PubMed Central

    Prchal-Murphy, Michaela; Witalisz-Siepracka, Agnieszka; Bednarik, Karoline T; Putz, Eva Maria; Gotthardt, Dagmar; Meissl, Katrin; Sexl, Veronika; Müller, Mathias; Strobl, Birgit

    2015-01-01

    Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2−/−) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2−/− mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2−/− and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2−/− NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2−/− NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2's kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy. PMID:26451322

  5. Studying NK cell responses to ectromelia virus infections in mice.

    PubMed

    Fang, Min; Sigal, Luis

    2010-01-01

    Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

  6. Mycoplasma arthritidis mitogen up-regulates human NK cell activity.

    PubMed Central

    D'Orazio, J A; Cole, B C; Stein-Streilein, J

    1996-01-01

    While the effects of superantigens on T lymphocytes are well characterized, how superantigens interact with other immune cells is less clear. This report examines the effects of Mycoplasma arthritidis mitogen (MAM) on human natural killer (NK) cell activity. Incubation of peripheral blood mononuclear cells (PBMC) with MAM for 16 to 20 h augmented NK cytotoxicity (against K562) in a dose-dependent manner (P < or = 0.05). Superantigen-dependent cellular cytotoxicity, an activity of superantigen-activated cytotoxic T cells, was not involved in lysis of K562 cells because the erythroleukemic tumor target cells expressed no class II major histocompatibility complex by fluorescence-activated cell sorter analysis. Kinetic experiments showed that the largest increase in NK activity induced by MAM occurred within 48 h. Incubation with MAM caused a portion of NK cells to become adherent to tissue culture flasks, a quality associated with activation, and augmented NK activity was found in both adherent and nonadherent subpopulations. Experiments using cytokine-specific neutralizing antibodies showed that interleukin-2 contributed to enhancement of the NK activity observed in superantigen-stimulated PBMC. Interestingly, MAM was able to augment NK lysis of highly purified NK (CD56+) cells in the absence of other immune cells in 9 of 12 blood specimens, with the augmented lytic activity ranging from 110 to 170% of unstimulated NK activity. In summary, data presented in this report show for the first time that MAM affects human NK cells directly by increasing their lytic capacity and indirectly in PBMC as a consequence of cytokines produced by T cells. Results of this work suggest that, in vivo, one consequence of interaction with superantigen-secreting microorganisms may be up-regulation of NK lytic activity. These findings may have clinical application as a means of generating augmented NK effector cells useful in the immunotherapy of parasitic infections or neoplasms. PMID

  7. Proinflammatory status influences NK cells subsets in the elderly.

    PubMed

    Campos, Carmen; Pera, Alejandra; Lopez-Fernandez, Isabel; Alonso, Corona; Tarazona, Raquel; Solana, Rafael

    2014-11-01

    Aging is associated to dysfunctional changes in the immune system, a process termed immunosenescence. Elderly individuals usually present chronic low level inflammation, likely as the consequence of continued exposure to antigens combined with poor immune function, increases in production of pro-inflammatory cytokines by effector memory and senescent T cells and macrophages. This condition not only results from, but also drives immunosenescence. Aging affects all cell components of the immune system, including NK cells and its different subsets (CD56dimCD16+, CD56brightCD16+/- and CD56-CD16+). In particular, the percentage of total NK cells is increased in healthy aging and centenarians, whereas there is a decrease in the CD56bright NK cell subset and an expansion of CD56-CD16+ NK cells. However, the causes of these alterations on NK cells in old donors are not fully understood. In this work we analyse NK cell subsets in the elderly in relation with markers of inflammation and health status. The results show that there is a positive correlation between the number of total NK cells and the body mass index (BMI), while the number of CD56bright NK cells negatively correlates with the levels of C-reactive protein (CRP), supporting that chronic inflammation is involved in the decrease of this NK cell subset.

  8. Effects of formaldehyde exposure on human NK cells in vitro.

    PubMed

    Li, Qi; Mei, Qibing; Huyan, Ting; Xie, Li; Che, Su; Yang, Hui; Zhang, Mingjie; Huang, Qingsheng

    2013-11-01

    Natural killer (NK) cells play a pivotal role in human immunologic surveillance. Formaldehyde (FA), a ubiquitous environmental contaminant, has been classified as a carcinogen to humans. Although it is known that immune cells are sensitive to FA, so far little is known about how it's affecting the activity of human NK cells. To probe it, the primary human NK cells were treated with different concentrations of FA (3200, 1600, 800, 400, 200, 100, 50, and 0 μM) in vitro. The morphology, viability, apoptosis, cytotoxicity (killing tumor cell activity) and cytokine and cytolytic proteins secretion of NK cells were evaluated respectively. Our results reveal that FA could induce NK cells death obviously in a concentration-dependent manner. With the decreased concentrations of FA from 3200 μM to 800 μM, accordingly, the viability of NK cells increased from 65. 2 ± 12.1% to 78.48 ± 10.3% (p<0.05), and the cytotoxicity of NK cells recovered from 29.2 ± 8.5% to 63.9 ± 5.9% (p<0.05). The secretion of perforin was affected significantly by FA, whereas the secretion of IFN-γ and granzyme-B altered slightly. It is concluded that human NK cell is sensitive to FA, 800 μM may be a critical concentration of FA inhibiting the activity of human NK cell.

  9. Effect of age and CMV on NK cell subpopulations.

    PubMed

    Campos, Carmen; Pera, Alejandra; Sanchez-Correa, Beatriz; Alonso, Corona; Lopez-Fernandez, Isabel; Morgado, Sara; Tarazona, Raquel; Solana, Rafael

    2014-06-01

    NK cells represent an important component of the innate immune response against infection and tumors. Age-associated changes in NK cell phenotype have been previously reported that can be responsible of functional NK cell deficiency. The aim of this work was to analyze the effect CMV seropositivity and aging on the distribution of NK cell subsets with a focus on the expression of cytotoxicity-related molecules and on the expression of CD94/NKG2 heterodimers and CD57 on these NK cell subsets. Our results show that CMV seropositivity in young individuals does not significantly affect peripheral blood NK cell percentage and NK cell subsets defined by the use of CD56 and CD16 markers. In contrast a significant increase in the percentage of NK cells is observed in elderly donors, all of them are CMV seropositive, when compared with young CMV seropositive subjects. A decrease in the percentage of CD56bright NK cells, either fully immature CD16 negative or CD16+ and an increase in the CD56-CD16+ subset are also found in the elderly. CMV seropositivity either in healthy young or elderly individuals is associated to the expression of CD94/NKG2C dimers and high expression of CD57on the CD56dimCD16+ NK cell subset. CD56-CD16+ NK cells, which are expanded in the elderly, show a decreased expression of granzymes A and B and an increased expression of CD94/NKG2C and CD57 in CMV seropositive young donors when compared with CMV seronegative young individuals. These results indicate that CMV and age have a different effect on NK cell phenotype and emphasize the relevance of including the determination of CMV serostatus in those studies addressed to analyze the immune response in the elderly.

  10. NK/T cell lymphoma associated with peripheral eosinophilia.

    PubMed

    Yap, E; Wan Jamaluddin, W F; Tumian, N R; Mashuri, F; Mohammed, F; Tan, G C; Masir, N; Abdul Wahid, F S

    2014-12-01

    NK/T cell lymphoma, nasal type is an aggressive and uncommon malignancy. Disease that occurs outside of the aerodigestive tract exhibits an even more aggressive clinical behaviour and does not respond as well to conventional therapy compared to its nasal counterpart. We report such a case of NK/T cell lymphoma, nasal type, that presented as an anterior chest wall mass, arising from the left pectoralis muscle. An interesting feature we wish to highlight is the associated eosinophilia that corresponded to disease activity, exhibiting fluctuations with surgical resection and chemotherapy. To the best of our knowledge this is the third reported case of NK/T cell lymphoma that is associated with peripheral eosinophilia. Our case highlights the role of certain NK cell subsets that play a major role in eosinophilic activation in NK/T lymphomas and calls for more research into further classification of this disease by virtue of its NK cell subsets. PMID:25500520

  11. Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

    PubMed Central

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient’s blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects. PMID:27014270

  12. A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells

    PubMed Central

    Nissen, Sara Konstantin; Rasmussen, Thomas A.; Østergaard, Lars; Denton, Paul W.; Søgaard, Ole Schmeltz

    2016-01-01

    ABSTRACT Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a “shock-and-kill” approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P = 0.005) and CXCL10 (P = 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56dim CD16+ NK cells (P = 0.001) as well as higher proportions of CD107a-positive (P = 0.002) and IFN-γ-producing (P = 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4+ and CD8+ T cells. Furthermore, CD4+ T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P = 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4+ T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P = 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4+ T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. IMPORTANCE We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the Cp

  13. NK cells infiltrating a MHC class I-deficient lung adenocarcinoma display impaired cytotoxic activity toward autologous tumor cells associated with altered NK cell-triggering receptors.

    PubMed

    Le Maux Chansac, Béatrice; Moretta, Alessandro; Vergnon, Isabelle; Opolon, Paule; Lécluse, Yann; Grunenwald, Dominique; Kubin, Marek; Soria, Jean-Charles; Chouaib, Salem; Mami-Chouaib, Fathia

    2005-11-01

    NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking beta2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I- tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition.

  14. Signaling pathways engaged by NK cell receptors: double concerto for activating receptors, inhibitory receptors and NK cells.

    PubMed

    Tomasello, E; Bléry, M; Vély, F; Vivier, E

    2000-04-01

    Despite the absence of antigen-specific receptors at their surface, NK cells can selectively eliminate virus-infected cells, tumor cells and allogenic cells. A dynamic and precisely coordinated balance between activating and inhibitory receptors governs NK cell activation programs. Multiple activating and inhibitory NK cell surface molecules have been described, a group of them acting as receptors for MHC class I molecules. In spite of their heterogeneity, activating NK cell receptors present remarkable structural and functional homologies with T cell- and B cell-antigen receptors. Inhibitory NK cell receptors operate at early stages of activating cascades by recruiting protein tyrosine phosphatases via intra- cytoplasmic motifs (ITIM), a strategy which is widely conserved in hematopoietic and non-hematopoietic cells.

  15. Role of the NK Cell-Activating Receptor CRACC in Periodontitis

    PubMed Central

    Krämer, Benjamin; Kebschull, Moritz; Nowak, Michael; Demmer, Ryan T.; Haupt, Manuela; Körner, Christian; Perner, Sven; Jepsen, Søren

    2013-01-01

    Periodontitis is a highly prevalent, biofilm-mediated chronic inflammatory disease that results in the loss of the tooth-supporting tissues. It features two major clinical entities: chronic periodontitis, which is more common, and aggressive periodontitis, which usually has an early onset and a rapid progression. Natural killer (NK) cells are a distinct subgroup of lymphocytes that play a major role in the ability of the innate immune system to steer immune responses. NK cells are abundant in periodontitis lesions, and NK cell activation has been causally linked to periodontal tissue destruction. However, the exact mechanisms of their activation and their role in the pathophysiology of periodontitis are elusive. Here, we show that the predominant NK cell-activating molecule in periodontitis is CD2-like receptor activating cytotoxic cells (CRACC). We show that CRACC induction was significantly more pronounced in aggressive than chronic periodontitis and correlated positively with periodontal disease severity, subgingival levels of specific periodontal pathogens, and NK cell activation in vivo. We delineate how Aggregatibacter actinomycetemcomitans, an oral pathogen that is causally associated with aggressive periodontitis, indirectly induces CRACC on NK cells via activation of dendritic cells and subsequent interleukin 12 (IL-12) signaling. In contrast, we demonstrate that fimbriae from Porphyromonas gingivalis, a principal pathogen in chronic periodontitis, actively attenuate CRACC induction on NK cells. Our data suggest an involvement of CRACC-mediated NK cell activation in periodontal tissue destruction and point to a plausible distinction in the pathobiology of aggressive and chronic periodontitis that may help explain the accelerated tissue destruction in aggressive periodontitis. PMID:23250953

  16. Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity.

    PubMed

    Shibina, Anastasia; Seidel, Diana; Somanchi, Srinivas S; Lee, Dean A; Stermann, Alexander; Maurer, Barry J; Lode, Holger N; Reynolds, C Patrick; Huebener, Nicole

    2013-04-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.

  17. Signatures of human NK cell development and terminal differentiation.

    PubMed

    Luetke-Eversloh, Merlin; Killig, Monica; Romagnani, Chiara

    2013-01-01

    Natural killer (NK) cells are part of the innate lymphoid cell (ILC) family and represent the main cytotoxic population. NK cells develop from bone marrow common lymphoid progenitors and undergo terminal differentiation in the periphery, where they finally gain their cytotoxic competence as well as the ability to produce IFN-γ in response to engagement of activating receptors. This process has been at least partially elucidated and several markers have been identified to discriminate different NK cell stages and other ILC populations. NK cell terminal differentiation is not only associated with progressive phenotypic changes but also with defined effector signatures. In this essay, we will describe the phenotypic and functional characteristics of the main stages of NK cell development and terminal differentiation and discuss them in light of recent discoveries of novel ILC populations.

  18. Signatures of Human NK Cell Development and Terminal Differentiation

    PubMed Central

    Luetke-Eversloh, Merlin; Killig, Monica; Romagnani, Chiara

    2013-01-01

    Natural killer (NK) cells are part of the innate lymphoid cell (ILC) family and represent the main cytotoxic population. NK cells develop from bone marrow common lymphoid progenitors and undergo terminal differentiation in the periphery, where they finally gain their cytotoxic competence as well as the ability to produce IFN-γ in response to engagement of activating receptors. This process has been at least partially elucidated and several markers have been identified to discriminate different NK cell stages and other ILC populations. NK cell terminal differentiation is not only associated with progressive phenotypic changes but also with defined effector signatures. In this essay, we will describe the phenotypic and functional characteristics of the main stages of NK cell development and terminal differentiation and discuss them in light of recent discoveries of novel ILC populations. PMID:24416035

  19. Cell-Extrinsic MHC Class I Molecule Engagement Augments Human NK Cell Education Programmed by Cell-Intrinsic MHC Class I.

    PubMed

    Boudreau, Jeanette E; Liu, Xiao-Rong; Zhao, Zeguo; Zhang, Aaron; Shultz, Leonard D; Greiner, Dale L; Dupont, Bo; Hsu, Katharine C

    2016-08-16

    The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by "self" MHC class I molecules in a process called "education." In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B(∗)27:05(+) transgenic mice and in patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34(+) stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1(+) NK cells gained reactivity after adoptive transfer to HLA-B(∗)27:05(+) mice or bone marrow chimeric mice where HLA-B(∗)27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell-intrinsic HLA in maintaining NK cell education.

  20. Human Parthenogenetic Embryonic Stem Cell–Derived Neural Stem Cells Express HLA-G and Show Unique Resistance to NK Cell–Mediated Killing

    PubMed Central

    Schmitt, Jessica; Eckardt, Sigrid; Schlegel, Paul G; Sirén, Anna-Leena; Bruttel, Valentin S; McLaughlin, K John; Wischhusen, Jörg; Müller, Albrecht M

    2015-01-01

    Parent-of-origin imprints have been implicated in the regulation of neural differentiation and brain development. Previously we have shown that, despite the lack of a paternal genome, human parthenogenetic (PG) embryonic stem cells (hESCs) can form proliferating neural stem cells (NSCs) that are capable of differentiation into physiologically functional neurons while maintaining allele-specific expression of imprinted genes. Since biparental (“normal”) hESC–derived NSCs (N NSCs) are targeted by immune cells, we characterized the immunogenicity of PG NSCs. Flow cytometry and immunocytochemistry revealed that both N NSCs and PG NSCs exhibited surface expression of human leukocyte antigen (HLA) class I but not HLA-DR molecules. Functional analyses using an in vitro mixed lymphocyte reaction assay resulted in less proliferation of peripheral blood mononuclear cells (PBMC) with PG compared with N NSCs. In addition, natural killer (NK) cells cytolyzed PG less than N NSCs. At a molecular level, expression analyses of immune regulatory factors revealed higher HLA-G levels in PG compared with N NSCs. In line with this finding, MIR152, which represses HLA-G expression, is less transcribed in PG compared with N cells. Blockage of HLA-G receptors ILT2 and KIR2DL4 on natural killer cell leukemia (NKL) cells increased cytolysis of PG NSCs. Together this indicates that PG NSCs have unique immunological properties due to elevated HLA-G expression. PMID:25811991

  1. Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

    PubMed

    Malviya, Gaurav; Nayak, Tapan; Gerdes, Christian; Dierckx, Rudi A J O; Signore, Alberto; de Vries, Erik F J

    2016-04-01

    A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice

  2. Effects of Simulated Microgravity on Primary Human NK Cells

    PubMed Central

    Li, Qi; Mei, Qibing; Huyan, Ting; Xie, Li; Che, Su; Yang, Hui; Zhang, Mingjie

    2013-01-01

    Abstract The deleterious effects of microgravity on lymphocytes have been demonstrated in previous studies. However, research on the effects of microgravity on human natural killer (NK) cells remains exceedingly limited. In this study, we demonstrated that NK cell cytotoxicity was significantly decreased under simulated microgravity (SMG) conditions (p<0.05). Several processes, including apoptosis, receptor expression, and cytokine secretion, were investigated in human NK cells under SMG. We observed decreased cytotoxicity, concurrent with increased apoptosis and necrosis, in NK cells after exposure to SMG (p<0.05). Additionally, interferon (IFN)-γ and perforin expression decreased significantly, and the expression of granzyme-B was only slightly reduced. Meanwhile, SMG selectively inhibited the expression of certain surface receptors on NK cells. Specifically, the expression of NKG2A and NKG2D were significantly downregulated under SMG, but the expression of NKp30 and NKp44 was not affected. We also found that interleukin (IL)–15 alone or in combination with IL-12 could counteract the inhibition of NK cell cytotoxicity under SMG. Our findings indicate that human NK cells were sensitive to SMG, as reflected by their decreased cytotoxicity. Factors such as increased early apoptosis and late apoptosis/necrosis and the decreased expression of INF-γ, cytolytic proteins, and cell surface receptors may be responsible for the loss of cytotoxicity in human NK cells under SMG. A combination of IL-12 and IL-15 may be useful as a therapeutic strategy for overcoming the effects of microgravity on human NK cells during long space missions. Key Words: Simulated microgravity (SMG)—Natural killer (NK) cells—Cytotoxicity. Astrobiology 13, 703–714. PMID:23919749

  3. Features of Memory-Like and PD-1(+) Human NK Cell Subsets.

    PubMed

    Della Chiesa, Mariella; Pesce, Silvia; Muccio, Letizia; Carlomagno, Simona; Sivori, Simona; Moretta, Alessandro; Marcenaro, Emanuela

    2016-01-01

    Human NK cells are distinguished into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56(bright) NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56(dim) NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56(dim) cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A(-)KIR(+)LIR-1(+)CD57(+)CD56(dim) NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain "memory-like" NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The "memory-like" NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.

  4. Features of Memory-Like and PD-1(+) Human NK Cell Subsets.

    PubMed

    Della Chiesa, Mariella; Pesce, Silvia; Muccio, Letizia; Carlomagno, Simona; Sivori, Simona; Moretta, Alessandro; Marcenaro, Emanuela

    2016-01-01

    Human NK cells are distinguished into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56(bright) NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56(dim) NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56(dim) cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A(-)KIR(+)LIR-1(+)CD57(+)CD56(dim) NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain "memory-like" NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The "memory-like" NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction. PMID:27683578

  5. Features of Memory-Like and PD-1+ Human NK Cell Subsets

    PubMed Central

    Della Chiesa, Mariella; Pesce, Silvia; Muccio, Letizia; Carlomagno, Simona; Sivori, Simona; Moretta, Alessandro; Marcenaro, Emanuela

    2016-01-01

    Human NK cells are distinguished into CD56brightCD16− cells and CD56dimCD16+ cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56bright NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56dim NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56dim cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A−KIR+LIR-1+CD57+CD56dim NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain “memory-like” NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The “memory-like” NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction. PMID:27683578

  6. Features of Memory-Like and PD-1+ Human NK Cell Subsets

    PubMed Central

    Della Chiesa, Mariella; Pesce, Silvia; Muccio, Letizia; Carlomagno, Simona; Sivori, Simona; Moretta, Alessandro; Marcenaro, Emanuela

    2016-01-01

    Human NK cells are distinguished into CD56brightCD16− cells and CD56dimCD16+ cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56bright NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56dim NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56dim cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A−KIR+LIR-1+CD57+CD56dim NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain “memory-like” NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The “memory-like” NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.

  7. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M.; Nyström, Sofia; Hinkula, Jorma

    2015-01-01

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  8. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  9. Human NK cells maintain licensing status and are subject to killer immunoglobulin-like receptor (KIR) and KIR-ligand inhibition following ex vivo expansion.

    PubMed

    Wang, Wei; Erbe, Amy K; Alderson, Kory A; Phillips, Emily; Gallenberger, Mikayla; Gan, Jacek; Campana, Dario; Hank, Jacquelyn A; Sondel, Paul M

    2016-09-01

    Infusion of allogeneic NK cells is a potential immunotherapy for both hematopoietic malignancies and solid tumors. Interactions between killer immunoglobulin-like receptors (KIR) on human NK cells and KIR-ligands on tumor cells influence the magnitude of NK function. To obtain sufficient numbers of activated NK cells for infusion, one potent method uses cells from the K562 human erythroleukemia line that have been transfected to express activating 41BB ligand (41BBL) and membrane-bound interleukin 15 (mbIL15). The functional importance of KIRs on ex vivo expanded NK cells has not been studied in detail. We found that after a 12-day co-culture with K562-mbIL15-41BBL cells, expanded NK cells maintained inhibition specificity and prior in vivo licensing status determined by KIR/KIR-ligand interactions. Addition of an anti-CD20 antibody (rituximab) induced NK-mediated antibody-dependent cellular cytotoxicity and augmented killing of CD20+ target cells. However, partial inhibition induced by KIR/KIR-ligand interactions persisted. Finally, we found that extended co-cultures of NK cells with stimulatory cells transduced to express various KIR-ligands modified both the inhibitory and activating KIR repertoires of the expanded NK cell product. These studies demonstrate that the licensing interactions known to occur during NK ontogeny also influence NK cell function following NK expansion ex vivo with HLA-null stimulatory cells. PMID:27392940

  10. Targeting NK Cells for Anticancer Immunotherapy: Clinical and Preclinical Approaches

    PubMed Central

    Carotta, Sebastian

    2016-01-01

    The recent success of checkpoint blockade has highlighted the potential of immunotherapy approaches for cancer treatment. Although the majority of approved immunotherapy drugs target T cell subsets, it is appreciated that other components of the immune system have important roles in tumor immune surveillance as well and thus represent promising additional targets for immunotherapy. Natural killer (NK) cells are the body’s first line of defense against infected or transformed cells, as they kill target cells in an antigen-independent manner. Although several studies have clearly demonstrated the active role of NK cells in cancer immune surveillance, only few clinically approved therapies currently exist that harness their potential. Our increased understanding of NK cell biology over the past few years has renewed the interest in NK cell-based anticancer therapies, which has lead to a steady increase of NK cell-based clinical and preclinical trials. Here, the role of NK cells in cancer immune surveillance is summarized, and several novel approaches to enhance NK cell cytotoxicity against cancer are discussed. PMID:27148271

  11. Natural killer (NK) cell cytotoxicity in athymic (nude) rats.

    PubMed

    Grzelak, I; Olszewski, W L; Fossum, S; Engeset, A

    1984-01-01

    The in vitro and in vivo natural killer (NK) cell activity of congenitally athymic, nude (ATH) rats and of normal, euthymic (EUTH) rats was compared. We found: a) a higher level of in vitro NK cell activity in blood, spleen and lymph nodes of ATH rats compared with their heterozygous littermates, b) in the spleen the number of NK lytic units per organ was not higher in ATH compared with EUTH whereas it was significantly higher in lymph nodes, c) a lack of age-dependence of in vitro NK cell activity tested in culture with heat inactivated fetal calf serum, d) a higher rate of in vivo elimination of target tumor cells in 4-week ATH rats compared with EUTH rats, e) an age-dependent decrease in the rate of in vivo target cell elimination in both groups, and finally, f) an age-dependent increase in the inhibitory effect of autologous serum on NK cell activity in vitro in both groups. These findings show that the blood and lymphoid organs of athymic rats contain a substantially higher proportion of NK cells, active both in vitro and in vivo against K562 tumor cells, than their euthymic littermates. In the spleen this increased proportion can be attributed to the lack of T cells, whereas in the ATH rat lymph nodes there is an absolute increase in NK cell activity, and that the decrease of cytotoxicity in vivo with age reflects the increasing inhibitory properties of autologous serum both in nude and in normal rats.

  12. The Effects of Age and Latent Cytomegalovirus Infection on NK-Cell Phenotype and Exercise Responsiveness in Man.

    PubMed

    Bigley, Austin B; Spielmann, Guillaume; Agha, Nadia; Simpson, Richard J

    2015-01-01

    The redeployment of NK-cells in response to an acute bout of exercise is thought to be an integral component of the "fight-or-flight" response, preparing the body for potential injury or infection. We showed previously that CMV seropositivity impairs the redeployment of NK-cells with exercise in the young. In the current study, we examined the effect of aging on the redeployment of NK-cells with exercise in the context of CMV. We show here that CMV blunts the exercise-induced redeployment of NK-cells in both younger (23-39 yrs) and older (50-64 yrs) subjects with older CMV(neg) subjects showing the largest postexercise mobilization and 1 h postexercise egress of NK-cells. The blunted exercise response in CMV(pos) individuals was associated with a decreased relative redeployment of the CD158a+ and CD57+ NK-cell subsets in younger and older individuals. In addition, we show that aging is associated with a CMV-independent increase in the proportion of NK-cells expressing the terminal differentiation marker CD57, while CMV is associated with an age-dependent decrease in the proportion of NK-cells expressing the inhibitory receptors KLRG1 (in the younger group) and CD158a (in the older group). Collectively, these data suggest that CMV may decrease NK-cell mediated immunosurveillance after exercise in both younger and older individuals. PMID:26583066

  13. Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

    SciTech Connect

    Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.

    1986-06-01

    Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

  14. Human NK Cell Diversity in Viral Infection: Ramifications of Ramification

    PubMed Central

    Strauss-Albee, Dara M.; Blish, Catherine A.

    2016-01-01

    Natural killer (NK) cells are a unique lymphocyte lineage with remarkable agility in the rapid destruction of virus-infected cells. They are also the most poorly understood class of lymphocyte. A spectrum of activating and inhibitory receptors at the NK cell surface leads to an unusual and difficult-to-study mechanism of cellular recognition, as well as a very high capacity for diversity at the single-cell level. Here, we review the evidence for the role of NK cells in the earliest stage of human viral infection, and in its prevention. We argue that single-cell diversity is a logical evolutionary adaptation for their position in the immune response and contributes to their ability to kill virus-infected cells. Finally, we look to the future, where emerging single-cell technologies will enable a new generation of rigorous and clinically relevant studies on NK cells accounting for all of their unique and diverse characteristics. PMID:26973646

  15. DNAM-1 controls NK cell activation via an ITT-like motif

    PubMed Central

    Zhang, Zhanguang; Wu, Ning; Lu, Yan; Davidson, Dominique; Colonna, Marco

    2015-01-01

    DNAM-1 (CD226) is an activating receptor expressed on natural killer (NK) cells, CD8+ T cells, and other immune cells. Upon recognition of its ligands, CD155 and CD112, DNAM-1 promotes NK cell–mediated elimination of transformed and virus-infected cells. It also has a key role in expansion and maintenance of virus-specific memory NK cells. Herein, the mechanism by which DNAM-1 controls NK cell–mediated cytotoxicity and cytokine production was elucidated. Cytotoxicity and cytokine production triggered by DNAM-1 were mediated via a conserved tyrosine- and asparagine-based motif in the cytoplasmic domain of DNAM-1. Upon phosphorylation by Src kinases, this motif enabled binding of DNAM-1 to adaptor Grb2, leading to activation of enzymes Vav-1, phosphatidylinositol 3′ kinase, and phospholipase C-γ1. It also promoted activation of kinases Erk and Akt, and calcium fluxes. Although, as reported, DNAM-1 promoted adhesion, this function was signal-independent and insufficient to promote cytotoxicity. DNAM-1 signaling was also required to enhance cytotoxicity, by increasing actin polymerization and granule polarization. We propose that DNAM-1 promotes NK cell activation via an immunoreceptor tyrosine tail (ITT)–like motif coupling DNAM-1 to Grb2 and other downstream effectors. PMID:26552706

  16. IL-18, but not IL-15, contributes to the IL-12-dependent induction of NK-cell effector functions by Leishmania infantum in vivo.

    PubMed

    Haeberlein, Simone; Sebald, Heidi; Bogdan, Christian; Schleicher, Ulrike

    2010-06-01

    Activation of NK cells is a hallmark of infections with intracellular pathogens. We previously showed that the protozoan parasite Leishmania infantum triggered a rapid NK-cell response in mice that required TLR9-positive myeloid DC and IL-12, but no IFN-alpha/beta. Here, we investigated whether IL-15 or IL-18 mediate the activity of IL-12 or function as independent activators of NK cells. In contrast to earlier studies that described IL-15 as crucial for NK-cell priming in response to TLR ligands, the expression of IFN-gamma, FasL, perforin and granzyme B by NK cells in L. infantum-infected mice was completely preserved in the absence of IL-15, whereas the proliferative capacity of NK cells was lower than in WT mice. IFN-gamma secretion, cytotoxicity and FasL expression of NK cells from infected IL-18(-/-) mice were significantly reduced compared with controls, but, unlike IL-12, IL-18 was not essential for NK-cell effector functions. Part of the NK-cell-stimulatory effect of IL-12 was dependent on IL-18. We conclude that IL-15 is not functioning as a universal NK-cell priming signal and that IL-18 contributes to the NK-cell response in visceral leishmaniasis. The cytokine requirements for NK-cell activation appear to differ contingent upon the infectious pathogen.

  17. Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

    PubMed Central

    Quoc Trung, Ly; Espinoza, J. Luis; Takami, Akiyoshi; Nakao, Shinji

    2013-01-01

    Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling. PMID:23372833

  18. Cord Blood Mononuclear Cells Have a Potential to Produce NK Cells Using IL2Rg Cytokines

    PubMed Central

    Khaziri, Nahid; Mohammadi, Momeneh; Aliyari, Zeinab; Soleimani Rad, Jafar; Tayefi Nasrabadi, Hamid; Nozad Charoudeh, Hojjatollah

    2016-01-01

    Purpose: Although bone marrow represents the main site for NK cell development and also distinct thymic-dependentNK cell pathway was identified, the cytokines effect on the NK cell generation from cord blood is unclear. Studies were identified the role of cytokines in the regulation of bone marrow and thymic NK cells. Previous studies reported that IL15 are critical for bone marrow dependent and IL7 is important for thymic NK cells. It is remain unclear the cytokines influence on the expantion of NK cells in cord blood mononuclear cells. Methods: We evaluated cultured cord blood mononuclear cells suplememnted with combinations of cytokines using FACS in distinct time points. In this study, we presented the role of IL2, IL7 and IL15 as members of the common gamma receptor -chain (Il2rg) on the expansion NK cells from cord blood cells. Results: By investigating cord blood mononuclear cells in vitro , we demonstrated that IL2 and IL15 are important for expansion of NK cells. IL2 in comparision with IL15 has more influences in NK cell expansion. In contrast IL-7 is dispensable for NK cell generation in cord blood. Conclusion: Thus,IL-2Rg cytokines play complementary roles and are indispensable for homeostasis of NK cell development in cord blood. Probably these cytokines could help to use NK beneficials in engrafment of transplanted cells and Anti tumor activity of NK cells. PMID:27123412

  19. Sera from patients with colon, breast and lung cancer induce resistance to lysis mediated by NK cytotoxic factors (NKCF).

    PubMed Central

    Marubayashi, M.; Solana, R.; Ramirez, R.; Aranda, E.; Galan, F.; Peña, J.

    1991-01-01

    Natural killer (NK) cells are involved in the antitumoral immunologic mechanism. These cells act through the release of cytotoxic molecules defined as NK cytotoxic factors (NKCF). Inhibitory factors of NK and NKCF mediated lysis have been described in in vitro assays. This study evaluates the induction of resistance to NKCF cytotoxicity by sera from 27 patients with colon, breast and lung cancer. Addition of these sera to the cytolytic assay where K562 cells and concentrated NKCF were used, induced resistance to NKCF mediated cytotoxicity in 21 cases (77%). The sera from the group with metastasis blocked NKCF lysis more markedly than the group with local tumours. However, no differences were observed when the groups with colon, breast and lung cancers were compared. This blocking effect was not found to be related to gamma interferon (IFN) levels. In a previous study, we described a tumour factor (NK-RIF) produced by human cell lines derived from metastatic adenocarcinomas. This factor blocked lysis of tumour target cells by NK cells. Consequently, it is proposed that the release of similar tumour factors with a capacity to induce resistance to NKCF may be involved in tumour growth and metastatic spreading in in vivo. PMID:1906292

  20. Generation and preclinical characterization of an NKp80-Fc fusion protein for redirected cytolysis of natural killer (NK) cells against leukemia.

    PubMed

    Deng, Gang; Zheng, Xiaodong; Zhou, Jing; Wei, Haiming; Tian, Zhigang; Sun, Rui

    2015-09-11

    The capacity of natural killer (NK) cells to mediate Fc receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for the NK-activating receptor NKp80, is frequently highly expressed on leukemia cells, the lack of therapeutic AICL-specific antibodies limits clinical application. Here we explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-Fc fusion proteins. The NKp80-Fc fusion protein we generated bound specifically to leukemia cells in an AICL-specific manner. Cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced the ADCC effect of NK cells. NKp80-Fc not only promoted NK-mediated leukemia cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in the late stage. The bifunctional NKp80-Fc could redirect NK cells toward leukemia cells and triggered NK cell killing in vitro. Moreover, NKp80-Fc enhanced the lysis of NK cells against tumors in leukemia xenograft non-obese diabetic/severe combined immunodeficiency mice. Taken together, our results demonstrate that NKp80-Fc potently amplifies NK cell anti-leukemia effects in vitro and in vivo through induction of the NK cell ADCC effect. This method could potentially be useful for molecular targeted therapy, and the fusion proteins may be a promising drug for immunotherapy of leukemia.

  1. Natural killer cell dysfunction in hepatocellular carcinoma and NK cell-based immunotherapy.

    PubMed

    Sun, Cheng; Sun, Hao-yu; Xiao, Wei-hua; Zhang, Cai; Tian, Zhi-gang

    2015-10-01

    The mechanisms linking hepatitis B virus (HBV) and hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) remain largely unknown. Natural killer (NK) cells account for 25%-50% of the total number of liver lymphocytes, suggesting that NK cells play an important role in liver immunity. The number of NK cells in the blood and tumor tissues of HCC patients is positively correlated with their survival and prognosis. Furthermore, a group of NK cell-associated genes in HCC tissues is positively associated with the prolonged survival. These facts suggest that NK cells and HCC progression are strongly associated. In this review, we describe the abnormal NK cells and their functional impairment in patients with chronic HBV and HCV infection, which contribute to the progression of HCC. Then, we summarize the association of NK cells with HCC based on the abnormalities in the numbers and phenotypes of blood and liver NK cells in HCC patients. In particular, the exhaustion of NK cells that represents lower cytotoxicity and impaired cytokine production may serve as a predictor for the occurrence of HCC. Finally, we present the current achievements in NK cell immunotherapy conducted in mouse models of liver cancer and in clinical trials, highlighting how chemoimmunotherapy, NK cell transfer, gene therapy, cytokine therapy and mAb therapy improve NK cell function in HCC treatment. It is conceivable that NK cell-based anti-HCC therapeutic strategies alone or in combination with other therapies will be great promise for HCC treatment.

  2. NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

    PubMed Central

    2013-01-01

    Background The capacity of patient’s Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling’s NKs were compared with the patient’s drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient’s autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling’s NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient’s autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for

  3. NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention.

    PubMed

    Prinz, Petra U; Mendler, Anna N; Brech, Dorothee; Masouris, Ilias; Oberneder, Ralph; Noessner, Elfriede

    2014-10-15

    The relevance of NK cells in tumor control is well established in mouse models and human hematologic malignancies; however, their contribution to the control of human solid tumors remains disputed due to problems with in situ detection and reports of functional inactivity in the tumor milieu. In this study, we established a reliable in situ detection method for NK cells. Moreover, we performed analysis to elucidate mechanisms that impair NK-cell function in the tumor milieu and thereby identify therapeutic targets that allow recovery of NK-cell functionality. It was observed that NK cells from clear cell renal cell carcinoma (ccRCC), compared to NK cells from nontumor kidney and peripheral blood lymphocytes (PBLs), displayed conjoint phenotypic alterations and dysfunction induced by the tumor milieu, which were associated mechanistically with high levels of signaling attenuator diacylglycerol kinase (DGK)-α and blunted mitogen-activated protein kinase pathway activation (ERK1/2, Jun kinase). Reinstating NK-cell functionality was possible by DGK inhibition or brief IL-2 culture, interventions that de-repressed the ERK pathway. The extent of alteration and magnitude of recovery could be linked to NK-cell frequency within ccRCC-infiltrating lymphocytes, possibly explaining the observed survival benefit of patients with NK(high) tumors. In conclusion, DGK-mediated dampening of the ERK pathway ensuing in NK-cell dysfunction was identified as an important escape mechanism in ccRCC. DGK and the ERK pathway thus emerge as promising therapeutic targets to restore suppressed NK-cell activity for the improvement of antitumor immunity.

  4. TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway.

    PubMed

    Viel, Sébastien; Marçais, Antoine; Guimaraes, Fernando Souza-Fonseca; Loftus, Roisin; Rabilloud, Jessica; Grau, Morgan; Degouve, Sophie; Djebali, Sophia; Sanlaville, Amélien; Charrier, Emily; Bienvenu, Jacques; Marie, Julien C; Caux, Christophe; Marvel, Jacqueline; Town, Liam; Huntington, Nicholas D; Bartholin, Laurent; Finlay, David; Smyth, Mark J; Walzer, Thierry

    2016-02-16

    Transforming growth factor-β (TGF-β) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-β signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-β in vitro blocked interleukin-15 (IL-15)-induced activation of mTOR. TGF-β and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-β signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-β receptor subunit TGF-βRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-β signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression.

  5. NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin.

    PubMed

    Esser, Ruth; Müller, Tina; Stefes, Dörthe; Kloess, Stephan; Seidel, Diana; Gillies, Stephen D; Aperlo-Iffland, Christel; Huston, James S; Uherek, Christoph; Schönfeld, Kurt; Tonn, Torsten; Huebener, Nicole; Lode, Holger N; Koehl, Ulrike; Wels, Winfried S

    2012-03-01

    Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.

  6. NK cells engineered to express a GD2-specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

    PubMed Central

    Esser, Ruth; Müller, Tina; Stefes, Dörthe; Kloess, Stephan; Seidel, Diana; Gillies, Stephen D; Aperlo-Iffland, Christel; Huston, James S; Uherek, Christoph; Schönfeld, Kurt; Tonn, Torsten; Huebener, Nicole; Lode, Holger N; Koehl, Ulrike; Wels, Winfried S

    2012-01-01

    Abstract Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD2, which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD2-specific chimeric antigen receptor (CAR) comprising an anti-GD2 ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD2 expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD2-specific antibody or anti-idiotypic antibody occupying the CAR’s cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD2-specific NK cells was also found against primary NB cells and GD2 expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells. PMID:21595822

  7. Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression

    PubMed Central

    Bellucci, Roberto; Martin, Allison; Bommarito, Davide; Wang, Kathy; Hansen, Steen H; Freeman, Gordon J; Ritz, Jerome

    2015-01-01

    Inhibition of JAK1 or JAK2 in human tumor cells was previously shown to increase susceptibility of these cells to NK cell lysis. In the present study, we examined the cellular mechanisms that mediate this effect in hematopoietic tumor cell lines and primary tumor cells. Incubation of tumor cells with supernatant from activated NK cells or interferon-gamma (IFNγ)-induced activation of pSTAT1 and increased expression of PD-L1 without altering expression of other activating or inhibitory NK cell ligands. These functional effects were blocked by chemical JAK inhibition or shRNAs targeting JAK1, JAK2 or STAT1. Inhibition of IFNγ signaling also prevented the upregulation of PD-L1 and blocking PD-L1 resulted in increased tumor lysis by NK cells. These results show that NK cell activation and secretion of IFNγ results in activation of JAK1, JAK2 and STAT1 in tumor cells, resulting in rapid up-regulation of PD-L1 expression. Increased expression of PD-L1 results in increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents increased PD-L1 expression resulting in increased susceptibility of tumor cells to NK cell activity. These observations suggest that JAK pathway inhibitors as well as PD-1 and PD-L1 antibodies may work synergistically with other immune therapies by preventing IFN-induced inhibition of NK cell-mediated tumor cell lysis. PMID:26155422

  8. Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection

    PubMed Central

    Guthrie, Brandon S.; Schmidt, Rebecca L.; Jamieson, Amanda; Merkel, Patricia; Knight, Vijaya; Cole, Caroline M.; Raulet, David H.; Lenz, Laurel L.

    2016-01-01

    Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility. PMID:27295349

  9. Functional Assessment of NK and LAK Cells Following Space Flight

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Space flight associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. PBMCs were collected from 10-ml blood specimens from 5 astronauts 10 days before launch, immediately after landing, and again at 3 days after landing and stored at -80 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4 hour Cr-51 release assay. K562 cells were used to assess NK cell cytotoxicity. Following 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 cells and Daudi cells as the target cells. NK cell cytotoxicity decreased at landing (p<.05) in 3/5 astronauts, and recovered to preflight levels by 3 days following landing; NK cell cytotoxicity was increased (p=0.1) in the remaining 2 astronauts at landing. In 4/5 astronauts, LAK cytotoxic activity was decreased at landing against K562 cells (p = 0.13) and Daudi cells (p = 0.08). Phenotyping of PBMC's and LAK cells showed alterations in some surface markers and adhesion molecules (CD11b, CD11c, CD11a, CD16, L-selectin, and CD3).

  10. Beyond NK cells: the expanding universe of innate lymphoid cells.

    PubMed

    Cella, Marina; Miller, Hannah; Song, Christina

    2014-01-01

    For a long time, natural killer (NK) cells were thought to be the only innate immune lymphoid population capable of responding to invading pathogens under the influence of changing environmental cues. In the last few years, an increasing amount of evidence has shown that a number of different innate lymphoid cell (ILC) populations found at mucosal sites rapidly respond to locally produced cytokines in order to establish or maintain homeostasis. These ILC populations closely mirror the phenotype of adaptive T helper subsets in their repertoire of secreted soluble factors. Early in the immune response, ILCs are responsible for setting the stage to mount an adaptive T cell response that is appropriate for the incoming insult. Here, we review the diversity of ILC subsets and discuss similarities and differences between ILCs and NK cells in function and key transcriptional factors required for their development.

  11. Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population.

    PubMed

    Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W

    2016-07-01

    Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.

  12. Selection and expansion of natural killer cells for NK cell-based immunotherapy.

    PubMed

    Becker, Petra S A; Suck, Garnet; Nowakowska, Paulina; Ullrich, Evelyn; Seifried, Erhard; Bader, Peter; Tonn, Torsten; Seidl, Christian

    2016-04-01

    Natural killer (NK) cells have been used in several clinical trials as adaptive immunotherapy. The low numbers of these cells in peripheral blood mononuclear cells (PBMC) have resulted in various approaches to preferentially expand primary NK cells from PBMC. While some clinical trials have used the addition of interleukin 2 (IL-2) to co-stimulate the expansion of purified NK cells from allogeneic donors, recent studies have shown promising results in achieving in vitro expansion of NK cells to large numbers for adoptive immunotherapy. NK cell expansion requires multiple cell signals for survival, proliferation and activation. Thus, expansion strategies have been focused either to substitute these factors using autologous feeder cells or to use genetically modified allogeneic feeder cells. Recent developments in the clinical use of genetically modified NK cell lines with chimeric antigen receptors, the development of expansion protocols for the clinical use of NK cell from human embryonic stem cells and induced pluripotent stem cells are challenging improvements for NK cell-based immunotherapy. Transfer of several of these protocols to clinical-grade production of NK cells necessitates adaptation of good manufacturing practice conditions, and the development of freezing conditions to establish NK cell stocks will require some effort and, however, should enhance the therapeutic options of NK cells in clinical medicine.

  13. The genetic and evolutionary balances in human NK cell receptor diversity.

    PubMed

    Parham, Peter

    2008-12-01

    In primates and cattle two ancient killer-cell immunoglobulin-like receptor (KIR) lineages independently evolved to become diverse NK cell receptors. In mice, KIR genes were sidelined to the X chromosome, a possible consequence of pathogen-mediated selection on the receptor for IgA-Fc. In humans, KIR uniquely form two omnipresent haplotype groups (A and B), postulated here to play complementary and necessary roles in immune defense and reproduction. The basis of KIR3DL1/S1 polymorphism is three ancient lineages maintained by long-term balancing selection and present in all human populations. Conserved and variable NK cell receptors produce structurally diverse NK cell receptor repertoires within a defined range of missing-self-response.

  14. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation.

    PubMed

    Shah, Nirali N; Baird, Kristin; Delbrook, Cynthia P; Fleisher, Thomas A; Kohler, Mark E; Rampertaap, Shakuntala; Lemberg, Kimberly; Hurley, Carolyn K; Kleiner, David E; Merchant, Melinda S; Pittaluga, Stefania; Sabatino, Marianna; Stroncek, David F; Wayne, Alan S; Zhang, Hua; Fry, Terry J; Mackall, Crystal L

    2015-01-29

    Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104. PMID:25452614

  15. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation.

    PubMed

    Shah, Nirali N; Baird, Kristin; Delbrook, Cynthia P; Fleisher, Thomas A; Kohler, Mark E; Rampertaap, Shakuntala; Lemberg, Kimberly; Hurley, Carolyn K; Kleiner, David E; Merchant, Melinda S; Pittaluga, Stefania; Sabatino, Marianna; Stroncek, David F; Wayne, Alan S; Zhang, Hua; Fry, Terry J; Mackall, Crystal L

    2015-01-29

    Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

  16. A novel bispecific protein (ULBP2-BB4) targeting the NKG2D receptor on natural killer (NK) cells and CD138 activates NK cells and has potent antitumor activity against human multiple myeloma in vitro and in vivo.

    PubMed

    von Strandmann, Elke Pogge; Hansen, Hinrich P; Reiners, Katrin S; Schnell, Roland; Borchmann, Peter; Merkert, Sabine; Simhadri, Venkateswara R; Draube, Andreas; Reiser, Marcel; Purr, Ingvill; Hallek, Michael; Engert, Andreas

    2006-03-01

    The inability of the immune system to recognize and kill malignant plasma cells in patients with multiple myeloma (MM) has been attributed in part to the ineffective activation of natural killer (NK) cells. In order to activate and target NK cells to the malignant cells in MM we designed a novel recombinant bispecific protein (ULBP2-BB4). While ULBP2 binds the activating NK receptor NKG2D, the BB4 moiety binds to CD138, which is overexpressed on a variety of malignancies, including MM. ULBP2-BB4 strongly activated primary NK cells as demonstrated by a significant increase in interferon-gamma (IFN-gamma) secretion. In vitro, ULBP2-BB4 enhanced the NK-mediated lysis of 2 CD138+ human MM cell lines, U-266 and RPMI-8226, and of primary malignant plasma cells in the allogenic and autologous setting. Moreover, in a nude mouse model with subcutaneously growing RPMI-8226 cells, the cotherapy with ULBP-BB4 and human peripheral blood lymphocytes abrogated the tumor growth. These data suggest potential clinical use of this novel construct in patients with MM. The use of recombinant NK receptor ligands that target NK cells to tumor cells might offer new approaches for other malignancies provided a tumor antigen-specific antibody is available.

  17. Using NK Cell Lipid Raft Fractionation to Understand the Role of Lipid Rafts in NK Cell Receptor Signaling.

    PubMed

    Serrano-Pertierra, Esther; López-Larrea, Carlos

    2016-01-01

    Lipid rafts were first defined as detergent-resistant membranes (DRMs) due to their relative insolubility in non-ionic detergents. Although they should not be confused with lipid rafts, DRMs are a valuable starting point for the study of these membrane domains and the interactions of proteins with rafts.Here we describe the isolation of DRMs by ultracentrifugation on a sucrose gradient, a method we have used to study the role of lipid rafts in NKG2D-mediated signaling. We also describe raft fractionation of NK cells involving the selective solubility of β-octylglucoside (β-OG). OG is a non-ionic detergent that efficiently dissolves DRMs but does not disrupt protein associations with the cytoskeleton. Using these two techniques may yield useful information about the proteins involved in receptor recruitment into lipid rafts and the interactions of the actin cytoskeleton with lipid rafts.

  18. The effect of pregnancy on the uterine NK cell KIR repertoire

    PubMed Central

    Male, Victoria; Sharkey, Andrew; Masters, Leanne; Kennedy, Philippa R; Farrell, Lydia E; Moffett, Ashley

    2011-01-01

    The major leukocyte population in the decidua during the first trimester of pregnancy consists of NK cells that express receptors capable of recognizing MHC class I molecules expressed by placental trophoblast. These include members of the killer immunoglobulin-like receptor (KIR) family, the two-domain KIR (KIR2D), which recognize HLA-C. Interactions between decidual NK (dNK) cell KIR2D and placental HLA-C contribute to the success of pregnancy and dNK cells express KIR2D at higher frequency than peripheral NK (pNK) cells. Thus, they are biased toward recognizing HLA-C. In order to investigate when this unusual KIR repertoire appears, we compared the phenotype of NK cells isolated from non-pregnant (endometrium) and pregnant (decidua) human uterine mucosa. Endometrial NK (eNK) cells did not express KIR2D at a higher level than matched pNK cells, so the bias toward HLA-C recognition occurs as a response to pregnancy. Furthermore, HLA-C expression was upregulated on uterine stromal cells as the mucosa transformed from endometrium to decidua at the onset of pregnancy. As uterine NK (uNK) cells can mature from NK precursors and acquire KIR expression in utero, the pregnancy-specific bias of uNK cells toward HLA-C recognition could arise as developing uNK cells interact with uterine stromal cells, which express higher levels of HLA-C during pregnancy. PMID:21739430

  19. Mouse NK cell–mediated rejection of bone marrow allografts exhibits patterns consistent with Ly49 subset licensing

    PubMed Central

    Sun, Kai; Alvarez, Maite; Ames, Erik; Barao, Isabel; Chen, Mingyi; Longo, Dan L.; Redelman, Doug

    2012-01-01

    Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49+ subsets in BMC rejection by using β2-microglobulin deficient (β2m−/−) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind “self-MHC” were found to be the dominant subset responsible for β2m−/− BMC rejection. This provides in vivo evidence for host MHC class I–dependent functional education. Interestingly, all H2d strain mice regardless of background were able to resist significantly greater amounts of β2m−/−, but not wild-type BMC than H2b mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability. PMID:22184406

  20. Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

    PubMed

    El Omar, Reine; Xiong, Yu; Dostert, Gabriel; Louis, Huguette; Gentils, Monique; Menu, Patrick; Stoltz, Jean-François; Velot, Émilie; Decot, Véronique

    2016-04-01

    Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1β, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive. PMID:26510892

  1. NK cells interfere with the generation of resistance against mycoplasma respiratory infection following nasal-pulmonary immunization1

    PubMed Central

    Bodhankar, Sheetal; Woolard, Mathew D.; Sun, Xiangle; Simecka, Jerry W.

    2009-01-01

    The purpose of the present study was to determine the impact of NK cells on the development of protective adaptive immunity in response to nasal-pulmonary immunization against mycoplasma. Depletion of NK cells prior to nasal-pulmonary immunization enhanced resistance to mycoplasma respiratory infection. The effect of NK cells on the generation of protective immunity in lungs was dependent on lymphoid cells, as immunization of either SCID mice or immunocompetent mice depleted of CD4+ T cells did not demonstrate any increased resistance in the presence or absence of NK cells. The presence of NK cells at the time of nasal-pulmonary immunization modulated mycoplasma-specific cytokine responses in lungs and lower respiratory nodes. In particular, NK cells skewed the mycoplasma-specific T cell cytokine responses in the draining lymph nodes to higher IL-4, IL-13 and IL-17 while lowering IFN-γ responses. Adoptive transfer of total lung lymphocytes isolated from immunized mice into naïve mice led to a significant reduction in the mycoplasma numbers in lungs, and the resistance was greater if cells were obtained from immunized mice which were depleted of NK cells. Similar results were obtained if purified B cells, T cells or CD4+ T cells were used. Interestingly, this is the first time that a favorable role of functional CD4+ T cells in mediating protection in mycoplasma respiratory disease was demonstrated. Thus, NK cells can influence the responses of multiple lymphocyte populations capable of mediating resistance to mycoplasma infection. PMID:19625649

  2. 17beta-estradiol suppresses cytotoxicity and proliferative capacity of murine splenic NK1.1+ cells.

    PubMed

    Hao, Sha; Li, Pengfei; Zhao, Junli; Hu, Yali; Hou, Yayi

    2008-10-01

    In order to clarify the effects of 17beta-estradiol (E2) on natural killer (NK) cells and the possibly regulatory mechanisms, we obtained highly purified and viable NK cells from C57BL/6J mouse spleen by a magnetic cell sorter (MACS). These cells were treated with E2 and then their cytotoxicity and proliferative capacity were examined. To further investigate the mechanisms on the effect of E2 on NK cells, expressions of activation-associated markers (CD69, CD122) and inhibitory receptors (CD94, Ly49), and intracellular cytokine production were analyzed. At last, we performed the cDNA microarray to explore the possible involved genes. We found that E2 could suppress NK cell cytotoxicity and proliferative capacity in vitro. E2 reduced NK cell cytotoxicity and proliferative capacity, which may be through influencing the phenotypes and cytokine expression of NK cells, mainly involving CD94 and IFN-gamma. Furthermore, regulation of Stat4, Fyn, Sh2d1a, Eat2, Cd244, Irf1, Runx1, Irf7, Irf5, Esrra and Nr5a1 genes may be related to the cytotoxicity, proliferation and cytokine production of E2-mediated purified NK cells.

  3. Extranodal NK/T Cell Lymphoma Causing Cardiorespiratory Failure

    PubMed Central

    2016-01-01

    Extranodal NK/T cell lymphoma is an uncommon malignancy usually involving the sinonasal area. We report an unusual case of extranodal NK/T cell lymphoma diagnosed in a 62-year-old Caucasian male who died of progressive cardiorespiratory failure but had no clinically detectable upper respiratory system lesions. The initial diagnosis was made cytologically on a sample of pericardial fluid that contained neoplastic lymphoid cells. These cells were positive for CD2, cytoplasmic CD3, and Epstein-Barr virus and negative for CD56. The diagnosis was confirmed at the autopsy, which disclosed lymphoma infiltrates in the myocardium, lungs, stomach, and pancreas. The death was caused by heart and lung failure due to uncontrollable arrhythmia and respiratory insufficiency due to the lymphoma infiltrates. To the best of our knowledge, this is the first case of extranodal NK/T cell lymphoma presenting with cardiopulmonary failure. PMID:27493813

  4. Extranodal NK/T Cell Lymphoma Causing Cardiorespiratory Failure.

    PubMed

    Li, Yiting; Damjanov, Ivan

    2016-01-01

    Extranodal NK/T cell lymphoma is an uncommon malignancy usually involving the sinonasal area. We report an unusual case of extranodal NK/T cell lymphoma diagnosed in a 62-year-old Caucasian male who died of progressive cardiorespiratory failure but had no clinically detectable upper respiratory system lesions. The initial diagnosis was made cytologically on a sample of pericardial fluid that contained neoplastic lymphoid cells. These cells were positive for CD2, cytoplasmic CD3, and Epstein-Barr virus and negative for CD56. The diagnosis was confirmed at the autopsy, which disclosed lymphoma infiltrates in the myocardium, lungs, stomach, and pancreas. The death was caused by heart and lung failure due to uncontrollable arrhythmia and respiratory insufficiency due to the lymphoma infiltrates. To the best of our knowledge, this is the first case of extranodal NK/T cell lymphoma presenting with cardiopulmonary failure. PMID:27493813

  5. Target-induced natural killer cell loss as a measure of NK cell responses.

    PubMed

    Warren, Hilary

    2013-01-01

    Natural killer (NK) cells are an important effector cell of innate immunity. Their interaction with susceptible target cells triggers NK cell cytotoxicity and the release of cytokines. Immunofluorescence flow cytometry-based assays are now the preferred methods for measuring NK cell responses. For these assays, assessment is made on NK cells (CD3(-)CD56(+) CD16(+)) within the viable lymphocyte gate, defined by the parameters of size (FSC) and granularity (SSC). Accordingly, NK cells that have not dissociated from target cells at the time of measurement, or that have undergone target cell-induced apoptosis, are excluded from the viable lymphocyte gate and therefore from analysis. This unit describes a protocol for assessing NK cell function in response to various target cells (natural killing, antibody-dependent cell cytotoxicity, and NK cell alloreactivity) based on the loss of NK cells from the lymphocyte gate. This target-induced NK loss (TINKL) should provide a sensitive measure of NK cell responses in a clinical laboratory setting.

  6. Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation

    PubMed Central

    Pedroza-Pacheco, Isabela; Shah, Divya; Domogala, Anna; Luevano, Martha; Blundell, Michael; Jackson, Nicola; Thrasher, Adrian; Madrigal, Alejandro; Saudemont, Aurore

    2016-01-01

    Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag-/- γc-/- mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions. PMID:26915707

  7. Candida albicans Induces Metabolic Reprogramming in Human NK Cells and Responds to Perforin with a Zinc Depletion Response

    PubMed Central

    Hellwig, Daniela; Voigt, Jessica; Bouzani, Maria; Löffler, Jürgen; Albrecht-Eckardt, Daniela; Weber, Michael; Brunke, Sascha; Martin, Ronny; Kurzai, Oliver; Hünniger, Kerstin

    2016-01-01

    As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin. PMID:27242763

  8. Increased frequency and function of KIR2DL1-3+ NK cells in primary HIV-1 infection are determined by HLA-C group haplotypes

    PubMed Central

    Körner, Christian; Granoff, Mitchell E.; Amero, Molly A.; Sirignano, Michael N.; Vaidya, Sagar A.; Jost, Stephanie; Allen, Todd M.; Rosenberg, Eric S.; Altfeld, Marcus

    2014-01-01

    Acquisition and maintenance of NK cell function is mediated by inhibitory killer-cell immunoglobulin-like receptors (KIR) through the interaction with HLA class I molecules. Recently, HLA-C expression levels were shown to be correlated with protection against multiple outcomes of HIV-1 infection; however the underlying mechanisms are poorly understood. As HLA-C is the natural ligand for the inhibitory receptors KIR2DL1 and KIR2DL2/3, we sought to determine whether HLA-C group haplotypes affect NK cell responses during primary HIV-1 infection. The phenotypes and functional capacity of NK cells derived from HIV-1(+) and HIV-1(-) individuals were assessed (N=42 and N=40, respectively). HIV-1 infection was associated with an increased frequency of KIR2DL1-3+ NK cells. Further analysis showed that KIR2DL1+ NK cells were selectively increased in individuals homozygous for HLA-C2, while HLA-C1-homozygous individuals displayed increased proportions of KIR2DL2/3+ NK cells. KIR2DL1-3+ NK cells were furthermore more polyfunctional during primary HIV-1 infection in individuals also encoding for their cognate HLA-C group haplotypes as measured by degranulation and cytokine production. These results identify a novel relationship between HLA-C and KIR2DL+ NK cell subsets and demonstrate that HLA-C-mediated licensing modulates NK cell responses to primary HIV-1 infection. PMID:25043727

  9. Advances in clinical NK cell studies: Donor selection, manufacturing and quality control

    PubMed Central

    Koehl, U.; Kalberer, C.; Spanholtz, J.; Lee, D. A.; Miller, J. S.; Cooley, S.; Lowdell, M.; Uharek, L.; Klingemann, H.; Curti, A.; Leung, W.; Alici, E.

    2016-01-01

    ABSTRACT Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013–2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies. PMID:27141397

  10. Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma.

    PubMed

    Kamath, Arun T; Sheasby, Christopher E; Tough, David F

    2005-01-15

    Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-alphabeta, secreted by dendritic cells (DCs), and IFN-gamma, secreted by NK cells; notably, IFN-gamma-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-gamma by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-gamma, maximal IFN-gamma production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-alphabeta, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-gamma, whereas IFN-alphabeta was shown to be important for induction of T cell bystander activation and NK cell IFN-gamma production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells. PMID:15634897

  11. The TGF-β/SMAD pathway is an important mechanism for NK cell immune evasion in childhood B acute lymphoblastic leukemia

    PubMed Central

    Rouce, Rayne H.; Shaim, Hila; Sekine, Takuya; Weber, Gerrit; Ballard, Brandon; Ku, Stephanie; Barese, Cecilia; Murali, Vineeth; Wu, Meng-Fen; Liu, Hao; Shpall, Elizabeth J.; Bollard, Catherine M.; Rabin, Karen R.; Rezvani, Katayoun

    2015-01-01

    Natural killer (NK) cells are key components of the innate immune system, providing potent antitumor immunity. Here, we show that the TGF-β/SMAD signaling pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia (ALL). We characterized NK cells in 50 consecutive children with B-ALL at diagnosis, end-Induction, and during maintenance therapy compared to age-matched controls. ALL-NK cells at diagnosis had an inhibitory phenotype associated with impaired function, most notably IFN-γ production and cytotoxicity. By maintenance, these phenotypic and functional abnormalities partially normalized, however, cytotoxicity against autologous blasts remained impaired. We identified ALL-derived TGF-β1 to be an important mediator of leukemia-induced NK cell dysfunction. The TGF-β/SMAD signaling pathway was constitutively activated in ALL-NK cells at diagnosis and end-induction when compared to healthy controls and patients during maintenance. Culture of ALL blasts with healthy NK cells induced NK dysfunction and an inhibitory phenotype, mediated by activation of the TGF-β/SMAD signaling pathway, and abrogated by blocking TGF-β. These data indicate that by regulating the TGF-β/SMAD pathway, ALL blasts induce changes in NK cells to evade innate immune surveillance, thus highlighting the importance of developing novel therapies to target this inhibitory pathway and restore antileukemic cytotoxicity. PMID:26621337

  12. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

    PubMed

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W; Olofsson, Per E; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  13. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

    PubMed

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W; Olofsson, Per E; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  14. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    PubMed Central

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  15. Absence of circulating natural killer (NK) cells in a child with erythrophagocytic lymphohistiocytosis lacking NK cell activity

    SciTech Connect

    Kawai, H.; Komiyama, A.; Aoyama, K.; Miyagawa, Y.; Akabane, T.

    1988-06-01

    A 5-year-old girl who was diagnosed as having erythrophagocytic lymphohistiocytosis died at age 9 years. Peripheral lymphocytes from the patient persistently lacked natural killer (NK) cell activity during the 4-year observation period: the percent lysis values as measured by a 4-hr /sup 51/Cr release assay at a 40:1 effector:target ratio were below 1.0% against K562 and Molt-4 cells as compared with the normal lymphocyte value (mean +/- SD) of 46.2% +/- 5.8% and 43.9% +/- 6.7%, respectively. The patient's lymphocytes never developed NK cell activity by their incubation with target cells for longer time periods or by their stimulation with interferon-alpha, interleukin-2, or polyinosinic-polycytidilic acid. Single cell-in-agarose assay showed the absence of target-binding cells (TBCs): TBC numbers were below 0.3% as compared with the normal lymphocyte value of 8.1% +/- 1.3% (mean +/- SD). Flow cytometry showed a marked decrease in Leu-7+ cells (1.7%) and the absence of Leu-11+ cells (0.4%) in the peripheral blood. These results first demonstrate a case of erythrophagocytic lymphohistiocytosis in which there is the lack of NK cell activity due to the absence of circulating NK cells.

  16. Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice

    PubMed Central

    Pernicone, Elizabeth; Korkes, Henri A.; Burke, Suzanne D.; Rajakumar, Augustine; Thadhani, Ravi I.; Roberts, Drucilla J.; Bhasin, Manoj; Karumanchi, S. Ananth

    2016-01-01

    Decidual NK (dNK) cells, a distinct type of NK cell, are thought to regulate uterine spiral artery remodeling, a process that allows for increased blood delivery to the fetal-placental unit. Impairment of uterine spiral artery remodeling is associated with decreased placental perfusion, increased uterine artery resistance, and obstetric complications such as preeclampsia and intrauterine growth restriction. Ex vivo manipulation of human peripheral blood NK (pNK) cells by a combination of hypoxia, TGFß-1 and 5-aza-2’-deoxycytidine yields cells with phenotypic and in vitro functional similarities to dNK cells, called idNK cells. Here, gene expression profiling shows that CD56Bright idNK cells derived ex vivo from human pNK cells, and to a lesser extent CD56Dim idNK cells, are enriched in the gene expression signature that distinguishes dNK cells from pNK cells. When injected into immunocompromised pregnant mice with elevated uterine artery resistance, idNK cells homed to the uterus and reduced the uterine artery resistance index, suggesting improved placental perfusion. PMID:27736914

  17. Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell–depleted stem cell transplantation

    PubMed Central

    Shah, Nirali N.; Baird, Kristin; Delbrook, Cynthia P.; Fleisher, Thomas A.; Kohler, Mark E.; Rampertaap, Shakuntala; Lemberg, Kimberly; Hurley, Carolyn K.; Kleiner, David E.; Merchant, Melinda S.; Pittaluga, Stefania; Sabatino, Marianna; Stroncek, David F.; Wayne, Alan S.; Zhang, Hua; Fry, Terry J.

    2015-01-01

    Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL–activated NK cells (aNK-DLI) following HLA-matched, T-cell–depleted (1-2 × 104 T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3+-depleted, CD56+-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL+IL-15Rα+ artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104. PMID:25452614

  18. NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype

    PubMed Central

    Ames, Erik; Canter, Robert J.; Grossenbacher, Steven K.; Mac, Stephanie; Chen, Mingyi; Smith, Rachel C.; Hagino, Takeshi; Perez-Cunningham, Jessica; Sckisel, Gail D.; Urayama, Shiro; Monjazeb, Arta M.; Fragoso, Ruben C.; Sayers, Thomas J.; Murphy, William J.

    2016-01-01

    Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24+/CD44+, CD133+, and aldehyde dehydrogenasebright) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies. PMID:26363055

  19. NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype.

    PubMed

    Ames, Erik; Canter, Robert J; Grossenbacher, Steven K; Mac, Stephanie; Chen, Mingyi; Smith, Rachel C; Hagino, Takeshi; Perez-Cunningham, Jessica; Sckisel, Gail D; Urayama, Shiro; Monjazeb, Arta M; Fragoso, Ruben C; Sayers, Thomas J; Murphy, William J

    2015-10-15

    Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

  20. Maternal obesity drives functional alterations in uterine NK cells

    PubMed Central

    Perdu, Sofie; Castellana, Barbara; Kim, Yoona; Chan, Kathy; DeLuca, Lauren; Beristain, Alexander G.

    2016-01-01

    Over one-fifth of North American women of childbearing age are obese, putting these women at risk for a variety of detrimental chronic diseases. In addition, obesity increases the risk for developing major complications during pregnancy. The mechanisms by which obesity contributes to pregnancy complications and loss remain unknown. Increasing evidence indicates that obesity results in major changes to adipose tissue immune cell composition and function; whether or not obesity also affects immune function in the uterus has not been explored. Here we investigated the effect of obesity on uterine natural killer (uNK) cells, which are essential for uterine artery remodeling and placental development. Using a cohort of obese or lean women, we found that obesity led to a significant reduction in uNK cell numbers accompanied with impaired uterine artery remodeling. uNK cells isolated from obese women had altered expression of genes and pathways associated with extracellular matrix remodeling and growth factor signaling. Specifically, uNK cells were hyper-responsive to PDGF, resulting in overexpression of decorin. Functionally, decorin strongly inhibited placental development by limiting trophoblast survival. Together, these findings establish a potentially new link between obesity and poor pregnancy outcomes, and indicate that obesity-driven changes to uterine-resident immune cells critically impair placental development. PMID:27699222

  1. Maternal obesity drives functional alterations in uterine NK cells

    PubMed Central

    Perdu, Sofie; Castellana, Barbara; Kim, Yoona; Chan, Kathy; DeLuca, Lauren; Beristain, Alexander G.

    2016-01-01

    Over one-fifth of North American women of childbearing age are obese, putting these women at risk for a variety of detrimental chronic diseases. In addition, obesity increases the risk for developing major complications during pregnancy. The mechanisms by which obesity contributes to pregnancy complications and loss remain unknown. Increasing evidence indicates that obesity results in major changes to adipose tissue immune cell composition and function; whether or not obesity also affects immune function in the uterus has not been explored. Here we investigated the effect of obesity on uterine natural killer (uNK) cells, which are essential for uterine artery remodeling and placental development. Using a cohort of obese or lean women, we found that obesity led to a significant reduction in uNK cell numbers accompanied with impaired uterine artery remodeling. uNK cells isolated from obese women had altered expression of genes and pathways associated with extracellular matrix remodeling and growth factor signaling. Specifically, uNK cells were hyper-responsive to PDGF, resulting in overexpression of decorin. Functionally, decorin strongly inhibited placental development by limiting trophoblast survival. Together, these findings establish a potentially new link between obesity and poor pregnancy outcomes, and indicate that obesity-driven changes to uterine-resident immune cells critically impair placental development.

  2. Tumors skew endothelial cells to disrupt NK cell, T-cell and macrophage functions

    PubMed Central

    Mulligan, Jennifer K.; Lathers, Deanne M. R.

    2012-01-01

    Introduction Patients and mice with solid tumors, such as Lewis lung carcinoma (LLC), have defects in functions of immune effector cells. Endothelial cells, a component of the tumor vasculature, are potential regulators of immune cell functions. Therefore, these studies examined the impact of exposure to LLC tumor on the ability of endothelial cells to modulate immune cell functions. Materials and methods Endothelial cells were pre-treated with LLC tumor-conditioned medium (EndoT-sup) for 24 h. Control endothelial cells that were exposed to medium (EndoMedia) or epithelial cell-conditioned medium (EndoEpi-sup). After the initial 24 h incubation, endothelial cells were washed and fresh media was added. Cells were allowed to incubate for an additional 24 h. Supernatants from EndoMedia, EndoEpi-sup or EndoT-sup were collected and assayed for immune modulatory products and for immune modulatory activity. Results Supernatant from EndoT-sup contained increased levels of PGE2, IL-6 and VEGF as compared to EndoMedia and EndoEpi-sup controls. NK cell activity, as measured by TNF-α and IFN-γ secretion, was increased following exposure to media conditioned by EndoMedia and EndoEpi-sup. Exposure of NK cells to supernatants of EndoT-sup, also increases TNF-α and IFN-γ secretion, but to a lesser extent than by EndoMedia and EndoEpi-sup. Examination of macrophage functions demonstrated that supernatant from EndoT-sup decreased microbead phagocytosis and increased production of the immune suppressive mediators, IL-10 and PGE2. Lastly, T-cell responses to stimulation with anti-CD3 in the presence of supernatants from EndoT-sup were examined. IFN-γ production by CD8+ T-cells was reduced after exposure to EndoT-sup-conditioned medium, as compared to cells treatments with medium or control conditioned medium. Production of IFN-γ by CD4+ T-cells exposed to EndoT-sup was not altered. Conclusions Taken together, these studies demonstrate that tumors skew endothelial cells to

  3. Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

    PubMed Central

    Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

    2012-01-01

    Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

  4. NK cells activated by Interleukin-4 in cooperation with Interleukin-15 exhibit distinctive characteristics.

    PubMed

    Kiniwa, Tsuyoshi; Enomoto, Yutaka; Terazawa, Natsumi; Omi, Ai; Miyata, Naoko; Ishiwata, Kenji; Miyajima, Atsushi

    2016-09-01

    Natural killer (NK) cells are known to be activated by Th1-type cytokines, such as IL-2, -12, or -18, and they secrete a large amount of IFN-γ that accelerates Th1-type responses. However, the roles of NK cells in Th2-type responses have remained unclear. Because IL-4 acts as an initiator of Th2-type responses, we examined the characteristics of NK cells in mice overexpressing IL-4. In this study, we report that IL-4 overexpression induces distinctive characteristics of NK cells (B220(high)/CD11b(low)/IL-18Rα(low)), which are different from mature conventional NK (cNK) cells (B220(low)/CD11b(high)/IL-18Rα(high)). IL-4 overexpression induces proliferation of tissue-resident macrophages, which contributes to NK cell proliferation via production of IL-15. These IL-4-induced NK cells (IL4-NK cells) produce higher levels of IFN-γ, IL-10, and GM-CSF, and exhibit high cytotoxicity compared with cNK cells. Furthermore, incubation of cNK cells with IL-15 and IL-4 alters their phenotype to that similar to IL4-NK cells. Finally, parasitic infection, which typically causes strong Th2-type responses, induces the development of NK cells with characteristics similar to IL4-NK cells. These IL4-NK-like cells do not develop in IL-4Rα KO mice by parasitic infection. Collectively, these results suggest a novel role of IL-4 in immune responses through the induction of the unique NK cells. PMID:27551096

  5. Memory-Like Antigen-Specific Human NK Cells from TB Pleural Fluids Produced IL-22 in Response to IL-15 or Mycobacterium tuberculosis Antigens

    PubMed Central

    Fu, Xiaoying; Yu, Sifei; Yang, Binyan; Lao, Suihua; Li, Baiqing; Wu, Changyou

    2016-01-01

    Our previous result indicated that memory-like human natural killer (NK) cells from TB pleural fluid cells (PFCs) produced large amounts of IFN-γ in response to Bacille Calmette Guerin (BCG). Furthermore, recent studies have shown that human lymphoid tissues harbored a unique NK cell subset that specialized in production of interleukin (IL)-22, a proinflammatory cytokine that mediates host defense against pathogens. Yet little information was available with regard to the properties of IL-22 production by memory-like human NK cells. In the present study, we found that cytokines IL-15 induced and IL-12 enhanced the levels of IL-22 by NK cells from TB PFCs. In addition, IL-22 but not IL-17 was produced by NK cells from PFCs in response to BCG and M.tb-related Ags. More importantly, the subset of specific IL-22-producing NK cells were distinct from IFN-γ-producing NK cells in PFCs. CD45RO+ or CD45RO- NK cells were sorted, co-cultured with autologous monocytes and stimulated with BCG for the production of IL-22. The result demonstrated that CD45RO+ but not CD45RO- NK cells produced significantly higher level of IL-22. Anti-IL-12Rβ1 mAbs (2B10) partially inhibit the expression of IL-22 by NK cells under the culture with BCG. Consistently, BCG specific IL-22-producing NK cells from PFCs expressed CD45ROhighNKG2Dhighgranzyme Bhigh. In conclusion, our data demonstrated that memory-like antigen-specific CD45RO+ NK cells might participate in the recall immune response for M. tb infection via producing IL-22, which display a critical role to fight against M. tb. PMID:27031950

  6. Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation.

    PubMed

    Killig, Monica; Friedrichs, Birte; Meisig, Johannes; Gentilini, Chiara; Blüthgen, Nils; Loddenkemper, Christoph; Labopin, Myriam; Basara, Nadezda; Pfrepper, Christian; Niederwieser, Dietger W; Uharek, Lutz; Romagnani, Chiara

    2014-09-01

    Haploidentical stem cell transplantation (haploSCT) offers an alternative treatment option for advanced leukemia patients lacking a HLA-compatible donor. Transfer of NK cells represents a promising therapeutic option in combination with SCT, as NK cells can promote graft versus leukemia with low risk of GVH disease. In this study, we show results from a phase I/II trial in which 24 acute myeloid leukemia patients underwent haploSCT in combination with early transfer of unmodified NK cells and observed a promising 2-year overall survival rate of 37%. By performing immunomonitoring and subsequent principal component analysis, we tracked donor NK-cell dynamics in the patients and distinguished between NK cells reconstituting from CD34(+) precursors, giving rise over time to a continuum of multiple differentiation stages, and adoptively transferred NK cells. Transferred NK cells displayed a mature phenotype and proliferated in vivo during the early days after haploSCT even in the absence of exogenous IL-2 administration. Moreover, we identified the NK-cell phenotype associated with in vivo expansion. Thus, our study indicates a promising path for adoptive transfer of unmodified NK cells in the treatment of high-risk acute myeloid leukemia.

  7. Human Amnion-Derived Stem Cells Have Immunosuppressive Properties on NK Cells and Monocytes.

    PubMed

    Li, Jiali; Koike-Soko, Chika; Sugimoto, Jun; Yoshida, Toshiko; Okabe, Motonori; Nikaido, Toshio

    2015-01-01

    Human amnion-derived cells are considered to be a promising alternative cell source for their potential clinical use in tissue engineering and regenerative medicine because of their proliferation and differentiation ability. The cells can easily be obtained from human amnion, offering a potential source without medical intervention. It has been proven that human amnion-derived cells express immunosuppressive factors CD59 and HLA-G, implying that they may have an immunosuppressive function. To assess the immunosuppressive activity, we investigated the effect of human amnion-derived cells on NK cell and monocyte function. Amnion-derived cells inhibited the cytotoxicity of NK cells to K562 cells. The inhibition depended on the NK/amnion-derived cell ratio. The inhibition of NK cytotoxicity was recovered by continuous culturing without amnion-derived cells. The inhibition of NK cytotoxicity was related to the downregulation of the expression of the activated NK receptors and the production of IFN-γ, as well as the upregulation of the expression of IL-10 and PGE2 in human amnion-derived cells. The addition of antibody to IL-10 or PGE2 inhibitor tended to increase NK cytotoxicity. IL-10 and PGE2 might be involved in the immunosuppressive activity of amniotic cells toward NK cells. Amniotic cells also suppressed the activity of cytokine production in monocytes analyzed with TNF-α and IL-6. These data suggested that amniotic cells have immunosuppressive activity.

  8. Impaired NK cells and increased T regulatory cell numbers during cytotoxic maintenance therapy in AML.

    PubMed

    Lichtenegger, Felix S; Lorenz, Robin; Gellhaus, Katharina; Hiddemann, Wolfgang; Beck, Barbara; Subklewe, Marion

    2014-08-01

    Cyclic cytotoxic maintenance therapy can be applied to patients with AML in post-remission. We studied the immune status of AML patients in complete remission and the effect of maintenance therapy on different immune cell populations. Patients in complete remission had reduced NK, TH and Treg counts and a reduced NK activation capacity. In the course of cytotoxic maintenance therapy, NK counts further declined, while TH and Treg cells increased, with lower proliferative potential of TH cells. We conclude that immunotherapeutic approaches in post-remission have to consider reduced NK cell function and further impairment of cellular immune responses during cytotoxic therapy.

  9. Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells

    PubMed Central

    Akazawa, Takashi; Ebihara, Takashi; Okuno, Manabu; Okuda, Yu; Shingai, Masashi; Tsujimura, Kunio; Takahashi, Toshitada; Ikawa, Masahito; Okabe, Masaru; Inoue, Norimitsu; Okamoto-Tanaka, Miki; Ishizaki, Hiroyoshi; Miyoshi, Jun; Matsumoto, Misako; Seya, Tsukasa

    2007-01-01

    Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1−/− or IFNAR−/− mice, whereas a normal NK antitumor response was induced in PKR−/−, MyD88−/−, IFN-β−/−, and wild-type mice. IFNAR was a prerequisite for the induction of IFN-α/β and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR−/− mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1−/− mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors. PMID:17190817

  10. Re-examining the origin and function of liver-resident NK cells.

    PubMed

    Peng, Hui; Tian, Zhigang

    2015-05-01

    Recent studies have identified a population of liver-resident innate lymphoid cells (ILCs) that, based on the expression of certain phenotypic markers, were termed 'liver-resident NK cells' and considered to be a new subset of conventional natural killer (cNK) cells. However, different transcriptional networks control the development of liver-resident NK cells and cNK cells and, furthermore, these cells exhibit features that characterize mucosal ILC1s. Here, we review findings providing insight into the origin, phenotype, and function of liver-resident NK cells, and discuss these in the context of the current understanding of lineage relations of ILC subsets. We propose that the similarities between liver-resident NK cells and mucosal ILC1s should be considered when revising the categorization framework for these cells, and discuss implications of this revision for other tissue-specific NK cells.

  11. CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment

    PubMed Central

    Matsuura, Toshiharu; Muraoka, Izumi; Tryphonopoulos, Panagiotis; Fan, Ji; Tekin, Akin; Selvaggi, Gennaro; Levi, David; Ruiz, Phillip; Ricordi, Camillo; Vianna, Rodrigo; Ohdan, Hideki; Waldmann, Herman; Tzakis, Andreas G.; Nishida, Seigo

    2016-01-01

    Background T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK) cell number was sustained. NK cells are important effectors of innate immunity. Since the effects of alemtuzumab on NK cell functions, especially those of liver NK cells, are unknown, this study aimed to investigate this in detail. Methods To assess the effect of alemtuzumab on NK cells, samples were obtained from 7 organ donors and examined by flow cytometry using Annexin V and propidium iodide. Phenotypical and functional differences within subsets of NK cells with different levels of CD52 expression were determined by flow cytometry and in vitro cytotoxicity assays. Results CD52 expression on NK cells was lower than that on other lymphocyte subsets. The liver contained a large number of CD52− NK cells compared with the peripheral blood. In vitro treatment of liver-derived NK cells with alemtuzumab did not result in cell death. In contrast, co-incubation with alemtuzumab induced cell death in peripheral blood mononuclear cells and non-NK cells in the liver. Furthermore, CD52− liver NK cells were more cytotoxic and produced more IFN-γ than CD52+ NK cells after cytokine activation. Conclusion The liver contains a large number of CD52− NK cells. These cells are refractory to alemtuzumab and have robust activity. These findings indicate that CD52− NK cells persist and could protect against infection after alemtuzumab-based lymphocyte depletion. PMID

  12. Licensed and Unlicensed NK Cells: Differential Roles in Cancer and Viral Control

    PubMed Central

    Tu, Megan M.; Mahmoud, Ahmad Bakur; Makrigiannis, Andrew P.

    2016-01-01

    Natural killer (NK) cells are known for their well characterized ability to control viral infections and eliminate tumor cells. Through their repertoire of activating and inhibitory receptors, NK cells are able to survey different potential target cells for various surface markers, such as MHC-I – which signals to the NK cell that the target is healthy – as well as stress ligands or viral proteins, which alert the NK cell to the aberrant state of the target and initiate a response. According to the “licensing” hypothesis, interactions between self-specific MHC-I receptors – Ly49 in mice and KIR in humans – and self-MHC-I molecules during NK cell development is crucial for NK cell functionality. However, there also exists a large proportion of NK cells in mice and humans, which lack self-specific MHC-I receptors and are consequentially “unlicensed.” While the licensed NK cell subset plays a major role in the control of MHC-I-deficient tumors, this review will go on to highlight the important role of the unlicensed NK cell subset in the control of MHC-I-expressing tumors, as well as in viral control. Unlike the licensed NK cells, unlicensed NK cells seem to benefit from the lack of self-specific inhibitory receptors, which could otherwise be exploited by some aberrant cells for immunoevasion by upregulating the expression of ligands or mimic ligands for these receptors. PMID:27199990

  13. HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells

    PubMed Central

    Formaglio, Pauline; Melki, Marie-Thérèse; Charbit, Bruno; Herbeuval, Jean-Philippe; Gougeon, Marie-Lise

    2016-01-01

    Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell–cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN-α-mediated

  14. Differential Effects of Interleukin-7 and Interleukin-15 on NK Cell Anti-Human Immunodeficiency Virus Activity

    PubMed Central

    Lum, Julian J.; Schnepple, David J.; Nie, Zilin; Sanchez-Dardon, Jaime; Mbisa, Georgina L.; Mihowich, Jennifer; Hawley, Nanci; Narayan, Shanil; Kim, John E.; Lynch, David H.; Badley, Andrew D.

    2004-01-01

    The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3− CD16+ CD56+) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01; P = 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior. PMID:15141001

  15. Deletion of PI3K-p85alpha gene impairs lineage commitment, terminal maturation, cytokine generation and cytotoxicity of NK cells.

    PubMed

    Awasthi, A; Samarakoon, A; Dai, X; Wen, R; Wang, D; Malarkannan, S

    2008-09-01

    Class IA phosphotidylinositol-3-kinases (PI3Ks) are a family of p85/p110 heterodimeric lipid kinases that are important in regulating signaling events in B and T cells. However, their role in natural killer (NK) cells is not understood. Here, using mice that lack the regulatory p85alpha subunit and its alternatively spliced variants p55alpha/p50alpha (collectively termed as p85alpha(-/-)), we defined the role of PI3K in NK cell development and function. p85alpha(-/-) mice had impaired lineage commitment leading to reduced NK cellularity in the bone marrow and liver. p85alpha(-/-) NK cells showed a defective Ly49 subset specification and a decreased expression of CD43. Lack of p85alpha severely reduced the NK-mediated cytotoxicity against tumor cells representing 'induced-self' and 'missing-self'. More importantly, NKG2D and NK1.1 receptor-mediated cytokine and chemokine generation was significantly compromised in p85alpha(-/-) NK cells. These results reveal a previously unrecognized role of p85alpha in the development, terminal maturation, cytokine/chemokine generation and tumor clearance of NK cells.

  16. NK Cells in Healthy Aging and Age-Associated Diseases

    PubMed Central

    Camous, Xavier; Pera, Alejandra; Solana, Rafael; Larbi, Anis

    2012-01-01

    NK cells exhibit the highest cytotoxic capacity within the immune system. Alteration of their number or functionality may have a deep impact on overall immunity. This is of particular relevance in aging where the elderly population becomes more susceptible to infection, cancer, autoimmune diseases, and neurodegenerative diseases amongst others. As the fraction of elderly increases worldwide, it becomes urgent to better understand the aging of the immune system to prevent and cure the elderly population. For this, a better understanding of the function and phenotype of the different immune cells and their subsets is necessary. We review here NK cell functions and phenotype in healthy aging as well as in various age-associated diseases. PMID:23251076

  17. NK cells in healthy aging and age-associated diseases.

    PubMed

    Camous, Xavier; Pera, Alejandra; Solana, Rafael; Larbi, Anis

    2012-01-01

    NK cells exhibit the highest cytotoxic capacity within the immune system. Alteration of their number or functionality may have a deep impact on overall immunity. This is of particular relevance in aging where the elderly population becomes more susceptible to infection, cancer, autoimmune diseases, and neurodegenerative diseases amongst others. As the fraction of elderly increases worldwide, it becomes urgent to better understand the aging of the immune system to prevent and cure the elderly population. For this, a better understanding of the function and phenotype of the different immune cells and their subsets is necessary. We review here NK cell functions and phenotype in healthy aging as well as in various age-associated diseases.

  18. Bone marrow-derived mesenchymal stem cells suppress NK cell recruitment and activation in PolyI:C-induced liver injury.

    PubMed

    Qu, Mengmeng; Cui, Jun; Zhu, Jun; Ma, Yuhong; Yuan, Xu; Shi, Jinming; Guo, Deyin; Li, Changyong

    2015-10-16

    Mesenchymal stem cells (MSCs) have been shown to have an immunomodulatory capability and clinical potential in immune diseases. However, it is unknown how MSCs may affect immunity in liver injury. This study was designed to explore the effect of bone marrow-derived MSCs (BM-MSCs) on hepatic natural killer (NK) cells in polyinosinic-polycytidylic acid (PolyI:C)-induced liver injury. Unlike in controls, adoptive transfer of BM-MSCs in mice ameliorated PolyI:C-induced liver injury, as shown by lower alanine aminotransferase levels and decreased lymphocyte infiltration in the liver. Importantly, BM-MSCs suppress NK cell accumulation and activation in the liver, which plays an important role in PolyI:C-induced liver injury. Furthermore, NK cells co-cultured with BM-MSCs reduced expression of sphingosine-1-phosphate receptor type 5 (S1PR5), an important receptor required for NK cell trafficking in vivo. BM-MSC administration suppressed the elevation of expression of S1PR5 in the liver induced by PolyI:C injection. Accordingly, BM-MSCs inhibited the chemotactic activity of NK cells induced by sphingosine-1-phosphate (S1P, the ligand of S1PR5). Our results provide an additional mechanism for the immunosuppressive effect of BM-MSCs on NK cells, which further supports the therapeutic potential of BM-MSCs in immune-mediated disorders, including those in which NK cells play a major role.

  19. Higher Frequency of NK and CD4+ T-Cells in Mucosa and Potent Cytotoxic Response in HIV Controllers

    PubMed Central

    Taborda, Natalia Andrea; González, Sandra Milena; Alvarez, Cristiam Mauricio; Correa, Luis Alfonso; Montoya, Carlos Julio; Rugeles, María Teresa

    2015-01-01

    HIV infection induces immune alterations, mainly in gut mucosa, where the main target cells reside. However, the evolution of the infection is variable among infected individuals, as evidenced by HIV controllers who exhibit low or undetectable viral load in the absence of treatment. The aim of this study was to evaluate the frequency, phenotype and activity of T and NK cells in peripheral blood and gut mucosa in a cohort of Colombian HIV controllers. Blood and gut biopsies were included. The frequency and the activation status of T and NK cells were performed by flow cytometry. In addition, Gag-stimulated CD8+ T-cells and cytokine-stimulated NK cells were tested for cytotoxic activity. Finally, microbial translocation was measured by plasma lipopolysaccharide quantification. Compared with HIV-progressors, HIV controllers exhibited higher frequency of CD4+ T and NK cells, and lower expression of activation molecules in blood and mucosal immune cells, as well as lower microbial translocation. An increased production of molecules associated with cytotoxic activity of CD8+ T-cells in blood and mucosa and a higher percentage of polyfunctional CD8+ T cells in blood were also observed in HIV controllers. In addition, an increased activity of NK cells was observed in blood. These findings suggest that HIV controllers have a potent immune response, mainly mediated by cytotoxic cells that control HIV replication, which contribute to reducing alterations at the gut mucosa. PMID:26291824

  20. Higher Frequency of NK and CD4+ T-Cells in Mucosa and Potent Cytotoxic Response in HIV Controllers.

    PubMed

    Taborda, Natalia Andrea; González, Sandra Milena; Alvarez, Cristiam Mauricio; Correa, Luis Alfonso; Montoya, Carlos Julio; Rugeles, María Teresa

    2015-01-01

    HIV infection induces immune alterations, mainly in gut mucosa, where the main target cells reside. However, the evolution of the infection is variable among infected individuals, as evidenced by HIV controllers who exhibit low or undetectable viral load in the absence of treatment. The aim of this study was to evaluate the frequency, phenotype and activity of T and NK cells in peripheral blood and gut mucosa in a cohort of Colombian HIV controllers. Blood and gut biopsies were included. The frequency and the activation status of T and NK cells were performed by flow cytometry. In addition, Gag-stimulated CD8+ T-cells and cytokine-stimulated NK cells were tested for cytotoxic activity. Finally, microbial translocation was measured by plasma lipopolysaccharide quantification. Compared with HIV-progressors, HIV controllers exhibited higher frequency of CD4+ T and NK cells, and lower expression of activation molecules in blood and mucosal immune cells, as well as lower microbial translocation. An increased production of molecules associated with cytotoxic activity of CD8+ T-cells in blood and mucosa and a higher percentage of polyfunctional CD8+ T cells in blood were also observed in HIV controllers. In addition, an increased activity of NK cells was observed in blood. These findings suggest that HIV controllers have a potent immune response, mainly mediated by cytotoxic cells that control HIV replication, which contribute to reducing alterations at the gut mucosa.

  1. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells

    PubMed Central

    Johansson, Maria A.; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4+ and CD8+ T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation. PMID:27462316

  2. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells.

    PubMed

    Johansson, Maria A; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4(+) and CD8(+) T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation.

  3. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells.

    PubMed

    Johansson, Maria A; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4(+) and CD8(+) T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation. PMID:27462316

  4. Lung cancer is associated with decreased expression of perforin, granzyme B and interferon (IFN)-γ by infiltrating lung tissue T cells, natural killer (NK) T-like and NK cells.

    PubMed

    Hodge, G; Barnawi, J; Jurisevic, C; Moffat, D; Holmes, M; Reynolds, P N; Jersmann, H; Hodge, S

    2014-10-01

    There is a limited understanding how of lung cancer cells evade cytotoxic attack. Previously, we have shown reduced production of the cytotoxic mediator granzyme B by CD8(+) T cells in lung cancer tissue. We hypothesized that lung cancer would be further associated with decreased production of granzyme B, perforin and proinflammatory cytokines by other cytotoxic lymphocytes, natural killer (NK) T-like and NK cells, and that this would result from soluble mediators released by the cancer cells. Lung cancer and non-cancer tissue from five patients was identified by experienced pathologists. Tumour necrosis factor (TNF)-α, interferon (IFN)-γ, granzyme B and perforin were measured in CD4 and CD8(+) T, NK T-like cells and NK cells by flow cytometry. Correlation between cancer stage and granzyme B was analysed retrospectively for 21 patients. The effects of soluble factors released by lung cancer cells on production of cytotoxic mediators and cytokines was assessed, and the role of prostaglandin E2 (PGE)2 /COX investigated using indomethacin inhibition. There were significantly decreased percentages of T, NK T-like and NK cells expressing perforin, TNF-α and IFN-γ in cancer versus non-cancer tissue, and of CD8(+) T cells and CD8(+) NK T-like cells expressing granzyme B (e.g. NK T-like cells: non-cancer 30% ± 7 versus cancer 6% ± 2·5). Cancer cells released soluble factors that inhibited granzyme B, perforin and IFN-γ production that was partially associated with the PGE2 /COX2 pathway. Thus, lung cancer is associated with decreased expression of granzyme B, perforin and IFN-γ by infiltrating T cells, NK T-like and NK cells, possibly as a result of soluble factors produced by the cancer cells including PGE2 . This may be an important immune evasion mechanism.

  5. Hepatitis C Virus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γ Production by Altering Cellular Metabolism via Arginase-1.

    PubMed

    Goh, Celeste C; Roggerson, Krystal M; Lee, Hai-Chon; Golden-Mason, Lucy; Rosen, Hugo R; Hahn, Young S

    2016-03-01

    The hepatitis C virus (HCV) infects ∼ 200 million people worldwide. The majority of infected individuals develop persistent infection, resulting in chronic inflammation and liver disease, including cirrhosis and hepatocellular carcinoma. The ability of HCV to establish persistent infection is partly due to its ability to evade the immune response through multiple mechanisms, including suppression of NK cells. NK cells control HCV replication during the early phase of infection and regulate the progression to chronic disease. In particular, IFN-γ produced by NK cells limits viral replication in hepatocytes and is important for the initiation of adaptive immune responses. However, NK cell function is significantly impaired in chronic HCV patients. The cellular and molecular mechanisms responsible for impaired NK cell function in HCV infection are not well defined. In this study, we analyzed the interaction of human NK cells with CD33(+) PBMCs that were exposed to HCV. We found that NK cells cocultured with HCV-conditioned CD33(+) PBMCs produced lower amounts of IFN-γ, with no effect on granzyme B production or cell viability. Importantly, this suppression of NK cell-derived IFN-γ production was mediated by CD33(+)CD11b(lo)HLA-DR(lo) myeloid-derived suppressor cells (MDSCs) via an arginase-1-dependent inhibition of mammalian target of rapamycin activation. Suppression of IFN-γ production was reversed by l-arginine supplementation, consistent with increased MDSC arginase-1 activity. These novel results identify the induction of MDSCs in HCV infection as a potent immune evasion strategy that suppresses antiviral NK cell responses, further indicating that blockade of MDSCs may be a potential therapeutic approach to ameliorate chronic viral infections in the liver. PMID:26826241

  6. Role of NK, NKT cells and macrophages in liver transplantation

    PubMed Central

    Fahrner, René; Dondorf, Felix; Ardelt, Michael; Settmacher, Utz; Rauchfuss, Falk

    2016-01-01

    Liver transplantation has become the treatment of choice for acute or chronic liver disease. Because the liver acts as an innate immunity-dominant organ, there are immunological differences between the liver and other organs. The specific features of hepatic natural killer (NK), NKT and Kupffer cells and their role in the mechanism of liver transplant rejection, tolerance and hepatic ischemia-reperfusion injury are discussed in this review. PMID:27468206

  7. NK Cell Maturation and Cytotoxicity Are Controlled by the Intramembrane Aspartyl Protease SPPL3.

    PubMed

    Hamblet, Corinne E; Makowski, Stefanie L; Tritapoe, Julia M; Pomerantz, Joel L

    2016-03-15

    NK cell maturation is critical for normal effector function and the innate immune response to tumors and pathogens. However, the molecular pathways that control NK cell maturation remain largely undefined. In this article, we investigate the role of SPPL3, an intramembrane aspartyl protease, in murine NK cell biology. We find that deletion of SPPL3 in the hematopoietic system reduces numbers of peripheral NK cells, clearance of MHC class I-deficient tumors in vivo, and cytotoxicity against tumor cells in vitro. This phenotype is concomitant with reduced numbers of CD27(+)CD11b(+) and CD27(-)CD11b(+) NK cells, indicating a requirement for SPPL3 in efficient NK cell maturation. NK cell-specific deletion of SPPL3 results in the same deficiencies, revealing a cell-autonomous role for SPPL3 in these processes. CRISPR/Cas9 genomic editing in murine zygotes was used to generate knockin mice with a catalytically compromised SPPL3 D271A allele. Mice engineered to express only SPPL3 D271A in NK cells phenocopy mice deleted for SPPL3, indicating a requirement for SPPL3 protease activity in NK cell biology. Our results identify SPPL3 as a cell-autonomous molecular determinant of NK cell maturation and expand the role of intramembrane aspartyl proteases in innate immunity. PMID:26851218

  8. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    PubMed

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-01

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  9. Anti-tumor activity of patient-derived NK cells after cell-based immunotherapy – a case report

    PubMed Central

    Milani, Valeria; Stangl, Stefan; Issels, Rolf; Gehrmann, Mathias; Wagner, Beate; Hube, Kathrin; Mayr, Doris; Hiddemann, Wolfgang; Molls, Michael; Multhoff, Gabriele

    2009-01-01

    Background Membrane-bound heat shock protein 70 (Hsp70) serves as a tumor-specific recognition structure for Hsp70-peptide (TKD) plus IL-2 activated NK cells. A phase I clinical trial has shown that repeated re-infusions of ex vivo TKD/IL-2-activated, autologous leukapheresis product is safe. This study investigated the maintenance of the cytolytic activity of NK cells against K562 cells and autologous tumor after 6 plus 3 infusions of TKD/IL-2-activated effector cells. Methods A stable tumor cell line was generated from the resected anastomotic relapse of a patient with colon carcinoma (pT3, N2, M0, G2). Two months after surgery, the patient received the first monthly i.v. infusion of his ex vivo TKD/IL-2-activated peripheral blood mononuclear cells (PBMNC). After 6 infusions and a pause of 3 months, the patient received another 3 cell infusions. The phenotypic characteristics and activation status of tumor and effector cells were determined immediately before and at times after each infusion. Results The NK cell ligands Hsp70, MICA/B, and ULBP-1,2,3 were expressed on the patient's anastomotic relapse. An increased density of activatory NK cell receptors following ex vivo stimulation correlated with an enhanced anti-tumoricidal activity. After 4 re-infusion cycles, the intrinsic cytolytic activity of non-stimulated PBMNC was significantly elevated and this heightened responsiveness persisted for up to 3 months after the last infusion. Another 2 re-stimulations with TKD/IL-2 restored the cytolytic activity after the therapeutic pause. Conclusion In a patient with colon carcinoma, repeated infusions of ex vivo TKD/IL-2-activated PBMNC initiate an intrinsic NK cell-mediated cytolytic activity against autologous tumor cells. PMID:19549307

  10. Polyclonal Expansion of NKG2C+ NK Cells in TAP-Deficient Patients

    PubMed Central

    Béziat, Vivien; Sleiman, Marwan; Goodridge, Jodie P.; Kaarbø, Mari; Liu, Lisa L.; Rollag, Halvor; Ljunggren, Hans-Gustaf; Zimmer, Jacques; Malmberg, Karl-Johan

    2015-01-01

    Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C+ NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C+ NK cell expansions. We demonstrate the expansion of NKG2C+ NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C+ NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C+ NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C+ NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients’ low incidence of severe viral infections. PMID:26500647

  11. Decreased NK-cell tumour immunosurveillance consequent to JAK inhibition enhances metastasis in breast cancer models

    PubMed Central

    Bottos, Alessia; Gotthardt, Dagmar; Gill, Jason W.; Gattelli, Albana; Frei, Anna; Tzankov, Alexandar; Sexl, Veronika; Wodnar-Filipowicz, Aleksandra; Hynes, Nancy E.

    2016-01-01

    The JAK/STAT pathway is an attractive target for breast cancer therapy due to its frequent activation, and clinical trials evaluating JAK inhibitors (JAKi) in advanced breast cancer are ongoing. Using patient biopsies and preclinical models of breast cancer, we demonstrate that the JAK/STAT pathway is active in metastasis. Unexpectedly, blocking the pathway with JAKi enhances the metastatic burden in experimental and orthotopic models of breast cancer metastasis. We demonstrate that this prometastatic effect is due to the immunosuppressive activity of JAKi with ensuing impairment of NK-cell-mediated anti-tumour immunity. Furthermore, we show that immunostimulation with IL-15 overcomes the enhancing effect of JAKi on metastasis formation. Our findings highlight the importance of evaluating the effect of targeted therapy on the tumour environment. The impact of JAKi on NK cells and the potential value of immunostimulators to overcome the weakened tumour immunosurveillance, are worthwhile considering in the clinical setting of breast cancer. PMID:27406745

  12. Genetic deletion of Cxcl14 in mice alters uterine NK cells

    SciTech Connect

    Cao, Qichen; Chen, Hua; Deng, Zhili; Yue, Jingwen; Chen, Qi; Cao, Yujing; Ning, Lina; Lei, Xiaohua; Duan, Enkui

    2013-06-14

    Highlights: •We first examined the expression of Cxcl14 in MLAp and DB of uterus. •We found the uNK cells in MLAp and decidua express Cxcl14. •In Cxcl14{sup −/−} placenta, we found significantly decreased uNK cells. •We first performed microarray to compare the gene expression in MLAp and DB. -- Abstract: The uterine natural killer cells (uNK cells) are the major immune cells in pregnant uterus and the number of uNK cells is dramatically increased during placentation and embryo development. The uNK cells are necessary for the immune tolerance, cytokine secretion and angiogenesis of placenta. Former studies indicated that the population expansion of uNK cells was accomplished through recruitment of NK cell precursors from the spleen and bone marrow, but not proliferation of NK cells. However, the necessary molecules within this process were little understood. Here in our study, we found the co-localized expression of Cxcl14 protein with uNK cells in E13.5 pregnant uterus. Moreover, we used Cxcl14 knockout mice to examine uNK cells in mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) of E13.5 pregnant uterus and found significantly decreased uNK cells in Cxcl14{sup −/−} pregnant uteri compared with Cxcl14{sup +/−} pregnant uteri. To further explorer the molecular change in MLAp and DB after Cxcl14 knockout, we isolated the MLAp and DB from Cxcl14{sup +/+} and Cxcl14{sup −/−} pregnant uteri and performed microarray analysis. We found many genes were up and down regulated after Cxcl14 knockout. In conclusion, our results suggested the important function of Cxcl14 in uNK cells and the proper level of Cxcl14 protein were required to recruit NK cells to pregnant uterus.

  13. NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation

    PubMed Central

    Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

    2016-01-01

    Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34+ cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT. PMID:27242795

  14. NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

    PubMed

    Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

    2016-01-01

    Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

  15. Flow Cytometry Analysis of NK Cell Phenotype and Function in Aging.

    PubMed

    Tarazona, Raquel; Campos, Carmen; Pera, Alejandra; Sanchez-Correa, Beatriz; Solana, Rafael

    2015-01-01

    Natural killer (NK) cells represent a subpopulation of lymphocytes involved in innate immunity, defined recently as group 1 of innate lymphoid cells (ILCs). NK cells are cytotoxic lymphocytes with a relevant role in the destruction of transformed cells as virus-infected or tumor cells, as well as the regulation of the immune response through cytokine and chemokine production that activates other cellular components of innate and adaptive immunity. In humans, NK cell subsets have been defined according to the level of expression of CD56. Aging differentially affects NK cell subsets and NK cell function. Here, we describe protocols for the delineation of NK cell subsets and the analysis of their functional capacity using multiparametric flow cytometry.

  16. Local Microenvironment Controls the Compartmentalization of NK Cell Responses during Systemic Inflammation in Mice.

    PubMed

    Rasid, Orhan; Ciulean, Ioana Sonya; Fitting, Catherine; Doyen, Noelle; Cavaillon, Jean-Marc

    2016-09-15

    Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell-intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo-transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell-intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner. PMID:27521338

  17. IL-21-Induced MHC Class II+ NK Cells Promote the Expansion of Human Uncommitted CD4+ Central Memory T Cells in a Macrophage Migration Inhibitory Factor-Dependent Manner.

    PubMed

    Loyon, Romain; Picard, Emilie; Mauvais, Olivier; Queiroz, Lise; Mougey, Virginie; Pallandre, Jean-René; Galaine, Jeanne; Mercier-Letondal, Patricia; Kellerman, Guillaume; Chaput, Nathalie; Wijdenes, John; Adotévi, Olivier; Ferrand, Christophe; Romero, Pedro; Godet, Yann; Borg, Christophe

    2016-07-01

    NK cells are critical for innate immunity-mediated protection. The main roles of NK cells rely on their cytotoxic functions or depend on the tuning of Th1 adaptive immunity by IFN-γ. However, the precise influence of inflammatory cytokines on NK cell and CD4 T lymphocyte interactions was never investigated. In this study, we provide evidence that IL-21, a cytokine produced during chronic inflammation or infectious diseases, promotes the differentiation of a specific subset of NK cells coexpressing CD86 and HLA-DR and lacking NKp44. More importantly, IL-21-propagated HLA-DR(+) NK cells produce macrophage migration inhibitory factor and provide costimulatory signaling during naive CD4(+) T cell priming inducing the differentiation of uncommitted central memory T cells. Central memory T cells expanded in the presence of HLA-DR(+) NK cells are CXCR3(+)CCR6(-)CCR4(-)CXCR5(-) and produce IL-2, as well as low levels of TNF-α. Costimulation of CD4(+) T cells by HLA-DR(+) NK cells prevents the acquisition of effector memory phenotype induced by IL-2. Moreover, we identified this population of NK HLA-DR(+) macrophage migration inhibitory factor(+) cells in inflammatory human appendix. Collectively, these results demonstrate a novel function for IL-21 in tuning NK and CD4(+) T cell interactions promoting a specific expansion of central memory lymphocytes. PMID:27233967

  18. Naive Donor NK Cell Repertoires Associated with Less Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation.

    PubMed

    Björklund, Andreas T; Clancy, Trevor; Goodridge, Jodie P; Béziat, Vivien; Schaffer, Marie; Hovig, Eivind; Ljunggren, Hans-Gustaf; Ljungman, Per T; Malmberg, Karl-Johan

    2016-02-01

    Acute and latent human CMV cause profound changes in the NK cell repertoire, with expansion and differentiation of educated NK cells expressing self-specific inhibitory killer cell Ig-like receptors. In this study, we addressed whether such CMV-induced imprints on the donor NK cell repertoire influenced the outcome of allogeneic stem cell transplantation. Hierarchical clustering of high-resolution immunophenotyping data covering key NK cell parameters, including frequencies of CD56(bright), NKG2A(+), NKG2C(+), and CD57(+) NK cell subsets, as well as the size of the educated NK cell subset, was linked to clinical outcomes. Clusters defining naive (NKG2A(+)CD57(-)NKG2C(-)) NK cell repertoires in the donor were associated with decreased risk for relapse in recipients with acute myeloid leukemia and myelodysplastic syndrome (hazard ratio [HR], 0.09; 95% confidence interval [CI]: 0.03-0.27; p < 0.001). Furthermore, recipients with naive repertoires at 9-12 mo after hematopoietic stem cell transplantation had increased disease-free survival (HR, 7.2; 95% CI: 1.6-33; p = 0.01) and increased overall survival (HR, 9.3; 95% CI: 1.1-77, p = 0.04). Conversely, patients with a relative increase in differentiated NK cells at 9-12 mo displayed a higher rate of late relapses (HR, 8.41; 95% CI: 6.7-11; p = 0.02), reduced disease-free survival (HR, 0.12; 95% CI: 0.12-0.74; p = 0.02), and reduced overall survival (HR, 0.07; 95% CI: 0.01-0.69; p = 0.02). Thus, our data suggest that naive donor NK cell repertoires are associated with protection against leukemia relapse after allogeneic HSCT. PMID:26746188

  19. Monomethyl fumarate augments NK cell lysis of tumor cells through degranulation and the upregulation of NKp46 and CD107a.

    PubMed

    Vego, Heidi; Sand, Kristin L; Høglund, Rune A; Fallang, Lars-Egil; Gundersen, Glenn; Holmøy, Trygve; Maghazachi, Azzam A

    2016-01-01

    Dimethyl fumarate (DMF) is a new drug used to treat multiple sclerosis (MS) patients. Here, we examined the effects of DMF and the DMF metabolite monomethyl fumarate (MMF) on various activities of natural killer (NK) cells. We demonstrated that MMF augments the primary CD56(+), but not CD56(-), NK cell lysis of K562 and RAJI tumor cells. MMF induced NKp46 expression on the surface of CD56(+), but not CD56(-), NK cells after incubation for 24 h. This effect was closely correlated with the upregulation of CD107a expression on the surface of CD56(+) NK cells and the induction of Granzyme B release from these cells through this metabolite. An anti-NKp46 antibody inhibited the MMF-induced upregulation of CD107a and the lysis of tumor cells through CD56(+) NK cells. Thus, these results are the first to show that MMF augments CD56(+) NK cell lysis of tumor target cells, an effect mediated through NKp46. This novel effect suggests the use of MMF for therapeutic and/or preventive protocols in cancer. PMID:25435072

  20. The Unique Neonatal NK Cells: A Critical Component Required for Neonatal Autoimmune Disease Induction by Maternal Autoantibody

    PubMed Central

    Rival, Claudia; Setiady, Yulius; Samy, Eileen T.; Harakal, Jessica; Tung, Kenneth S. K.

    2014-01-01

    Human maternal autoantibodies can trigger autoimmune diseases such as congenital heart block (CHB) in the progeny of women with lupus or Sjogren’s disease. The pathogenic effect of early autoantibody (autoAb) exposure has been investigated in a murine neonatal autoimmune ovarian disease (nAOD) model triggered by a unique ZP3 antibody. Although immune complexes (IC) are formed in adult and neonatal ovaries, ZP3 antibody triggers severe nAOD only in <7-day-old neonatal mice. Propensity to nAOD is due to the uniquely hyper-responsive neonatal natural killer (NK) cells that lack the inhibitory Ly49C/I receptors. In nAOD, the neonatal NK cells directly mediate ovarian inflammation and oocyte depletion while simultaneously promoting de novo pathogenic ovarian-specific T cell responses. Resistance to nAOD in older mice results from the emergence of the Ly49C/I+ NK cells that regulate effector NK cells and from CD25+ regulatory T cell control. In preliminary studies, FcγRIII+ NK cells as well as the ovarian resident FcγRIII+ macrophages and/or dendritic cells were found to be as indispensable players. Activated by ovarian IC, they migrate to lymphoid organs where NK cell priming occurs. Remarkably, the findings in nAOD are very similar to those reported for neonatal responses to a retrovirus and its cognate antibody that lead to long-lasting immunity. Studies on nAOD therefore provide insights into maternal autoAb-mediated neonatal autoimmunity, including CHB, while simultaneously uncovering new properties of the neonatal innate and adaptive responses, lethality of premature infant infection, and novel neonatal antiviral vaccine design. PMID:24904590

  1. A novel TLR7 agonist reverses NK cell anergy and cures RMA-S lymphoma-bearing mice.

    PubMed

    Wiedemann, Gabriela Maria; Jacobi, Severin Johannes; Chaloupka, Michael; Krächan, Angelina; Hamm, Svetlana; Strobl, Stefan; Baumgartner, Roland; Rothenfusser, Simon; Duewell, Peter; Endres, Stefan; Kobold, Sebastian

    2016-07-01

    Toll-like receptor 7 (TLR7) agonists are potent immune stimulants able to overcome cancer-associated immune suppression. Due to dose-limiting systemic toxicities, only the topically applied TLR7 agonist (imiquimod) has been approved for therapy of skin tumors. There is a need for TLR7-activating compounds with equivalent efficacy but less toxicity. SC1, a novel small molecule agonist for TLR7, is a potent type-1 interferon inducer, comparable to the reference TLR7 agonist resiquimod, yet with lower induction of proinflammatory cytokines. In vivo, SC1 activates NK cells in a TLR7-dependent manner. Mice bearing the NK cell-sensitive lymphoma RMA-S are cured by repeated s. c. administrations of SC1 as efficiently as by the administration of resiquimod. No relevant toxicities were observed. Mechanistically, SC1 reverses NK cell anergy and restores NK cell-mediated tumor cell killing in an IFN-α-dependent manner. TLR7 targeting by SC1-based compounds may form an attractive strategy to activate NK cell responses for cancer therapy. PMID:27622045

  2. A fatal cytokine-induced systemic inflammatory response reveals a critical role for NK cells.

    PubMed

    Carson, W E; Yu, H; Dierksheide, J; Pfeffer, K; Bouchard, P; Clark, R; Durbin, J; Baldwin, A S; Peschon, J; Johnson, P R; Ku, G; Baumann, H; Caligiuri, M A

    1999-04-15

    The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-alpha, IFN-gamma, macrophage-inflammatory protein-1alpha, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans.

  3. Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications

    PubMed Central

    Carlsten, Mattias; Childs, Richard W.

    2015-01-01

    Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression in vivo. Improving NK cell persistence in vivo via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors in vivo, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic. PMID:26113846

  4. Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications.

    PubMed

    Carlsten, Mattias; Childs, Richard W

    2015-01-01

    Given their rapid and efficient capacity to recognize and kill tumor cells, natural killer (NK) cells represent a unique immune cell to genetically reprogram in an effort to improve the outcome of cell-based cancer immunotherapy. However, technical and biological challenges associated with gene delivery into NK cells have significantly tempered this approach. Recent advances in viral transduction and electroporation have now allowed detailed characterization of genetically modified NK cells and provided a better understanding for how these cells can be utilized in the clinic to optimize their capacity to induce tumor regression in vivo. Improving NK cell persistence in vivo via autocrine IL-2 and IL-15 stimulation, enhancing tumor targeting by silencing inhibitory NK cell receptors such as NKG2A, and redirecting tumor killing via chimeric antigen receptors, all represent approaches that hold promise in preclinical studies. This review focuses on available methods for genetic reprograming of NK cells and the advantages and challenges associated with each method. It also gives an overview of strategies for genetic reprograming of NK cells that have been evaluated to date and an outlook on how these strategies may be best utilized in clinical protocols. With the recent advances in our understanding of the complex biological networks that regulate the ability of NK cells to target and kill tumors in vivo, we foresee genetic engineering as an obligatory pathway required to exploit the full potential of NK-cell based immunotherapy in the clinic.

  5. (19)F-MRI for monitoring human NK cells in vivo.

    PubMed

    Bouchlaka, Myriam N; Ludwig, Kai D; Gordon, Jeremy W; Kutz, Matthew P; Bednarz, Bryan P; Fain, Sean B; Capitini, Christian M

    2016-05-01

    The availability of clinical-grade cytokines and artificial antigen-presenting cells has accelerated interest in using natural killer (NK) cells as adoptive cellular therapy (ACT) for cancer. One of the technological shortcomings of translating therapies from animal models to clinical application is the inability to effectively and non-invasively track these cells after infusion in patients. We have optimized the nonradioactive isotope fluorine-19 ((19)F) as a means to label and track NK cells in preclinical models using magnetic resonance imaging (MRI). Human NK cells were expanded with interleukin (IL)-2 and labeled in vitro with increasing concentrations of (19)F. Doses as low as 2 mg/mL (19)F were detected by MRI. NK cell viability was only decreased at 8 mg/mL (19)F. No effects on NK cell cytotoxicity against K562 leukemia cells were observed with 2, 4 or 8 mg/mL (19)F. Higher doses of (19)F, 4 mg/mL and 8 mg/mL, led to an improved (19)F signal by MRI with 3 × 10(11) (19)F atoms per NK cell. The 4 mg/mL (19)F labeling had no effect on NK cell function via secretion of granzyme B or interferon gamma (IFNγ), compared to NK cells exposed to vehicle alone. (19)F-labeled NK cells were detectable immediately by MRI after intratumoral injection in NSG mice and up to day 8. When (19)F-labeled NK cells were injected subcutaneously, we observed a loss of signal through time at the site of injection suggesting NK cell migration to distant organs. The (19)F perfluorocarbon is a safe and effective reagent for monitoring the persistence and trafficking of NK cell infusions in vivo, and may have potential for developing novel imaging techniques to monitor ACT for cancer. PMID:27467963

  6. Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

    PubMed Central

    Huergo-Zapico, Leticia; Acebes-Huerta, Andrea; Gonzalez-Rodriguez, Ana Pilar; Contesti, Juan; Gonzalez-García, Esther; Payer, Angel R.; Villa-Alvarez, Monica; Fernández-Guizán, Azahara; López-Soto, Alejandro; Gonzalez, Segundo

    2014-01-01

    The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL. PMID:25286418

  7. Combination of NK Cells and Cetuximab to Enhance Anti-Tumor Responses in RAS Mutant Metastatic Colorectal Cancer

    PubMed Central

    Spanholtz, Jan; Tordoir, Marleen; Thijssen, Victor L.; Heideman, Daniëlle A. M.; Verheul, Henk M. W.; de Gruijl, Tanja D.; van der Vliet, Hans J.

    2016-01-01

    The ability of Natural Killer (NK) cells to kill tumor targets has been extensively studied in various hematological malignancies. However, NK cell therapy directed against solid tumors is still in early development. Epidermal Growth Factor Receptor (EGFR) targeted therapies using monoclonal antibodies (mAbs) such as cetuximab and panitumumab are widely used for the treatment of metastatic colorectal cancer (mCRC). Still, the clinical efficacy of this treatment is hampered by mutations in RAS gene, allowing tumors to escape from anti-EGFR mAb therapy. It is well established that NK cells kill tumor cells by natural cytotoxicity and can in addition be activated upon binding of IgG1 mAbs through Fc receptors (CD16/FcγRIIIa) on their surface, thereby mediating antibody dependent cellular cytotoxicity (ADCC). In the current study, activated Peripheral Blood NK cells (PBNK) were combined with anti-EGFR mAbs to study their effect on the killing of EGFR+/- cancer cell lines, including those with RAS mutations. In vitro cytotoxicity experiments using colon cancer primary tumors and cell lines COLO320, Caco-2, SW620, SW480 and HT-29, demonstrated that PBNK cells are cytotoxic for a range of tumor cells, regardless of EGFR, RAS or BRAF status and at low E:T ratios. Cetuximab enhanced the cytotoxic activity of NK cells on EGFR+ tumor cells (either RASwt, RASmut or BRAFmut) in a CD16 dependent manner, whereas it could not increase the killing of EGFR- COLO320. Our study provides a rationale to strengthen NK cell immunotherapy through a combination with cetuximab for RAS and BRAF mutant mCRC patients. PMID:27314237

  8. Genotyping Single Nucleotide Polymorphisms and Copy Number Variability of the FCGRs Expressed on NK Cells.

    PubMed

    Erbe, Amy K; Wang, Wei; Gallenberger, Mikayla; Hank, Jacquelyn A; Sondel, Paul M

    2016-01-01

    Natural killer (NK) cells are one of the main effector immune cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Upon recognition of cell-bound IgG antibodies, which occurs through Fc gamma receptors (FCGRs) expressed on the cell surface of NK cells, NK cells become activated and lyse target tumor or infected cells. The FCGRs, FCGR3A and FCGR2C, expressed on the surface of NK cells have single nucleotide polymorphisms (SNPs) that result in differential activity of NK cells. In addition to SNP genetic variation within each of these genes, the FCGRs are subject to copy number variation (CNV), which leads to variable protein expression levels on the cell surface. Studies have found that FCGR genotype for FCGR3A and FCGR2C is associated with variation in the response to immunotherapy.Due to high sequence homology within FCGR3 and FCGR2 families, there are difficulties associated with genotyping these specific receptors related to cross-amplification of non-targeted FCGRs. To improve specificity for both FCGR3A and FCGR2C, Rnase-H (RH) primers were designed to amplify specifically FCGR3A (while not co-amplifying FCGR3B) and FCGR2C (while not co-amplifying FCGR2B). In addition, fluorescently labeled locked nucleic acid (LNA) probes provide additional precision for determination of the SNPs within both FCGR3A and FCGR2C. For CNV determination, separate fluorescently labeled probes for FCGR3A, and for FCGR2C, can be used with the same RH primers for each gene. These probes can be combined in the same well with control primers/probe for a known diploid gene and used to calculate the copy number of both FCGR3A and FCGR2C. Here we provide new detailed methodology that allows for the specific amplification of these FCGRs in a single PCR reaction, allowing for genotyping of both the SNPs and CNVs using real-time PCR.

  9. Tim-3 pathway affects NK cell impairment in patients with active tuberculosis.

    PubMed

    Wang, Feng; Hou, Hongyan; Wu, Shiji; Tang, Qing; Huang, Min; Yin, Botao; Huang, Jing; Liu, Weiyong; Mao, Lie; Lu, Yanfang; Sun, Ziyong

    2015-12-01

    Active tuberculosis (TB) patients show impaired NK cell function, and the underlying mechanism remains largely unknown. In this study, we confirmed the decrease in activation, cytokine secretion, and degranulation potential of NK cells in active TB patients. We further investigated whether coinhibitory receptor Tim-3 was involved with impairment of NK cells. Our results revealed that the expression of Tim-3 on NK cells was increased in active TB patients. Tim-3 expression was inversely correlated with IL-12-stimualted IFN-γ production. Moreover, blocking the Tim-3 pathway restored IFN-γ secretion and degranulation of NK cells. Blocking this pathway also increased NK cell cytotoxicity against K562 target cells, and improved the ability of NK cells to control Mtb growth in monocyte-derived macrophages. The Tim-3 expression on NK cells was also observed to be significantly decreased in TB patients post-treatment. In this study, we have identified that Tim-3 is involved with NK cell impairment in TB patients.

  10. Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing.

    PubMed

    Kearney, Conor J; Ramsbottom, Kelly M; Voskoboinik, Ilia; Darcy, Phillip K; Oliaro, Jane

    2016-08-01

    Acute myeloid leukemia (AML) is associated with poor natural killer (NK) cell function through aberrant expression of NK-cell-activating receptors and their ligands on tumor cells. These alterations are thought to promote formation of inhibitory NK-target cell synapses, in which killer cell degranulation is attenuated. Allogeneic stem cell transplantation can be effective in treating AML, through restoration of NK cell lytic activity. Similarly, agents that augment NK-cell-activating signals within the immunological synapse may provide some therapeutic benefit. However, the receptor-ligand interactions that critically dictate NK cell function in AML remain undefined. Here, we demonstrate that CD112/CD155 expression is required for DNAM-1-dependent killing of AML cells. Indeed, the low, or absent, expression of CD112/CD155 on multiple AML cell lines resulted in failure to stimulate optimal NK cell function. Importantly, isolated clones with low CD112/155 expression were resistant to NK cell killing while those expressing abundant levels of CD112/155 were highly susceptible. Attenuated NK cell killing in the absence of CD112/CD155 originated from decreased NK-target cell conjugation. Furthermore, we reveal by time-lapse microscopy, a significant increase in NK cell 'failed killing' in the absence of DNAM-1 ligands. Consequently, NK cells preferentially lysed ligand-expressing cells within heterogeneous populations, driving clonal selection of CD112/CD155-negative blasts upon NK cell attack. Taken together, we identify reduced CD155 expression as a major NK cell escape mechanism in AML and an opportunity for targeted immunotherapy. PMID:27622064

  11. Laminin: a possible role as a mediator of natural cell-mediated functions

    SciTech Connect

    Laybourn, K.A.

    1986-01-01

    Natural Cell-mediated cytotoxicity is clearly important in regulating host response during tumorgenicity. Natural killer (NK), and Natural Cytotoxic (NC) lymphocytes responsible for mediating cytolysis, are subpopulations of non-B, non-T, nonphagocytic, nonadherent lymphocytes capable of spontaneously lysing a variety of tumor targets. Evidence is presented that laminin, a high molecular weight glycoprotein, is present on the surface of murine NK cells. In addition, NK sensitive tumor targets are able to bind laminin. This suggest that laminin participates as a ligand in the binding of NK cells to their tumor targets. The saturable binding of laminin by NK and NC sensitive tumor targets was shown by laminin-induced cell-cell aggregation/adherence, and /sup 125/I-laminin binding studies. Exogenous laminin inhibited NK cytotoxicity but not CTL cytotoxicity in vitro. In comparison, NK-resistant tumor cells bound little, if any exogenous laminin. Modulating NK activity in vivo prior to challenging mice with an inoculation of a murine fibrosarcoma, showed that elimination or activation of NK activity resulted in an increase or decrease in pulmonary metastases, respectively.

  12. Folate-conjugated immunoglobulin targets melanoma tumor cells for NK cell effector functions

    PubMed Central

    Skinner, Cassandra C.; McMichael, Elizabeth L.; Jaime-Ramirez, Alena C.; Abrams, Zachary B.; Lee, Robert J.; Carson, William E.

    2016-01-01

    The folate receptor (FR) is over-expressed on the vascular side of cancerous cells including those of the breast, ovaries, testes, and cervix. We hypothesized that a folate-conjugated immunoglobulin (F-IgG) would bind to the FR that is over-expressed on melanoma tumor cells to target these cells for lysis by natural killer (NK) cells. Folate receptor expression was confirmed in the Mel-39 (human melanoma) cell line by flow cytometry and immunoblot analysis, using KB (human oral epithelial) and F01 (human melanoma) as a positive and negative control, respectively. FR-positive and negative cell lines were treated with F-IgG or control immunoglobulin G (C-IgG) in the presence or absence of cytokines in order to determine NK cell ability to lyse FR-positive cell lines. NK cell activation was significantly upregulated and lysis of Mel 39 tumor cells enhanced following treatment with F-IgG, as compared to C-IgG at all effector:target (E:T) ratios (p<0.01). This trend was further enhanced by NK cell stimulation with the activating cytokine interleukin-12 (IL-12). NK cell production of cytokines such as interferon-gamma (IFN-γ), macrophage inflammatory protein 1 alpha (MIP-1α), and regulated on activation normal T-cell expressed and secreted (RANTES) were also significantly increased in response to co-stimulation with IL-12 stimulation and F-IgG-coated Mel 39 target cells, as compared to controls (p<0.01). In contrast, F-IgG did not bind to the FR-negative cell line F01 and had no significant effect on NK cell lysis or cytokine production. This research indicates the potential use of F-IgG for its ability to induce an immune response from NK cells against FR-positive melanoma tumor cells which can be further enhanced by the addition of cytokines. PMID:27035691

  13. A role for pre-mNK cells in tumor progression.

    PubMed

    Rosinsky, Carolyn; Antony, Paul Andrew

    2016-01-01

    The innate and adaptive immune systems have evolved together to fight infection and cancerous tissues. The innate immune system emerges first with the adaptive immune system following, both ostensibly being bridged by dendritic cells (DC). Recently cells have emerged that possess characteristics of both innate and adaptive immune cell qualities, termed interferon-producing killer dendritic cells (IKDCs). These cells have an indistinct origin that is not well understood. They appear to have more NK cell attributes than DC but purportedly can regulate the immune system similar to immunoregulatory NK cells. Because of this, they have been renamed pre-mNK cells (pre-mature NK cells). We argue in this commentary that pre-mNK cells may contribute to cancer recurrence. PMID:26981246

  14. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    PubMed

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response. PMID:27127520

  15. Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib

    PubMed Central

    Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

    2015-01-01

    There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8+ T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses. PMID:26405606

  16. Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

    PubMed

    Kelly, Michelle N; Zheng, Mingquan; Ruan, Sanbao; Kolls, Jay; D'Souza, Alain; Shellito, Judd E

    2013-01-01

    Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

  17. Influenza Vaccination Generates Cytokine-Induced Memory-like NK Cells: Impact of Human Cytomegalovirus Infection

    PubMed Central

    Goodier, Martin R.; Rodriguez-Galan, Ana; Lusa, Chiara; Nielsen, Carolyn M.; Darboe, Alansana; Moldoveanu, Ana L.; White, Matthew J.; Behrens, Ron

    2016-01-01

    Human NK cells are activated by cytokines, immune complexes, and signals transduced via activating ligands on other host cells. After vaccination, or during secondary infection, adaptive immune responses can enhance both cytokine-driven and Ab-dependent NK cell responses. However, induction of NK cells for enhanced function after in vitro exposure to innate inflammatory cytokines has also been reported and may synergize with adaptive signals to potentiate NK cell activity during infection or vaccination. To test this hypothesis, we examined the effect of seasonal influenza vaccination on NK cell function and phenotype in 52 previously unvaccinated individuals. Enhanced, IL-2–dependent, NK cell IFN-γ responses to Influenza A/California/7/2009 virus were detected up to 4 wk postvaccination and higher in human CMV (HCMV)-seronegative (HCMV−) individuals than in HCMV-seropositive (HCMV+) individuals. By comparison, robust NK cell degranulation responses were observed both before and after vaccination, due to high titers of naturally occurring anti-influenza Abs in human plasma, and did not differ between HCMV+ and HCMV− subjects. In addition to these IL-2–dependent and Ab-dependent responses, NK cell responses to innate cytokines were also enhanced after influenza vaccination; this was associated with proliferation of CD57− NK cells and was most evident in HCMV+ subjects. Similar enhancement of cytokine responsiveness was observed when NK cells were cocultured in vitro with Influenza A/California/7/2009 virus, and this was at least partially dependent upon IFN-αβR2. In summary, our data indicate that attenuated or live viral vaccines promote cytokine-induced memory-like NK cells and that this process is influenced by HCMV infection. PMID:27233958

  18. Influenza Vaccination Generates Cytokine-Induced Memory-like NK Cells: Impact of Human Cytomegalovirus Infection.

    PubMed

    Goodier, Martin R; Rodriguez-Galan, Ana; Lusa, Chiara; Nielsen, Carolyn M; Darboe, Alansana; Moldoveanu, Ana L; White, Matthew J; Behrens, Ron; Riley, Eleanor M

    2016-07-01

    Human NK cells are activated by cytokines, immune complexes, and signals transduced via activating ligands on other host cells. After vaccination, or during secondary infection, adaptive immune responses can enhance both cytokine-driven and Ab-dependent NK cell responses. However, induction of NK cells for enhanced function after in vitro exposure to innate inflammatory cytokines has also been reported and may synergize with adaptive signals to potentiate NK cell activity during infection or vaccination. To test this hypothesis, we examined the effect of seasonal influenza vaccination on NK cell function and phenotype in 52 previously unvaccinated individuals. Enhanced, IL-2-dependent, NK cell IFN-γ responses to Influenza A/California/7/2009 virus were detected up to 4 wk postvaccination and higher in human CMV (HCMV)-seronegative (HCMV(-)) individuals than in HCMV-seropositive (HCMV(+)) individuals. By comparison, robust NK cell degranulation responses were observed both before and after vaccination, due to high titers of naturally occurring anti-influenza Abs in human plasma, and did not differ between HCMV(+) and HCMV(-) subjects. In addition to these IL-2-dependent and Ab-dependent responses, NK cell responses to innate cytokines were also enhanced after influenza vaccination; this was associated with proliferation of CD57(-) NK cells and was most evident in HCMV(+) subjects. Similar enhancement of cytokine responsiveness was observed when NK cells were cocultured in vitro with Influenza A/California/7/2009 virus, and this was at least partially dependent upon IFN-αβR2. In summary, our data indicate that attenuated or live viral vaccines promote cytokine-induced memory-like NK cells and that this process is influenced by HCMV infection. PMID:27233958

  19. Lysis of HIV-1 Infected Autologous CD4+ Primary T cells by Interferon-alpha Activated NK cells Requires NKp46 and NKG2D

    PubMed Central

    Tomescu, Costin; Mavilio, Domenico; Montaner, Luis J.

    2015-01-01

    Objective Autologous HIV-1 infected CD4+ primary T cells (aHIV+CD4) have been shown to be largely resistant to Natural Killer (NK) cell mediated lysis due to viral strategies of immune evasion. We have previously shown that a pre-activation of NK cells with Plasmacytoid Dendritic Cells can significantly augment lysis of aHIV+CD4 through a mechanism dependent on Interferon-alpha (IFN-α). Design The goal of the present study is to identify the specific NK activating receptors involved in NK lysis of aHIV+CD4 following IFN-α activation. Methods PBMC were incubated with aHIV+CD4 to induce the secretion of endogenous levels of IFN-α and drive NK activation. We then utilized a standard chromium lysis assay to assess the degree of IFN-α activated lysis of aHIV+CD4 in the presence or absence of masking antibodies to a panel of NK activating receptors and co-receptors. Results Direct recognition of HIV-1 infected, but not uninfected, autologous CD4+ primary T cells by PBMC induced the secretion IFN-α (Median 2280 pg/ml, p<0.001, n=9) that, in turn, activated NK cells (p<0.001, n=12) and significantly increased their cytolytic potential against aHIV+CD4 (p<0.01, n=12). The masking of NKp46 (p<0.01, n=8) and NKG2D (p<0.05, n=8), but not 2B4, NTBA, NKp30 or NKp44, significantly reduced IFN-α activated lysis of aHIV+CD4. Conclusions Taken together, these results demonstrate that endogenous levels of IFN-α secreted by pDCs induce NK cells to lyse aHIV+CD4 via the engagement of NKp46 and NKG2D. PMID:26372382

  20. Deletion of Slam locus in mice reveals inhibitory role of SLAM family in NK cell responses regulated by cytokines and LFA-1.

    PubMed

    Guo, Huaijian; Cranert, Stacey A; Lu, Yan; Zhong, Ming-Chao; Zhang, Shaohua; Chen, Jun; Li, Rui; Mahl, Sarah E; Wu, Ning; Davidson, Dominique; Waggoner, Stephen N; Veillette, André

    2016-09-19

    Signaling lymphocytic activation molecule (SLAM) family receptors (SFRs) can mediate either activating or inhibitory effects during natural killer cell (NK cell) activation. In this study, we addressed the global role, regulation, and mechanism of action of the SLAM family in NK cells by analyzing a mouse lacking the entire ∼400-kilobase Slam locus, which encodes all six SFRs and CD48, the ligand of SFR 2B4. This mouse displayed enhanced NK cell activation responses toward hematopoietic target cells. Analyses of mice lacking individual SFRs showed that the inhibitory function of the Slam locus was due solely to 2B4 and was not influenced positively or negatively by other SFRs. Differences in NK cell responses between recognition of targets expressing or lacking ligands for SFRs were enhanced by IL-12 but suppressed by type I interferon. Cytokines also changed the levels of SLAM-associated protein adaptors, which prevent the inhibitory function of SFRs. The enhanced activation responses of SFR-deficient NK cells were dependent on integrin LFA-1 but not on DNAM-1 or NKG2D. SFR-mediated inhibition prevented the generation of activated forms of LFA-1. Hence, the Slam locus has an overall inhibitory role during NK cell activation that is solely dependent on 2B4. This effect is influenced by cytokines and leads to suppression of LFA-1 activity.

  1. Chronic HCV Infection Affects the NK Cell Phenotype in the Blood More than in the Liver

    PubMed Central

    Kroy, Daniela C.; Cheney, Patrick C.; Ghebremichael, Musie; Aneja, Jasneet; Tomlinson, Michelle; Kim, Arthur Y.; Lauer, Georg M.; Alter, Galit

    2014-01-01

    Although epidemiological and functional studies have implicated NK cells in protection and early clearance of HCV, the mechanism by which they may contribute to viral control is poorly understood, particularly at the site of infection, the liver. We hypothesized that a unique immunophenotypic/functional NK cell signature exists in the liver that may provide insights into the contribution of NK cells to viral control. Intrahepatic and blood NK cells were profiled from chronically infected HCV-positive and HCV-negative individuals. Baseline expression of activating and inhibitory receptors was assessed, as well as functional responses following stimulation through classic NK cell pathways. Independent of HCV infection, the liver was enriched for the immunoregulatory CD56bright NK cell population, which produced less IFNγ and CD107a but comparable levels of MIP1β, and was immunophenotypically distinct from their blood counterparts. This profile was mostly unaltered in chronic HCV infection, though different expression levels of NKp46 and NKG2D were associated with different grades of fibrosis. In contrast to the liver, chronic HCV infection associated with an enrichment of CD161lowperforinhigh NK cells in the blood correlated with increased AST and 2B4 expression. However, the association of relatively discrete changes in the NK cell phenotype in the liver with the fibrosis stage nevertheless suggests an important role for the NK response. Overall these data suggest that tissue localization has a more pervasive effect on NK cells than the presence of chronic viral infection, during which these cells might be mostly attuned to limiting immunopathology. It will be important to characterize NK cells during early HCV infection, when they should have a critical role in limiting infection. PMID:25148254

  2. NK-cell enteropathy: a benign NK-cell lymphoproliferative disease mimicking intestinal lymphoma: clinicopathologic features and follow-up in a unique case series.

    PubMed

    Mansoor, Adnan; Pittaluga, Stefania; Beck, Paul L; Wilson, Wyndham H; Ferry, Judith A; Jaffe, Elaine S

    2011-02-01

    Intestinal T-cell and natural killer (NK)-cell lymphomas are clinically aggressive and can be challenging to diagnose in small endoscopic biopsies. We describe 8 patients in whom atypical NK-cell lymphoproliferative lesions mimicked NK- or T-cell lymphoma. The patients (2 men; 6 women; ages 27-68 years) presented with vague gastrointestinal symptoms with lesions involving stomach, duodenum, small intestine, and colon. At endoscopy, the lesions exhibited superficial ulceration, edema, and hemorrhage. Biopsies revealed a mucosal infiltrate of atypical cells with an NK-cell phenotype (CD56(+)/TIA-1(+)/Granzyme B(+)/cCD3(+)), which displaced but did not invade the glandular epithelium. Epstein-Barr virus-encoded RNA in situ hybridization was negative, and T-cell receptor-γ gene rearrangement showed no evidence of a clonal process. Based on an original diagnosis of lymphoma, 3 patients received aggressive chemotherapy followed by autologous bone marrow transplantation in 2. Five patients were followed without treatment. However, no patient developed progressive disease or died of lymphoma (median follow-up, 30 months). Repeat endoscopies in 6 of 8 patients showed persistence or recurrence of superficial gastrointestinal lesions. This unique entity mimics intestinal and NK-/T-cell lymphomas on endoscopic biopsies and can result in erroneous diagnosis, leading to aggressive chemotherapy. We propose the term "NK-cell enteropathy" for this syndrome of as yet unknown etiology.

  3. Epigenetic suppression of the antitumor cytotoxicity of NK cells by histone deacetylase inhibitor valproic acid

    PubMed Central

    Shi, Xiumin; Li, Min; Cui, Meizi; Niu, Chao; Xu, Jianting; Zhou, Lei; Li, Wei; Gao, Yushun; Kong, Weisheng; Cui, Jiuwei; Hu, Jifan; Jin, Haofan

    2016-01-01

    Natural killer (NK) cells play an essential role in the fight against tumor development. The therapeutic use of autologous NK cells has been exploited to treat human malignancies, yet only limited antitumor activity is observed in cancer patients. In this study, we sought to augment the antitumor activity of NK cells using epigenetic approaches. Four small molecules that have been known to promote epigenetic reprogramming were tested for their ability to enhance the activity of NK cells. Using a tumor cell lysis assay, we found that the DNA demethylating agent 5-azacytidine and vitamin C did not significantly affect the tumor killing ability of NK cells. The thyroid hormone triiodothyronine (T3) slightly increased the activity of NK cells. The histone deacetylase inhibitor valproic acid (VPA), however, inhibited NK cell lytic activity against leukemic cells in a dose-dependent manner. Pretreatment using VPA reduced IFNγ secretion, impaired CD107a degranulation, and induced apoptosis by activating the PD-1/PD-L1 pathway. VPA downregulated the expression of the activating receptor NKG2D (natural-killer group 2, member D) by inducing histone K9 hypermethylation and DNA methylation in the gene promoter. Histone deacetylase inhibitors have been developed as anticancer agents for use as monotherapies or in combination with other anticancer therapies. Our data suggest that the activity of histone deacetylase inhibitors on NK cell activity should be considered in drug development. PMID:27152238

  4. Murine viral hepatitis involves NK cell depletion associated with virus-induced apoptosis

    PubMed Central

    LEHOUX, M; JACQUES, A; LUSIGNAN, S; LAMONTAGNE, L

    2004-01-01

    Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of immunological disorders related to acute and chronic hepatitis. In this study, we have verified if the fulminant hepatitis induced by MHV3 could be related to an impairment of innate immunity. Groups of three C57BL/6 mice were infected with the pathogenic L2-MHV3 or attenuated YAC-MHV3 viruses, and the natural killer (NK) cell populations from liver, spleen and bone marrow were analysed. The percentage of intrahepatic NK1·1+T cell receptor (TCR)− cells did not increase while NK1·1+TCRinter cells decreased in both L2-MHV3- and YAC-MHV3-infected mice. Concurrently, splenic and myeloid NK1·1+ cells decreased in L2-MHV3-infected mice. However, the cytotoxic activity of NK cells increased in liver and decreased in bone marrow from pathogenic L2-MHV3-infected mice while no modification was detected in YAC-MHV3-infected mice. Flow cytometric analysis revealed that both normal and larger splenic or myeloid NK cells decreased more in pathogenic L2-MHV3-infected mice than in attenuated YAC-MHV3-infected mice. In vitro viral infections of interleukin (IL)-15-stimulated lymphoid cells from liver and bone marrow revealed that L2-MHV3 induced higher decreases in cell viability of NK1·1+ cells than the YAC-MHV3 variant. The NK cell decreases were due to the viral permissivity leading to cytopathic effects characterized by cell rounding, syncytia formation and apoptosis. Larger NK+ syncytia were observed in L2-MHV3-infected cells than in YAC-MHV3-infected cells. These results suggest that NK cell production is impaired by viral infection favouring fulminant hepatitis. PMID:15196242

  5. Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry

    PubMed Central

    Horowitz, Amir; Strauss-Albee, Dara M.; Leipold, Michael; Kubo, Jessica; Nemat-Gorgani, Neda; Dogan, Ozge C.; Dekker, Cornelia L.; Mackey, Sally; Maecker, Holden; Swan, Gary E.; Davis, Mark M.; Norman, Paul J.; Guethlein, Lisbeth A.; Desai, Manisha; Parham, Peter; Blish, Catherine A.

    2013-01-01

    Natural Killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 35 parameters, including 28 NK cell receptors, on peripheral blood NK cells from five sets of monozygotic twins and twelve unrelated donors of defined HLA and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6,000-30,000 phenotypic populations within an individual and >100,000 phenotypes in this population. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors, while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation. PMID:24154599

  6. NK cells lacking FcεRIγ are associated with reduced liver damage in chronic hepatitis C virus infection.

    PubMed

    Oh, Jun S; Ali, Alaa K; Kim, Sungjin; Corsi, Daniel J; Cooper, Curtis L; Lee, Seung-Hwan

    2016-04-01

    A novel subset of human natural killer (NK) cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently uncovered in cytomegalovirus (CMV)+ individuals. This NK-cell subset (g-NK) was characterized by a deficiency in the expression of FcεRIγ adaptor protein and the long-lasting memory-like NK-cell phenotype, suggesting a role in chronic infections. This study investigates whether the g-NK-cell subset is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK-cell proportions and function in the PBMCs of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of the g-NK-cell subset having similarly enhanced antibody-dependent cellular cytotoxicity responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56(neg) NK cell population often found in chronic HCV patients, suggesting their involvement in immune response during HCV infection. For the first time, our findings indicate that the presence of the g-NK cells in CMV+ individuals is associated with amelioration of liver disease in chronic HCV infection, suggesting the beneficial roles of g-NK cells during a chronic infection.

  7. Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol.

    PubMed

    Koehl, Ulrike; Brehm, Claudia; Huenecke, Sabine; Zimmermann, Stefanie-Yvonne; Kloess, Stephan; Bremm, Melanie; Ullrich, Evelyn; Soerensen, Jan; Quaiser, Andrea; Erben, Stephanie; Wunram, Claudia; Gardlowski, Tanja; Auth, Eileen; Tonn, Torsten; Seidl, Christian; Meyer-Monard, Sandrine; Stern, Martin; Passweg, Jakob; Klingebiel, Thomas; Bader, Peter; Schwabe, Dirk; Esser, Ruth

    2013-01-01

    Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

  8. T-bet and Eomesodermin in NK Cell Development, Maturation, and Function

    PubMed Central

    Simonetta, Federico; Pradier, Amandine; Roosnek, Eddy

    2016-01-01

    Recent reports give insights into the role of the T-box transcription factors, T-bet and Eomesodermin (Eomes), in NK cell biology. In this mini-review, we recapitulate the initial reports that delineate T-bet and Eomes as master regulators of NK cell development, maturation, and function. We discuss how T-bet and Eomes expression is regulated during NK cell development and peripheral maturation. Furthermore, we summarize the current literature on the role of T-bet and Eomes in the transcriptional regulation of NK cell function and review possible effects of T-box transcription factor anomalies during aging, infection, cancer, and after hematopoietic stem cell transplantation. We discuss how the current data argue in favor of a model of T-bet and Eomes synergy in transcriptional regulation of NK cell function and identify T-box transcription factors as potential targets for therapeutic interventions. PMID:27379101

  9. Know Thyself: NK-Cell Inhibitory Receptors Prompt Self-Tolerance, Education, and Viral Control

    PubMed Central

    Nash, William T.; Teoh, Jeffrey; Wei, Hairong; Gamache, Awndre; Brown, Michael G.

    2014-01-01

    Natural killer (NK) cells provide essential protection against viral infections. One of the defining features of this lymphocyte population is the expression of a wide array of variable cell surface stimulatory and inhibitory NK receptors (sNKR and iNKR, respectively). The iNKR are particularly important in terms of NK-cell education. As receptors specific for MHC class I (MHC I) molecules, they are responsible for self-tolerance and adjusting NK-cell reactivity based on the expression level of self-MHC I. The end result of this education is twofold: (1) inhibitory signaling tunes the functional capacity of the NK cell, endowing greater potency with greater education, and (2) education on self allows the NK cell to detect aberrations in MHC I expression, a common occurrence during many viral infections. Many studies have indicated an important role for iNKR and MHC I in disease, making these receptors attractive targets for manipulating NK-cell reactivity in the clinic. A greater understanding of iNKR and their ability to regulate NK cells will provide a basis for future attempts at translating their potential utility into benefits for human health. PMID:24795719

  10. Early Murine Cytomegalovirus (MCMV) Infection Induces Liver Natural Killer (NK) Cell Inflammation and Protection Through Macrophage Inflammatory Protein 1α (MIP-1α)–dependent Pathways

    PubMed Central

    Salazar-Mather, Thais P.; Orange, Jordan S.; Biron, Christine A.

    1998-01-01

    Natural killer (NK) cells mediate defense against early murine cytomegalovirus (MCMV) infections in liver. The chemokine, macrophage inflammatory protein 1α (MIP-1α), can promote inflammatory responses. Our studies evaluated contributions of NK cells to early MCMV-induced liver inflammation and MIP-1α requirements for inflammation and delivery of antiviral defenses. NK cells were shown to be responsible for focal inflammation, and to be induced to migrate at high levels, in MCMV-infected livers. MIP-1α gene expression was elevated at coinciding times, and mice deficient in MIP-1α function were dramatically inhibited in both inflammatory and protective liver responses. The results precisely define MIP-1α–dependent steps required to achieve NK cell inflammation during, and mechanisms promoting defense against, viral infections in tissues. PMID:9419206

  11. B1a cells enhance susceptibility to infection with virulent Francisella tularensis via modulation of NK/NKT cell responses*

    PubMed Central

    Crane, Deborah D.; Griffin, Amanda J.; Wehrly, Tara D.; Bosio, Catharine M.

    2013-01-01

    B1a cells are an important source of natural antibodies, antibodies directed against T-independent antigens, and are a primary source of IL-10. Bruton's tyrosine kinase (btk) is a cytoplasmic kinase that is essential for mediating signals from the B cell receptor and is critical for development of B1a cells. Consequentially, animals lacking btk have few B1a cells, minimal antibody responses, and can preferentially generate Th1 type immune responses following infection. B1a cells have been shown to aid in protection against infection with attenuated Francisella tularensis but their role in infection mediated by fully virulent F. tularensis is not known. Therefore, we utilized mice with defective btk (XID mice) to determine the contribution of B1a cells in defense against the virulent, F. tularensis ssp. tularensis strain SchuS4. Surprisingly, XID mice displayed increased resistance to pulmonary infection with F. tularensis. Specifically, XID mice had enhanced clearance of bacteria from the lung and spleen and significantly greater survival of infection compared to wild type controls. We revealed that resistance to infection in XID mice was associated with decreased numbers of IL-10 producing B1a cells and concomitant increased numbers of IL-12 producing macrophages and IFN-γ producing NK/NKT cells. Adoptive transfer of wild type B1a cells into XID mice reversed the control of bacterial replication. Similarly, depletion of NK/NKT cells also increased bacterial burdens in XID mice. Together, our data suggest B cell-NK/NKT cell crosstalk is a critical pivot controlling survival of infection with virulent F. tularensis. PMID:23378429

  12. Conventional NK cells can produce IL-22 and promote host defense in Klebsiella pneumoniae pneumonia.

    PubMed

    Xu, Xin; Weiss, Ido D; Zhang, Hongwei H; Singh, Satya P; Wynn, Thomas A; Wilson, Mark S; Farber, Joshua M

    2014-02-15

    It was reported that host defense against pulmonary Klebsiella pneumoniae infection requires IL-22, which was proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22(-/-) mice had decreased survival compared with wild-type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2(-/-) mice did not differ from wild-type mice in survival or levels of IL-22 in the lungs postinfection with K. pneumoniae. In contrast, K. pneumoniae-infected Rag2(-/-)Il2rg(-/-) mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells in host defense and production of IL-22. Unlike NK cell-like innate lymphoid cells that produce IL-22 and display a surface phenotype of NK1.1(-)NKp46(+)CCR6(+), lung NK cells showed the conventional phenotype, NK1.1(+)NKp46(+)CCR6(-). Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver, compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within 1 d postinfection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild-type and Rag2(-/-) mice. Our data suggest that, during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.

  13. Effects of a cloned cell line with NK activity on bone marrow transplants, tumour development and metastasis in vivo

    NASA Astrophysics Data System (ADS)

    Warner, John F.; Dennert, Gunther

    1982-11-01

    Natural killer (NK) cells cloned in vitro have been transferred into NK-deficient hosts. These cells have been shown to have a role in the rejection of allogeneic bone marrow grafts, resistance to both radiation-induced thymic leukaemia and challenge with melanoma tumour cells. It appears that NK cells have an important role in immune surveillance.

  14. NK Cell Responses to Human Tick-Borne Encephalitis Virus Infection.

    PubMed

    Blom, Kim; Braun, Monika; Pakalniene, Jolita; Lunemann, Sebastian; Enqvist, Monika; Dailidyte, Laura; Schaffer, Marie; Lindquist, Lars; Mickiene, Aukse; Michaëlsson, Jakob; Ljunggren, Hans-Gustaf; Gredmark-Russ, Sara

    2016-10-01

    Tick-borne encephalitis virus (TBEV) is a flavivirus that is transferred to humans by infected ticks. The virus causes tick-borne encephalitis, a severe infection of the CNS with a high risk for long-lasting sequelae. Currently, no treatment exists for the disease. Understanding the cellular immune response to this infection is important to gain further understanding into the pathogenesis, treatment, and prevention of the disease. NK cells are known to participate in the control of viral infections. We performed a longitudinal analysis of the human NK cell response to TBEV infection in a cohort of infected individuals from the onset of severe clinical symptoms to the convalescence phase. NK cell activation, as measured by expression of Ki67, was apparent at the time of hospitalization. By 3 wk after hospitalization, it decreased to levels seen in healthy controls. Concomitant with the increase in NK cell activation, augmented levels of IL-12, IL-15, IL-18, IFN-γ, and TNF were detected in patient plasma. This TBEV-induced NK cell activation was restricted predominantly to differentiated CD57(+)CD56(dim) NK cells. Functionally, CD56(dim) NK cells responded poorly to target cells at the time of hospitalization, but they recovered functional capacity to control levels during the convalescent phase. In contrast, the responsiveness of NK cells to cytokine stimulation remained intact throughout the disease. These findings demonstrate that NK cells respond to TBEV infection with characteristics that are distinct from those of other human viral infections and provide insights into the NK cell response to clinical TBEV infection. PMID:27543616

  15. Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

    PubMed Central

    Kyei, Stephen K.; Ogbomo, Henry; Li, ShuShun; Timm-McCann, Martina; Xiang, Richard F.; Huston, Shaunna M.; Ganguly, Anutosh; Colarusso, Pina; Gill, M. John

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans. Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. PMID:27555306

  16. Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.

    PubMed

    Peppa, Dimitra; Micco, Lorenzo; Javaid, Alia; Kennedy, Patrick T F; Schurich, Anna; Dunn, Claire; Pallant, Celeste; Ellis, Gidon; Khanna, Pooja; Dusheiko, Geoffrey; Gilson, Richard J; Maini, Mala K

    2010-01-01

    NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-β blockade. PMID:21187913

  17. Tetrabromobisphenol A Decreases Cell Surface Proteins Involved in Human Natural Killer (NK) Cell-Dependent Target Cell Lysis

    PubMed Central

    Hurd, Tasia; Whalen, Margaret M.

    2011-01-01

    Human natural killer (NK) lymphocytes are able to destroy tumor cells and virally-infected cells. Interference with their function can leave an individual with increased susceptibility to cancer development and/or viral infection. We have shown that the tumor destroying (lytic) function of NK cells can be dramatically decreased by exposure to the environmental contaminant tetra-bromobisphenol A (TBBPA). TBBPA is a flame retardant used in a variety of materials including circuit boards, carpeting, and upholstery and has been found in human blood samples. TBBPA interferes with NK cell lytic function, in part, by decreasing the ability of NK cells to bind to target cells. This study examines the effects of exposures to concentrations of TBBPA (i.e., that were able to decrease the binding capacity of NK cells) on the expression of cell-surface proteins (CD2, CD11a, CD16, CD18, and CD56) that are needed for NK cells to bind target cells. NK cells were exposed to TBBPA for 24 hr, 48 hr, and 6 d or for 1 hr followed by 24 hr, 48 hr, and 6 d in TBBPA-free media. Twenty-four hr exposures to 5 µM TBBPA caused decreases in four of the cell surface proteins examined. CD16 was decreased by > 35%. The decreases in cell surface proteins after a 48 hr exposure were similar to those seen after 24 hr. The results indicate that TBBPA exposures that decrease the binding function of human NK cells do so by decreasing the expression of cell surface proteins needed for attachment of NK cells to targets cells. PMID:21623697

  18. Dysregulation of Chemokine/Chemokine Receptor Axes and NK Cell Tissue Localization during Diseases

    PubMed Central

    Bernardini, Giovanni; Antonangeli, Fabrizio; Bonanni, Valentina; Santoni, Angela

    2016-01-01

    Chemokines are small chemotactic molecules that play key roles in physiological and pathological conditions. Upon signaling via their specific receptors, chemokines regulate tissue mobilization and trafficking of a wide array of immune cells, including natural killer (NK) cells. Current research is focused on analyzing changes in chemokine/chemokine receptor expression during various diseases to interfere with pathological trafficking of cells or to recruit selected cell types to specific tissues. NK cells are a heterogeneous lymphocyte population comprising several subsets endowed with distinct functional properties and mainly representing distinct stages of a linear development process. Because of their different functional potential, the type of subset that accumulates in a tissue drives the final outcome of NK cell-regulated immune response, leading to either protection or pathology. Correspondingly, chemokine receptors, including CXCR4, CXCR3, and CX3CR1, are differentially expressed by NK cell subsets, and their expression levels can be modulated during NK cell activation. At first, this review will summarize the current knowledge on the contribution of chemokines to the localization and generation of NK cell subsets in homeostasis. How an inappropriate chemotactic response can lead to pathology and how chemokine targeting can therapeutically affect tissue recruitment/localization of distinct NK cell subsets will also be discussed. PMID:27766097

  19. Chemokine Receptor Expression on Normal Blood CD56+ NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

    PubMed Central

    Lima, Margarida; Leander, Magdalena; Santos, Marlene; Santos, Ana Helena; Lau, Catarina; Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João; Teixeira, Maria dos Anjos; Orfao, Alberto

    2015-01-01

    Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56+low CD16+ and CD56+high  CD16−/+low NK-cells. Conventional CD56+low and CD56+high NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56+low NK-cells are mainly CXCR1/CXCR2+ and CXCR3/CCR5−/+, whereas mostly CD56+high NK-cells are CXCR1/CXCR2− and CXCR3/CCR5+. Both NK-cell subsets have variable CXCR4 expression and are CCR4− and CCR6−. The CKR repertoire of the CD56+low NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56+high NK-cells mimics that of Th1+ T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56+int NK-cells. These NK-cells are CXCR3/CCR5+, they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57− and CD158a−. In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56+high and CD56+low NK-cells populations. PMID:26543875

  20. NK Cell Activation in the Antitumor Response Induced by IFN-α Dendritic Cells Loaded with Apoptotic Cells from Follicular Lymphoma Patients.

    PubMed

    Lapenta, Caterina; Donati, Simona; Spadaro, Francesca; Castaldo, Paolo; Belardelli, Filippo; Cox, Maria C; Santini, Stefano M

    2016-08-01

    Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity. PMID:27357153

  1. NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

    PubMed Central

    Azeredo, E L; De Oliveira-Pinto, L M; Zagne, S M; Cerqueira, D I S; Nogueira, R M R; Kubelka, C F

    2006-01-01

    During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3−) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3−, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease. PMID:16412060

  2. Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition

    PubMed Central

    Kwon, Hyung-Joon; Choi, Go-Eun; Ryu, Sangryeol; Kwon, Soon Jae; Kim, Sun Chang; Booth, Claire; Nichols, Kim E.; Kim, Hun Sik

    2016-01-01

    NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses. PMID:27221592

  3. IFN‐λ3 polymorphism indirectly influences NK cell phenotype and function during acute HCV infection

    PubMed Central

    Depla, Marion; Pelletier, Sandy; Bédard, Nathalie; Brunaud, Camille; Bruneau, Julie

    2016-01-01

    Abstract Introduction Polymorphisms in the type III interferon IFN‐λ3 and the killer cell immunoglobulin‐like receptor (KIR) genes controlling the activity of natural killer (NK) cells can predict spontaneous resolution of acute hepatitis C virus (HCV) infection. We hypothesized that IFN‐λ3 polymorphism may modulate NK cell function during acute HCV. Methods We monitored the plasma levels of type III IFNs in relation to the phenotype and the function of NK cells in a cohort of people who inject drugs (PWID) during acute HCV infection with different outcomes. Results Early acute HCV was associated with high variability in type III IFNs plasma levels and the favorable IFN‐λ3 CC genotype was associated with higher viral loads. Reduced expression of Natural Killer Group Protein 2A (NKG2A) was associated with lower IFN‐λ3 plasma levels and the CC genotype. IFN‐γ production by NK cells was higher in individuals with the CC genotype during acute infection but this did not prevent viral persistence. IFN‐λ3 plasma levels did not correlate with function of NK cells and IFN‐λ3 prestimulation did not affect NK cell activation and function. Conclusions These results suggest that IFN‐λ3 polymorphism indirectly influences NK cell phenotype and function during acute HCV but other factors may act in concert to determine the outcome of the infection. PMID:27621819

  4. IFN‐λ3 polymorphism indirectly influences NK cell phenotype and function during acute HCV infection

    PubMed Central

    Depla, Marion; Pelletier, Sandy; Bédard, Nathalie; Brunaud, Camille; Bruneau, Julie

    2016-01-01

    Abstract Introduction Polymorphisms in the type III interferon IFN‐λ3 and the killer cell immunoglobulin‐like receptor (KIR) genes controlling the activity of natural killer (NK) cells can predict spontaneous resolution of acute hepatitis C virus (HCV) infection. We hypothesized that IFN‐λ3 polymorphism may modulate NK cell function during acute HCV. Methods We monitored the plasma levels of type III IFNs in relation to the phenotype and the function of NK cells in a cohort of people who inject drugs (PWID) during acute HCV infection with different outcomes. Results Early acute HCV was associated with high variability in type III IFNs plasma levels and the favorable IFN‐λ3 CC genotype was associated with higher viral loads. Reduced expression of Natural Killer Group Protein 2A (NKG2A) was associated with lower IFN‐λ3 plasma levels and the CC genotype. IFN‐γ production by NK cells was higher in individuals with the CC genotype during acute infection but this did not prevent viral persistence. IFN‐λ3 plasma levels did not correlate with function of NK cells and IFN‐λ3 prestimulation did not affect NK cell activation and function. Conclusions These results suggest that IFN‐λ3 polymorphism indirectly influences NK cell phenotype and function during acute HCV but other factors may act in concert to determine the outcome of the infection.

  5. Cetuximab Reconstitutes Pro-Inflammatory Cytokine Secretions and Tumor-Infiltrating Capabilities of sMICA-Inhibited NK Cells in HNSCC Tumor Spheroids

    PubMed Central

    Klöss, Stephan; Chambron, Nicole; Gardlowski, Tanja; Weil, Sandra; Koch, Joachim; Esser, Ruth; Pogge von Strandmann, Elke; Morgan, Michael A.; Arseniev, Lubomir; Seitz, Oliver; Köhl, Ulrike

    2015-01-01

    Immunosuppressive factors, such as soluble major histocompatibility complex class I chain-related peptide A (sMICA) and transforming growth factor beta 1 (TGF-β1), are involved in tumor immune escape mechanisms (TIEMs) exhibited by head and neck squamous cell carcinomas (HNSCCs) and may represent opportunities for therapeutic intervention. In order to overcome TIEMs, we investigated the antibody-dependent cellular cytotoxicity (ADCC), cytokine release and retargeted tumor infiltration of sMICA-inhibited patient NK cells expressing Fcγ receptor IIIa (FcγRIIIa, CD16a) in the presence of cetuximab, an anti-epidermal growth factor receptor (HER1) monoclonal antibody (mAb). Compared to healthy controls, relapsed HNSCC patients (n = 5), not currently in treatment revealed decreased levels of circulating regulatory NK cell subsets in relation to increased cytotoxic NK cell subpopulations. Elevated sMICA and TGF-β1 plasma levels correlated with diminished TNFα and IFN-γ release and decreased NKG2D (natural killer group 2 member D)-dependent killing of HNSCC cells by NK cells. Incubation of IL-2-activated patient NK cells with patient plasma containing elevated sMICA or sMICA analogs (shed MICA and recombinant MICA) significantly impaired NKG2D-mediated killing by down-regulation of NKG2D surface expression. Of note, CD16 surface expression levels, pro-apoptotic and activation markers, and viability of patient and healthy donor NK cell subpopulations were not affected by this treatment. Accordingly, cetuximab restored killing activity of sMICA-inhibited patient NK cells against cetuximab-coated primary HNSCC cells via ADCC in a dose-dependent manner. Rapid reconstitution of anti-tumor recognition and enhanced tumor infiltration of treated NK cells was monitored by 24 h co-incubation of HNSCC tumor spheroids with cetuximab (1 μg/ml) and was characterized by increased IFN-γ and TNFα secretion. This data show that the impaired NK cell-dependent tumor

  6. CD62L expression identifies a unique subset of polyfunctional CD56dim NK cells.

    PubMed

    Juelke, Kerstin; Killig, Monica; Luetke-Eversloh, Merlin; Parente, Eliana; Gruen, Joachim; Morandi, Barbara; Ferlazzo, Guido; Thiel, Andreas; Schmitt-Knosalla, Isabela; Romagnani, Chiara

    2010-08-26

    Human natural killer (NK) cells comprise 2 main subsets, CD56(bright) and CD56(dim) cells, that differ in function, phenotype, and tissue localization. To further dissect the heterogeneity of CD56(dim) cells, we have performed transcriptome analysis and functional ex vivo characterization of human NK-cell subsets according to the expression of markers related to differentiation, migration or competence. Here, we show for the first time that the ability to respond to cytokines or to activating receptors is mutually exclusive in almost all NK cells with the exception of CD56(dim) CD62L(+) cells. Indeed, only these cells combine the ability to produce interferon-gamma after cytokines and proliferate in vivo during viral infection with the capacity to kill and produce cytokines upon engagement of activating receptors. Therefore, CD56(dim) CD62L(+) cells represent a unique subset of polyfunctional NK cells. Ex vivo analysis of their function, phenotype, telomere length, frequencies during ageing as well as transfer experiments of NK-cell subsets into immunodeficient mice suggest that CD56(dim) CD62L(+) cells represent an intermediate stage of NK-cell maturation, which after restimulation can accomplish multiple tasks and further develop into terminally differentiated effectors.

  7. Tethering of ICAM on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization

    PubMed Central

    Gross, Catharina C.; Brzostowski, Joseph A.; Liu, Dongfang; Long, Eric O.

    2013-01-01

    αLβ2 integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. Here we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. PMID:20675589

  8. Intrahepatic endothelial and Kupffer cells involved in immunosuppressive cytokines and natural killer (NK)/NK T cell disorders in viral acute hepatitis

    PubMed Central

    Jacques, A; Bleau, C; Martin, J-P; Lamontagne, L

    2008-01-01

    During acute viral hepatitis, the intrahepatic tolerance sustained by immunosuppressive cytokines such as interleukin (IL)-4, IL-10, transforming growth factor (TGF)-β and prostaglandin E2 (PGE2), produced by Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), natural killer (NK) T cells and natural regulatory T cells may be disturbed. NK cells are recruited normally in the liver and produce interferon (IFN)-γ to control viral replication. The use of mouse hepatitis virus type 3 (MHV3) attenuated variants showing selected tropisms for KC or LSEC have allowed determining their roles in the disturbances of immune tolerance during viral hepatitis. Groups of C57BL/6 mice were infected with the pathogenic L2-MHV3 (KC+, LSEC+), low attenuated 51·6-MHV3 (KC+, LSEC−) or high attenuated CL12-MHV3 (KC−, LSEC−) variants for the first 3 days. Results showed that IL-10, TGF-β and PGE2 production in the liver decreased in L2-MHV3-infected mice and increased in 51·6-MHV3- and CL12-MHV3-infected mice. The ratio of IFN-γ/IL-4 in liver decreased in L2-MHV3-infected mice, while it was not (or low) altered in mice infected with the attenuated MHV3 variant mice. Phenotypic analysis of intrahepatic mononuclear cells revealed that apoptotic NK and NK T cells increased in mice infected with the L2-MHV3, but were minor in 51·6-MHV3- and CL12-MHV3-infected mice. The numbers of CD4+ forkhead box P3+ cells increased in the livers from low pathogenic CL12-MHV3 and YAC-MHV3-infected mice. These results indicate that viral permissivity of KC and LSEC is involved in the decrease of IL-10 and PGE2, while KC may play an additional role in the apoptosis of NK and NK T cells during acute viral hepatitis. PMID:18336588

  9. Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts.

    PubMed

    Zhu, Yin; Cheng, Ming; Yang, Zhen; Zeng, Chun-Yan; Chen, Jiang; Xie, Yong; Luo, Shi-Wen; Zhang, Kun-He; Zhou, Shu-Feng; Lu, Nong-Hua

    2014-01-01

    Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of tumors. Gastric cancer is the third leading cause of worldwide cancer mortality, and novel treatment modalities are urgently needed. NK4 is an antagonist of hepatocyte growth factor receptors (Met) which are often aberrantly activated in gastric cancer and thus represent a useful candidate for targeted therapies. This study investigated MSC-delivered NK4 gene therapy in nude mice bearing gastric cancer xenografts. MSCs were transduced with lentiviral vectors carrying NK4 complementary DNA or enhanced green fluorescent protein (GFP). Such transduction did not change the phenotype of MSCs. Gastric cancer xenografts were established in BALB/C nude mice, and the mice were treated with phosphate-buffered saline (PBS), MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tropism of MSCs toward gastric cancer cells was determined by an in vitro migration assay using MKN45 cells, GES-1 cells and human fibroblasts and their presence in tumor xenografts. Tumor growth, tumor cell apoptosis and intratumoral microvessel density of tumor tissue were measured in nude mice bearing gastric cancer xenografts treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 via tail vein injection. The results showed that MSCs migrated preferably to gastric cancer cells in vitro. Systemic MSCs-NK4 injection significantly suppressed the growth of gastric cancer xenografts. MSCs-NK4 migrated and accumulated in tumor tissues after systemic injection. The microvessel density of tumor xenografts was decreased, and tumor cellular apoptosis was significantly induced in the mice treated with MSCs-NK4 compared to control mice. These findings demonstrate that MSC-based NK4 gene therapy can obviously inhibit the growth of gastric cancer xenografts, and MSCs are a better vehicle for NK4 gene therapy than lentiviral vectors. Further studies are warranted to explore the efficacy and safety of the MSC-based NK4 gene therapy in

  10. Mesenchymal stem cell-based NK4 gene therapy in nude mice bearing gastric cancer xenografts.

    PubMed

    Zhu, Yin; Cheng, Ming; Yang, Zhen; Zeng, Chun-Yan; Chen, Jiang; Xie, Yong; Luo, Shi-Wen; Zhang, Kun-He; Zhou, Shu-Feng; Lu, Nong-Hua

    2014-01-01

    Mesenchymal stem cells (MSCs) have been recognized as promising delivery vehicles for gene therapy of tumors. Gastric cancer is the third leading cause of worldwide cancer mortality, and novel treatment modalities are urgently needed. NK4 is an antagonist of hepatocyte growth factor receptors (Met) which are often aberrantly activated in gastric cancer and thus represent a useful candidate for targeted therapies. This study investigated MSC-delivered NK4 gene therapy in nude mice bearing gastric cancer xenografts. MSCs were transduced with lentiviral vectors carrying NK4 complementary DNA or enhanced green fluorescent protein (GFP). Such transduction did not change the phenotype of MSCs. Gastric cancer xenografts were established in BALB/C nude mice, and the mice were treated with phosphate-buffered saline (PBS), MSCs-GFP, Lenti-NK4, or MSCs-NK4. The tropism of MSCs toward gastric cancer cells was determined by an in vitro migration assay using MKN45 cells, GES-1 cells and human fibroblasts and their presence in tumor xenografts. Tumor growth, tumor cell apoptosis and intratumoral microvessel density of tumor tissue were measured in nude mice bearing gastric cancer xenografts treated with PBS, MSCs-GFP, Lenti-NK4, or MSCs-NK4 via tail vein injection. The results showed that MSCs migrated preferably to gastric cancer cells in vitro. Systemic MSCs-NK4 injection significantly suppressed the growth of gastric cancer xenografts. MSCs-NK4 migrated and accumulated in tumor tissues after systemic injection. The microvessel density of tumor xenografts was decreased, and tumor cellular apoptosis was significantly induced in the mice treated with MSCs-NK4 compared to control mice. These findings demonstrate that MSC-based NK4 gene therapy can obviously inhibit the growth of gastric cancer xenografts, and MSCs are a better vehicle for NK4 gene therapy than lentiviral vectors. Further studies are warranted to explore the efficacy and safety of the MSC-based NK4 gene therapy in

  11. TLR4 plays a crucial role in MSC-induced inhibition of NK cell function

    SciTech Connect

    Lu, Ying; Liu, Jin; Liu, Yang; Qin, Yaru; Luo, Qun; Wang, Quanli; Duan, Haifeng

    2015-08-21

    Mesenchymal stem cells (MSC) are a kind of stromal cell within the tumor microenvironment. In our research, MSC derived from acute myeloid leukemia patients' bone marrow (AML-MSC) and lung cancer tissues (LC-MSC) as well as normal bone marrow-derived MSC (BM-MSC) cultured in conditioned medium of HeLa cells were found to have higher expressions of Toll-like receptor (TLR4) mRNA compared with BM-MSC. The sorted TLR4-positive MSC (TLR4+ MSC) differed in cytokine (interleukin-6, interleukin-8, and monocyte chemoattractant protein-1) secretion from those of unsorted MSC. MSC was reported to inhibit natural killer (NK) cell proliferation and function. In this research, we confirmed that TLR4+ MSC aggravate this suppression. Furthermore, when TLR4 in the sorted cells were stimulated by LPS or following blocked by antibody, the suppression on NK cell proliferation and cytotoxicity were more intensive or recovered respectively. Compared to unsorted MSC, NKG2D receptor expression on NK cells were also inhibited by TLR4+ MSC. These findings suggest that activation of TLR4 pathway is important for TLR4+ MSC and MSC to obstruct anti-tumor immunity by inhibiting NK cell function, which may provide a potential stroma-targeted tumor therapy. - Highlights: • TLR4+ MSC inhibit NK cell proliferation in vivo and in vitro. • TLR4+ MSC inhibit NKG2D expression on NK cells and NK cell cytotoxicity. • The distinguished cytokine expression of TLR4+ MSC may contribute to the inhibition on NK cell function.

  12. CAR-pNK Cell Immunotherapy in CD7 Positive Leukemia and Lymphoma

    ClinicalTrials.gov

    2016-07-11

    Acute Myeloid Leukemia; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; T-cell Prolymphocytic Leukemia; T-cell Large Granular Lymphocytic Leukemia; Peripheral T-cell Lymphoma, NOS; Angioimmunoblastic T-cell Lymphoma; Extranodal NK/T-cell Lymphoma, Nasal Type; Enteropathy-type Intestinal T-cell Lymphoma; Hepatosplenic T-cell Lymphoma

  13. Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies.

    PubMed

    Legris, Tristan; Picard, Christophe; Todorova, Dilyana; Lyonnet, Luc; Laporte, Cathy; Dumoulin, Chloé; Nicolino-Brunet, Corinne; Daniel, Laurent; Loundou, Anderson; Morange, Sophie; Bataille, Stanislas; Vacher-Coponat, Henri; Moal, Valérie; Berland, Yvon; Dignat-George, Francoise; Burtey, Stéphane; Paul, Pascale

    2016-01-01

    Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1(+) cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of

  14. Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies

    PubMed Central

    Legris, Tristan; Picard, Christophe; Todorova, Dilyana; Lyonnet, Luc; Laporte, Cathy; Dumoulin, Chloé; Nicolino-Brunet, Corinne; Daniel, Laurent; Loundou, Anderson; Morange, Sophie; Bataille, Stanislas; Vacher-Coponat, Henri; Moal, Valérie; Berland, Yvon; Dignat-George, Francoise; Burtey, Stéphane; Paul, Pascale

    2016-01-01

    Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of

  15. Age-related changes in natural killer cell repertoires: impact on NK cell function and immune surveillance.

    PubMed

    Manser, Angela R; Uhrberg, Markus

    2016-04-01

    A key feature of human natural killer (NK) cells, which enables efficient recognition of infected and malignant target cells, is the expression of HLA class I-specific receptors of the KIR and NKG2 gene families. Cell-to-cell variability in receptor expression leads to the formation of complex NK cell repertoires. As outlined here, NK cells go through major changes from newborns to adults characterized by downregulation of the inhibitory NKG2A receptor and concomitant upregulation of KIR family members. This process is completed in young adults, and in the majority of individuals, KIR/NKG2A repertoires remain remarkably stable until old age. Nonetheless, age-related factors have the potential to majorly influence the complexity of NK cell repertoires: Firstly infection with HCMV is associated with major clonal expansions of terminally differentiated NKG2C- and KIR-expressing NK cells in certain individuals. Secondly, ineffective hematopoiesis can lead to immature and less diversified NK cell repertoires as observed in myelodysplastic syndrome (MDS), a malignant disease of the elderly. Thus, whereas in the majority of elderly the NK cell compartment appears to be highly stable in terms of function and phenotype, in a minority of subjects a breakdown of NK cell repertoire diversity is observed that might influence immune surveillance and healthy aging.

  16. Analysis of donor NK and T cells infused in patients undergoing MHC-matched allogeneic hematopoietic transplantation.

    PubMed

    Pascal, V; Brunet, C; Pradel, V; Thirion, X; Andre, P; Faucher, C; Sampol, J; Dignat-George, F; Blaise, D; Vivier, E; Chabannon, C

    2002-11-01

    We retrospectively analyzed the percentages and absolute numbers of T cells, natural killer (NK) cells and NK cell subsets in cryopreserved samples of either bone marrow or blood non-T cell-depleted allogeneic MHC-matched hematopoietic grafts. Using flow cytometry, we found higher numbers of NK cells in aphereses than in bone marrow collections. We further investigated the distribution of NK cell subsets, defined by the cell surface expression of MHC class I-specific receptors, in these allogeneic grafts. The distribution of NK cell subsets from the two different origins were similar, with the exception of the CD158a/h(+) NK cell subset, whose size appeared to be smaller in bone marrow. The search for relations between the numbers of infused cells and post-transplantation events demonstrated that increasing numbers of infused T cells but not NK cells are related with decreased overall survival. Our study highlights the toxicity of infused T cells but not NK cells in allogeneic MHC-matched hematopoietic grafts. These data pave the way for further trials to investigate the effect of NK cell infusion in MHC-matched allogeneic transplantation, and in particular whether ex vivo NK cell expansion and activation may enhance the anti-tumoral effect of the procedure and decrease its morbidity.

  17. Toll-like receptors expression and interferon-γ production by NK cells in human sepsis

    PubMed Central

    2012-01-01

    Introduction During the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production. Methods We investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples. Results We first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced. Conclusions This study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ. PMID:23098236

  18. Tumor-associated macrophages promote tumor cell proliferation in nasopharyngeal NK/T-cell lymphoma

    PubMed Central

    Liu, Yixiong; Fan, Linni; Wang, Yingmei; Li, Peifeng; Zhu, Jin; Wang, Lu; Zhang, Weichen; Zhang, Yuehua; Huang, Gaosheng

    2014-01-01

    Objective: To explore the relationship between the number of tumor-associated macrophages (TAMs) and proliferative activity of tumor cells and the relationship between two macrophage biomarkers CD68 and CD163 in nasopharyngeal NK/T-cell lymphoma. Methods: Immunohistochemistry was used to reconfirm the diagnosis of nasal NK/T-cell lymphoma and detect the numbers of TAMs and the ki-67 label index of the tumor cells in all 31 cases. In addition, 12 cases of inflammatory cases were collected as controls, for which the immunostaining of CD68 and CD163 were done as well. Then staining results were analyzed with Pearson correlation and t test. Results: The number of TAMs was positively correlated with tumor proliferative activity (P = 0.024) in nasopharyngeal NK/T-cell lymphoma. The expression of CD68 and CD163 was closely related (P = 0.009), and the positive rate of CD68 was generally higher than CD163, however there is no statistical significance. Conclusion: The increase in numbers of TAMs in nasopharyngeal NK/T-cell lymphoma is related to higher proliferative index, indicating the TAMs play an important role in tumor proliferation. Meanwhile both CD68 and CD163 might be the markers for TAMs but CD163 would be the better one. PMID:25337185

  19. A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction

    PubMed Central

    Carbone, Ennio; Ruggiero, Giuseppina; Terrazzano, Giuseppe; Palomba, Carmen; Manzo, Ciro; Fontana, Silvia; Spits, Hergen; Kärre, Klas; Zappacosta, Serafino

    1997-01-01

    NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies. PMID:9182676

  20. Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases

    PubMed Central

    Quintieri, L; Rosato, A; Amboldi, N; Vizler, C; Ballinari, D; Zanovello, P; Collavo, D

    1999-01-01

    The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 μg kg−1 MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 μg kg−1 free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection. © 1999 Cancer Research Campaign PMID:10098738

  1. IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion.

    PubMed

    Huenecke, Sabine; Zimmermann, Stefanie Yvonne; Kloess, Stephan; Esser, Ruth; Brinkmann, Andrea; Tramsen, Lars; Koenig, Melanie; Erben, Stephanie; Seidl, Christian; Tonn, Torsten; Eggert, Angelika; Schramm, Alexander; Bader, Peter; Klingebiel, Thomas; Lehrnbecher, Thomas; Passweg, Jakob Robert; Soerensen, Jan; Schwabe, Dirk; Koehl, Ulrike

    2010-01-01

    To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56CD3 donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16 and CD16 subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR) NK cells were overgrown by KIR proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2-stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

  2. Activation of NK cells in subjects exposed to mild hyper- or hypothermic load.

    PubMed

    Lackovic, V; Borecký, L; Vigas, M; Rovenský, J

    1988-06-01

    The effect of mild hyper- and hypothermic stress on release of selected hormones (somatotropin, noradrenaline, etc.), interferon (IFN), and activity of NK cells in the blood was examined in groups of young males during a 30 min exposure to 39 degrees C and 4 degrees C. A quick release of somatotropin was registered in 44% of examinees in the hyperthermic group, while the persons exposed to 4 degrees C reacted with a release of noradrenaline only. Concurrently, an elevation of NK cell activity was observed both in the subgroup releasing somatotropin after hyperthermic stress and in the group exposed to cold. Since these forms of mild stress did not lead to an appearance of IFN in the serum, the possibility of an NK cell activating effect of somatotropin and/or the adrenal hormones was tested. While the adrenal hormones stimulated the NK cell activity in vitro, no support for a similar role for somatotropin was found. PMID:2457640

  3. Circadian rhythms of circulating NK cells in healthy and human immunodeficiency virus-infected men.

    PubMed

    Bourin, P; Mansour, I; Doinel, C; Roué, R; Rouger, P; Levi, F

    1993-08-01

    Antiviral immunity involves NK cells, which circulate rhythmically every 24 hours. We have investigated circadian and 12-hour rhythms in the peripheral count of circulating NK cells in 15 men infected with human immunodeficiency virus (HIV) and 13 healthy controls. We analyzed three phenotypes using double-labeling with monoclonal antibodies and flow cytometry assessment: CD3- CD16+, CD3-CD57+, and CD2+CD3-. A statistical validation of time-dependent differences was achieved if significance (p < 0.05) was validated both with analysis of variance and cosinor. The circadian rhythm had a similar asymmetric waveform for the three phenotypes and is homogeneous on an individual basis. The circulating NK cell count peaked in the early morning and was low at night. A circadian rhythm and a circahemidian harmonic characterized all phenotypes in healthy subjects. We considered two groups of HIV-infected men: those who were asymptomatic (eight) and those with acquired immune deficiency syndrome (AIDS) (seven). Circadian changes in NK cell count were similar in both subgroups and in healthy controls. The circadian pattern was also consistent among individual patients. Asymptomatic HIV-infected men (early-stage disease) exhibited more pronounced 12-hour rhythmicity than did patients with AIDS or controls. The circulation of NK cells does not appear to share the same synchronizer(s) as other circulating T- or B-lymphocyte subsets. Thus, HIV infection gradually abolished circadian rhythmicity in circulating T and B cells, whereas it did not disturb that in NK cells.

  4. Metabolic Reprogramming Supports IFN-γ Production by CD56bright NK Cells.

    PubMed

    Keating, Sinéad E; Zaiatz-Bittencourt, Vanessa; Loftus, Roisín M; Keane, Ciara; Brennan, Kiva; Finlay, David K; Gardiner, Clair M

    2016-03-15

    Human NK cells can be classified into phenotypically and functionally distinct subsets based on levels of CD56 receptor. CD56(dim) cells are generally considered more cytotoxic, whereas the CD56(bright) cells are potent producers of IFN-γ. In this study, we define the metabolic changes that occur in peripheral blood NK cells in response to cytokine. Metabolic analysis showed that NK cells upregulate glycolysis and oxidative phosphorylation in response to either IL-2 or IL-12/15 cytokine combinations. Despite the fact that both these cytokine combinations robustly upregulated mammalian Target of Rapamycin Complex 1 in human NK cells, only the IL-2-induced metabolic changes were sensitive to mammalian Target of Rapamycin Complex 1 inhibition by rapamycin. Interestingly, we found that CD56(bright) cells were more metabolically active compared with CD56(dim) cells. They preferentially upregulated nutrient receptors and also differed substantially in terms of their glucose metabolism. CD56(bright) cells expressed high levels of the glucose uptake receptor, Glut1 (in the absence of any cytokine), and had higher rates of glucose uptake compared with CD56(dim) cells. Elevated levels of oxidative phosphorylation were required to support both cytotoxicity and IFN-γ production in all NK cells. Finally, although elevated glycolysis was not required directly for NK cell degranulation, limiting the rate of glycolysis significantly impaired IFN-γ production by the CD56(bright) subset of cells. Overall, we have defined CD56(bright) NK cells to be more metabolically active than CD56(dim) cells, which supports their production of large amounts of IFN-γ during an immune response. PMID:26873994

  5. Viral MHC class I-like molecule allows evasion of NK cell effector responses in vivo.

    PubMed

    Pyzik, Michal; Dumaine, Anne; Dumaine, Anne A; Charbonneau, Benoît; Fodil-Cornu, Nassima; Jonjic, Stipan; Vidal, Silvia M

    2014-12-15

    The outcome of mouse CMV (MCMV) infection varies among different inbred mouse strains depending on NK cell effector functions governed through recognition receptor triggering. NK cells from different mouse strains possess diverse repertoires of activating or inhibitory Ly49 receptors, which share some of their polymorphic MHC class I (MHC-I) ligands. By examining the NK cell response to MCMV infection in novel BALB substrains congenic for different MHC (or H-2 in mice) haplotypes, we show that recognition of viral MHC-I-like protein m157 by inhibitory Ly49C receptor allows escape from NK cell control of viral replication. Dominant inhibition by Ly49C bound to self-H-2(b) encoded MHC-I molecules masks this effect, which only becomes apparent in distinct H-2 haplotypes, such as H-2(f). The recognition of m157-expressing cells by Ly49C resulted in both decreased NK cell killing in vitro and reduced rejection in vivo. Further, control of infection with m157-deletant (Δm157) MCMV was improved in mice carrying H-2 molecules unrecognized by Ly49C but allowing expansion of NK cell effectors expressing activating Ly49L receptors. Hence, our study is the first, to our knowledge, to demonstrate that MHC-I mimicry strategies used by MCMV to avoid NK cell control are biologically relevant during in vivo viral infection. Of value for human studies is that only a few genetic assortments conditional on the repertoires of viral MHC-I-like proteins/host NK receptors/MHC haplotypes should allow efficient protection against CMV infection.

  6. The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells.

    PubMed

    Textor, Sonja; Bossler, Felicitas; Henrich, Kai-Oliver; Gartlgruber, Moritz; Pollmann, Julia; Fiegler, Nathalie; Arnold, Annette; Westermann, Frank; Waldburger, Nina; Breuhahn, Kai; Golfier, Sven; Witzens-Harig, Mathias; Cerwenka, Adelheid

    2016-07-01

    Natural Killer (NK) cells are innate effector cells that are able to recognize and eliminate tumor cells through engagement of their surface receptors. NKp30 is a potent activating NK cell receptor that elicits efficient NK cell-mediated target cell killing. Recently, B7-H6 was identified as tumor cell surface expressed ligand for NKp30. Enhanced B7-H6 mRNA levels are frequently detected in tumor compared to healthy tissues. To gain insight in the regulation of expression of B7-H6 in tumors, we investigated transcriptional mechanisms driving B7-H6 expression by promoter analyses. Using luciferase reporter assays and chromatin immunoprecipitation we mapped a functional binding site for Myc, a proto-oncogene overexpressed in certain tumors, in the B7-H6 promoter. Pharmacological inhibition or siRNA/shRNA-mediated knock-down of c-Myc or N-Myc significantly decreased B7-H6 expression on a variety of tumor cells including melanoma, pancreatic carcinoma and neuroblastoma cell lines. In tumor cell lines from different origin and primary tumor tissues of hepatocellular carcinoma (HCC), lymphoma and neuroblastoma, mRNA levels of c-Myc positively correlated with B7-H6 expression. Most importantly, upon inhibition or knock-down of c-Myc in tumor cells impaired NKp30-mediated degranulation of NK cells was observed. Thus, our data imply that Myc driven tumors could be targets for cancer immunotherapy exploiting the NKp30/B7-H6 axis.

  7. The proto-oncogene Myc drives expression of the NK cell-activating NKp30 ligand B7-H6 in tumor cells.

    PubMed

    Textor, Sonja; Bossler, Felicitas; Henrich, Kai-Oliver; Gartlgruber, Moritz; Pollmann, Julia; Fiegler, Nathalie; Arnold, Annette; Westermann, Frank; Waldburger, Nina; Breuhahn, Kai; Golfier, Sven; Witzens-Harig, Mathias; Cerwenka, Adelheid

    2016-07-01

    Natural Killer (NK) cells are innate effector cells that are able to recognize and eliminate tumor cells through engagement of their surface receptors. NKp30 is a potent activating NK cell receptor that elicits efficient NK cell-mediated target cell killing. Recently, B7-H6 was identified as tumor cell surface expressed ligand for NKp30. Enhanced B7-H6 mRNA levels are frequently detected in tumor compared to healthy tissues. To gain insight in the regulation of expression of B7-H6 in tumors, we investigated transcriptional mechanisms driving B7-H6 expression by promoter analyses. Using luciferase reporter assays and chromatin immunoprecipitation we mapped a functional binding site for Myc, a proto-oncogene overexpressed in certain tumors, in the B7-H6 promoter. Pharmacological inhibition or siRNA/shRNA-mediated knock-down of c-Myc or N-Myc significantly decreased B7-H6 expression on a variety of tumor cells including melanoma, pancreatic carcinoma and neuroblastoma cell lines. In tumor cell lines from different origin and primary tumor tissues of hepatocellular carcinoma (HCC), lymphoma and neuroblastoma, mRNA levels of c-Myc positively correlated with B7-H6 expression. Most importantly, upon inhibition or knock-down of c-Myc in tumor cells impaired NKp30-mediated degranulation of NK cells was observed. Thus, our data imply that Myc driven tumors could be targets for cancer immunotherapy exploiting the NKp30/B7-H6 axis. PMID:27622013

  8. Variable CD52 Expression in Mature T Cell and NK Cell Malignancies: Implications for Alemtuzumab Therapy

    PubMed Central

    Jiang, Liuyan; Yuan, Constance; Hubacheck, Julia; Janik, John E.; Wilson, Wyndham; Morris, John C.; Jasper, Gregory A; Stetler-Stevenson, Maryalice

    2012-01-01

    Summary The anti-CD52 antibody alemtuzumab has been explored as a novel targeted therapy in T-cell malignancies. To assess the suitability of alemtuzumab therapy we carried out a comprehensive study of CD52 expression using flow cytometry (FC) in 78 untreated patients diagnosed with mature T/NK cell neoplasms, including 34 adult T-cell leukemia /lymphomas (ATLL), two anaplastic large cell lymphomas (ALCL), three angioimmunoblastic T-cell lymphomas (AITL), 16 cutaneous T-cell lymphomas (CTCL), four extra-nodal T/NK cell lymphomas (ENT/NKCL), four hepatosplenic T-cell lymphomas (HSTCL), 13 peripheral T-cell lymphomas, unspecified (PTCL-NOS), and two T-prolymphocytic leukemia (T-PLL). The level of CD52 expression was quantitated using QuantiBRITE standard beads. The level of CD52 expression varied widely within each diagnostic category. All AITL, HSTCL, and T-PLL cases were CD52 positive and the frequency of CD52 expression was high in PTCL-NOS (92.3%), ATLL (94.1%) and CTCL (87.5%), implying a rational role for alemtuzumab in the treatment of these diseases; however, CD52 expression was low in ALCL (50%) and ENT/NKCL (25%). FC testing for cell surface expression of CD52 is indicated in patients with T/NK cell malignancies being considered for alemtuzumab therapy. Further studies are necessary to determine if the level of CD52 expression correlates with response to therapy. PMID:19236377

  9. PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells

    PubMed Central

    Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra

    2016-01-01

    Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2. PMID:27413259

  10. Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease.

    PubMed

    Nalle, Sam C; Kwak, H Aimee; Edelblum, Karen L; Joseph, Nora E; Singh, Gurminder; Khramtsova, Galina F; Mortenson, Eric D; Savage, Peter A; Turner, Jerrold R

    2014-07-01

    Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)-matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

  11. NK cells are intrinsically functional in pigs with Severe Combined Immunodeficiency (SCID) caused by spontaneous mutations in the Artemis gene.

    PubMed

    Powell, Ellis J; Cunnick, Joan E; Knetter, Susan M; Loving, Crystal L; Waide, Emily H; Dekkers, Jack C M; Tuggle, Christopher K

    2016-07-01

    We have identified Severe Combined Immunodeficiency (SCID) in a line of Yorkshire pigs at Iowa State University. These SCID pigs lack B-cells and T-cells, but possess Natural Killer (NK) cells. This SCID phenotype is caused by recessive mutations in the Artemis gene. Interestingly, two human tumor cell lines, PANC-1 and A375-SM, survived after injection into these SCID pigs, but, as we demonstrate here, these cells, as well as K562 tumor cells, can be lysed in vitro by NK cells from SCID and non-SCID pigs. NK cells from both SCID and non-SCID pigs required activation in vitro with either recombinant human IL-2 or the combination of recombinant porcine IL-12 and IL-18 to kill tumor targets. We also showed that SCID NK cells could be activated to produce perforin, and perforin production was greatly enhanced in NK cells from both SCID and non-SCID pigs after IL-2 cytokine treatment. While CD16+, CD172- NK cells constituted an average of only 4% in non-SCID pigs, NK cells averaged 27% of the peripheral blood mononuclear cell population in SCID pigs. We found no significant differences in killing activity per NK cell between SCID and non-SCID pigs. We conclude that survival of human cancer cells in these SCID pigs is not due to an intrinsic defect in NK cell killing ability.

  12. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

    PubMed Central

    Andersson, M; Gunne, H; Agerberth, B; Boman, A; Bergman, T; Sillard, R; Jörnvall, H; Mutt, V; Olsson, B; Wigzell, H

    1995-01-01

    A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells. Images PMID:7737114

  13. The interaction of NK cells and dendritic cells in the tumor environment: how to enforce NK cell & DC action under immunosuppressive conditions?

    PubMed

    Jacobs, B; Ullrich, E

    2012-01-01

    The crosstalk of natural killer (NK) and dendritic cells (DCs) plays an important role in the induction of the tumor-specific immune response against cancer. During the last decade, our advanced understanding of the immune system led to the development of new therapeutic strategies in the field of immunotherapy and cellular immunology. However, these immunotherapeutic concepts have not been as successful as initially expected because of their inability to counteract cancer-induced immunosuppressive pathways. Some of the major difficulties of effective cellular immunotherapy are the highly immunosuppressive factors induced by tumor cells themselves or by their microenvironment. Therefore, one major challenge in immunotherapy is the question: "How to enforce NK cell & DC action under immunosuppressive conditions?" This review focuses on the current knowledge on the tumor microenvironment, the crosstalk of NK cells and DCs, as well as their deregulation in the complex interplay with the immunosuppressive tumor microenvironment. We further discuss possible strategies to minimize the negative impact of the tumor microenvironment on the immune system.

  14. Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity

    PubMed Central

    Shah, Nina; Martin-Antonio, Beatriz; Yang, Hong; Ku, Stephanie; Lee, Dean A.; Cooper, Laurence J. N.; Decker, William K.; Li, Sufang; Robinson, Simon N.; Sekine, Takuya; Parmar, Simrit; Gribben, John; Wang, Michael; Rezvani, Katy; Yvon, Eric; Najjar, Amer; Burks, Jared; Kaur, Indreshpal; Champlin, Richard E.; Bollard, Catherine M.; Shpall, Elizabeth J.

    2013-01-01

    Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM. PMID:24204673

  15. Natural killer (NK) activity of pit cells perfused from livers of rats treated with ethanol

    SciTech Connect

    Albornoz, L.; Jones, J.M.; Crutchfield, C.; Veech, R.L. Univ. of Arkansas Medical Sciences, Little Rock )

    1991-03-11

    The liver is the major site of ethanol (ETOH) metabolism. Liver sinusoids contain lymphocytes with NK activity. The authors treated LEW rats for 2 weeks with i.p. injection of 1.25 ml 25% ETOH/kg 3 times/week and 5% ETOH in drinking water. Livers were perfused at 5-fold physiological pressure and cells obtained were banded on 1.077 density Ficoll. Their cytotoxicity was tested against {sup 51}Cr-labeled YAC-1 or U937 and compared to spleen and blood lymphocytes. In untreated rats, pit cell NK activity was 2-fold that of splenic lymphocytes and 4-fold that of blood lymphocytes. Compared to controls, ETOH-treated rats exhibited a 30 to 90% rise in pit cell NK activity detected with YAC-1 or U937 targets. The pit cell enhanced NK activity in ETOH-treated rats was further increased if polyinosinicpolycytidilic acid was injection i.p. 18 hours before the assay. Blood and spleen lymphocyte NK activity of ETOH-treated rats was also greater than in controls. There was no evidence that ETOH merely redistributed lymphocytes among the tissues. Although ETOH acutely inhibits NK activity in vitro, chronic ETOH increases in vivo.

  16. Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility

    PubMed Central

    Strauss-Albee, Dara M.; Fukuyama, Julia; Liang, Emily C.; Yao, Yi; Jarrell, Justin A.; Drake, Alison L.; Kinuthia, John; Montgomery, Ruth R.; John-Stewart, Grace; Holmes, Susan; Blish, Catherine A.

    2015-01-01

    Innate natural killer (NK) cells are diverse at the single-cell level because of variegated expressions of activating and inhibitory receptors, yet the developmental roots and functional consequences of this diversity remain unknown. Because NK cells are critical for antiviral and antitumor responses, a better understanding of their diversity could lead to an improved ability to harness them therapeutically. We found that NK diversity is lower at birth than in adults. During an antiviral response to either HIV-1 or West Nile virus, NK diversity increases, resulting in terminal differentiation and cytokine production at the cost of cell division and degranulation. In African women matched for HIV-1 exposure risk, high NK diversity is associated with increased risk of HIV-1 acquisition. Existing diversity may therefore decrease the flexibility of the antiviral response. Collectively, the data reveal that human NK diversity is a previously undefined metric of immune history and function that may be clinically useful in forecasting the outcomes of infection and malignancy. PMID:26203083

  17. NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

    SciTech Connect

    Ogbomo, Henry; Hahn, Anke; Geiler, Janina; Michaelis, Martin; Doerr, Hans Wilhelm; Cinatl, Jindrich . E-mail: Cinatl@em.uni-frankfurt.de

    2006-01-06

    The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.

  18. Interleukin-22: a novel T- and NK-cell derived cytokine that regulates the biology of tissue cells.

    PubMed

    Wolk, Kerstin; Sabat, Robert

    2006-10-01

    Interleukin (IL)-22, discovered in 2000, is a member of the IL-10 family of cytokines. The major sources of IL-22 are activated T1- and NK-cells. IL-22 acts via a heterodimeric receptor complex consisting of IL-22R1 and IL-10R2. Neither resting nor activated immune cells express IL-22R1 or respond to IL-22. In contrast, tissue cells at outer body barriers, i.e. of the skin, kidney, and the digestive and respiratory systems are targets of this cytokine. IL-22 functions by promoting the anti-microbial defense, protecting against damage, and re-organizing non-immune tissues. Furthermore, IL-22 induces acute phase reactants. These findings indicate that IL-22 represents a novel type of immune mediator that, although produced by immune cells, regulates tissue protection and homeostasis.

  19. Polymethoxylated flavones potentiate the cytolytic activity of NK leukemia cell line KHYG-1 via enhanced expression of granzyme B.

    PubMed

    Saito, Takeshi; Abe, Daigo; Nogata, Yoichi

    2015-01-16

    Polymethoxylated flavones (PMFs) are found in the peel tissues of some citrus species. Here, we report that PMFs, such as nobiletin, potentiate the cytolytic activity of KHYG-1 natural killer (NK) leukemia cells. Nobiletin markedly enhanced the expression of granzyme B, a serine protease that plays critical roles in the cytolytic activity of NK cells. The potentiated cytolytic activity induced by nobiletin was canceled by the granzyme B inhibitor Z-AAD-CMK. Nobiletin also increased the levels of phosphorylated CREB, ERK1/2, and p38 MAPK in KHYG-1 cells, which are known to participate in NK cell function. Inhibition of an upstream kinase of ERK1/2 failed to reduce the granzyme B expression and KHYG-1 cytolytic activity. Meanwhile, inhibition of p38 MAPK attenuated both granzyme B expression and KHYG-1 cytolytic activity. These results suggest that the primary role of nobiletin in KHYG-1 cytolytic activity lies in upregulation of granzyme B expression, at least in part, mediated through p38 MAPK function.

  20. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.

  1. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.

    PubMed

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T C; Imren, Suzan; Lam, Vivian; Poon, Grace F T; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William; Krystal, Gerald

    2016-05-26

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light-inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation-dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell-depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

  2. Substance P activates responses correlated with tumour growth in human glioma cell lines bearing tachykinin NK1 receptors

    PubMed Central

    Palma, C; Nardelli, F; Manzini, S; Maggi, C A

    1999-01-01

    The neuropeptide substance P (SP), by stimulating tachykinin NK1receptors (NK1R), triggers a number of biological responses in human glioma cells which are potentially relevant for tumour growth. First, radioligand binding studies demonstrated the presence of tachykinin NK1R on SNB-19, DBTRG-05 MG and U373 MG, but not on U138 MG and MOG-G-GCM human glioma cell lines. Second, application of SP or neurokinin A (NKA) to NK1R+glioma cell lines increased the secretion of interleukin 6 (IL-6) and potentiated IL-6 secretion induced by IL-1β. SP also up-regulated the release of transforming growth factor β1 (TGF-β1) by the U373 MG glioma cell line. Third, SP induced new DNA synthesis and enhanced the proliferation rate of NK1R+, but not of NK1R−glioma cell lines. Also, NKA stimulated the proliferation and cytokine secretion in NK1R+glioma cell lines. All the stimulant effects of SP/NKA on NK1R+glioma cell lines were completely blocked by a specific tachykinin NK1R antagonist, MEN 11467. These data support the potential use of tachykinin NK1R antagonist for controlling the proliferative rate of human gliomas. © 1999 Cancer Research Campaign PMID:9888463

  3. Involvement of NK Cells and NKp30 Pathway in Antisynthetase Syndrome.

    PubMed

    Hervier, Baptiste; Perez, Mikaël; Allenbach, Yves; Devilliers, Hervé; Cohen, Fleur; Uzunhan, Yurdagül; Ouakrim, Hanane; Dorgham, Karim; Méritet, Jean-François; Longchampt, Elisabeth; Stenzel, Werner; Cremer, Isabelle; Benveniste, Olivier; Vieillard, Vincent

    2016-09-01

    Antisynthetase syndrome (aSS) is characterized by the association of interstitial lung disease and myositis with anti-tRNA synthetase autoantibodies. Immune mechanisms leading to aSS could be initiated in the lungs, but the role of NK cells has not yet been studied. Both extensive NK cell phenotype and functions were compared between 33 patients and 26 controls. Direct and redirected polyfunctionality assays (degranulation and intracellular production of TNF-α and IFN-γ) were performed spontaneously or after IL-12 plus IL-18 stimulation in the presence of K562 or P815 target cells, respectively. NK cells from inactive patients showed normal phenotype, whereas active aSS revealed a differentiated NK cell profile, as indicated by increased CD57 and Ig-like transcript 2 and an inability to produce IFN-γ (p = 0.002) compared with controls. Importantly, active aSS was more specifically associated with a significant NKp30 decrease (p = 0.009), although levels of mRNA and intracellular protein were similar in aSS and healthy controls. This NKp30 decrease was strongly correlated with reduced NK cell polyfunctionality in both direct and redirected killing assays with anti-NKp30 Abs (p = 0.009 and p = 0.03, respectively), confirming its important impact in aSS. Histological studies revealed massive infiltrations of NK cells inside the lungs of aSS patients (148 versus 11/mm(2)). Taken together, these data suggest that NK cells and NKp30 could play a role in aSS pathogenesis. PMID:27511738

  4. Involvement of NK Cells and NKp30 Pathway in Antisynthetase Syndrome.

    PubMed

    Hervier, Baptiste; Perez, Mikaël; Allenbach, Yves; Devilliers, Hervé; Cohen, Fleur; Uzunhan, Yurdagül; Ouakrim, Hanane; Dorgham, Karim; Méritet, Jean-François; Longchampt, Elisabeth; Stenzel, Werner; Cremer, Isabelle; Benveniste, Olivier; Vieillard, Vincent

    2016-09-01

    Antisynthetase syndrome (aSS) is characterized by the association of interstitial lung disease and myositis with anti-tRNA synthetase autoantibodies. Immune mechanisms leading to aSS could be initiated in the lungs, but the role of NK cells has not yet been studied. Both extensive NK cell phenotype and functions were compared between 33 patients and 26 controls. Direct and redirected polyfunctionality assays (degranulation and intracellular production of TNF-α and IFN-γ) were performed spontaneously or after IL-12 plus IL-18 stimulation in the presence of K562 or P815 target cells, respectively. NK cells from inactive patients showed normal phenotype, whereas active aSS revealed a differentiated NK cell profile, as indicated by increased CD57 and Ig-like transcript 2 and an inability to produce IFN-γ (p = 0.002) compared with controls. Importantly, active aSS was more specifically associated with a significant NKp30 decrease (p = 0.009), although levels of mRNA and intracellular protein were similar in aSS and healthy controls. This NKp30 decrease was strongly correlated with reduced NK cell polyfunctionality in both direct and redirected killing assays with anti-NKp30 Abs (p = 0.009 and p = 0.03, respectively), confirming its important impact in aSS. Histological studies revealed massive infiltrations of NK cells inside the lungs of aSS patients (148 versus 11/mm(2)). Taken together, these data suggest that NK cells and NKp30 could play a role in aSS pathogenesis.

  5. UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

    PubMed Central

    Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

    2016-01-01

    Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

  6. A blessing and a curse: is high NK cell activity good for health and bad for reproduction?

    PubMed

    Templer, Sophie; Sacks, Gavin

    2016-09-01

    Few topics in recent reproductive medicine have been the subject of as much controversy, media attention and passionate debate as natural killer (NK) cells and their role in reproductive failure. The question of whether elevated NK cell levels are a cause of infertility and pregnancy loss, and whether they provide a potential target for therapy to improve reproductive outcomes, lacks a definitive answer. It is clear, however, that a significant number of women with reproductive failure have abnormal NK cell parameters reflecting high immunological activity. Amongst all the debate, the wider implications of NK cell overactivity - and attempts to suppress it - have not yet been considered. The literature suggests that although elevated NK cell activity may not be conducive to reproduction, it could in fact be beneficial in other areas of health and disease such as cancer and infection. Further research is needed to determine whether this hypothesis holds true in women with NK cell-related reproductive failure. PMID:27662415

  7. Establishment, characterization, and successful adaptive therapy against human tumors of NKG cell, a new human NK cell line.

    PubMed

    Cheng, Min; Ma, Juan; Chen, Yongyan; Zhang, Jianhua; Zhao, Weidong; Zhang, Jian; Wei, Haiming; Ling, Bin; Sun, Rui; Tian, Zhigang

    2011-01-01

    Natural killer (NK) cells play important roles in adoptive cellular immunotherapy against certain human cancers. This study aims to establish a new human NK cell line and to study its role for adoptive cancer immunotherapy. Peripheral blood samples were collected from 54 patients to establish the NK cell line. A new human NK cell line, termed as NKG, was established from a Chinese male patient with rapidly progressive non-Hodgkin's lymphoma. NKG cells showed LGL morphology and were phenotypically identified as CD56(bright) NK cell with CD16(-), CD27(-), CD3(-), αβTCR(-), γδTCR(-), CD4(-), CD8(-), CD19(-), CD161(-), CD45(+), CXCR4(+), CCR7(+), CXCR1(-), and CX3CR1(-). NKG cells showed high expression of adhesive molecules (CD2, CD58, CD11a, CD54, CD11b, CD11c), an array of activating receptors (NKp30, NKp44, NKp46, NKG2D, NKG2C), and cytolysis-related receptors and molecules (TRAIL, FasL, granzyme B, perforin, IFN-γ). The cytotoxicity of NKG cells against tumor cells was higher than that of the established NK cell lines NK-92, NKL, and YT. NKG cell cytotoxicity depended on the presence of NKG2D and NKp30. When irradiated with 8 Gy, NKG cells were still with high cytotoxicity and activity in vitro and with safety in vivo, but without proliferation. Further, the irradiated NKG cells exhibited strong cytotoxicity against human primary ovarian cancer cells in vitro, and against human ovarian cancer in a mouse xenograft model. The adoptive transfer of NKG cells significantly inhibited the ovarian tumor growth, decreased the mortality rate and prolonged the survival, even in cases of advanced diseases. A number of NKG cells were detected in the ovarian tumor tissues during cell therapy. In use of the new human NK cell line, NKG would a promising cellular candidate for adoptive immunotherapy of human cancer. PMID:21669033

  8. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections.

    PubMed

    Meier, Ute-Christiane; Owen, Rachel E; Taylor, Elizabeth; Worth, Andrew; Naoumov, Nikolai; Willberg, Christian; Tang, Kwok; Newton, Phillipa; Pellegrino, Pierre; Williams, Ian; Klenerman, Paul; Borrow, Persephone

    2005-10-01

    Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections. PMID:16160163

  9. [Commemorative lecture of receiving Imamura Memorial Prize. NK cell in pulmonary tuberculosis from basic and clinical point of view].

    PubMed

    Yoneda, T

    1996-11-01

    Although natural killer (NK) cells, which lyse certain tumors in vitro, have been shown to provide early defense mechanism against cancer growth and viral infection, possible role in the host defense against pulmonary tuberculosis remains undefined. A series of my studies have recently provided several evidence supporting the involvement of NK cells in the immunopathology of pulmonary tuberculosis. NK cell activity in patients with active pulmonary tuberculosis was significantly augmented compared with that in age-, sex- matched healthy controls, which suggests NK cells are activated in vivo in pulmonary tuberculosis. Lung NK cells from BCG-infected mice also are shown to be activated. Asialo GM 1 was demonstrated to be a novel surface marker of mice NK cells, which inhibited activation of NK cells by interferon. Chronic intractable tuberculosis was classified with a combination of NK cell activity and delayed-type hypersensitivity reaction to 2, 4-dinitrochrolbenzene. Subgroup defined with high NK cell activity and normal delayed-type hypersensitivity was characterized with moderate radiographical lesions and stable clinical course, suggesting the immune-spectrum classification was associated with clinical manifestations. Malnutrition has been suggested to be a risk factor associated with the development and reactivation of pulmonary tuberculosis. NK cell activity was significantly correlated with visceral proteins. IL-2 producing capability was significantly decreased in patients with serum albumin less than 3.5 g/dl. More recently, I established an in vitro system evaluating quantitative capability for intracellular killing by human monocytes, in which monocyte phagocytize Mycobacterium tuberculosis and subsequently inhibit intracellular replication of the organisms by adding some cytokines or cells. Purified NK cells by using discontinuous gradient centrifugation and magnetic separation technique were added to M. tuberculosis-infected monocytes monolayer

  10. [Commemorative lecture of receiving Imamura Memorial Prize. NK cell in pulmonary tuberculosis from basic and clinical point of view].

    PubMed

    Yoneda, T

    1996-11-01

    Although natural killer (NK) cells, which lyse certain tumors in vitro, have been shown to provide early defense mechanism against cancer growth and viral infection, possible role in the host defense against pulmonary tuberculosis remains undefined. A series of my studies have recently provided several evidence supporting the involvement of NK cells in the immunopathology of pulmonary tuberculosis. NK cell activity in patients with active pulmonary tuberculosis was significantly augmented compared with that in age-, sex- matched healthy controls, which suggests NK cells are activated in vivo in pulmonary tuberculosis. Lung NK cells from BCG-infected mice also are shown to be activated. Asialo GM 1 was demonstrated to be a novel surface marker of mice NK cells, which inhibited activation of NK cells by interferon. Chronic intractable tuberculosis was classified with a combination of NK cell activity and delayed-type hypersensitivity reaction to 2, 4-dinitrochrolbenzene. Subgroup defined with high NK cell activity and normal delayed-type hypersensitivity was characterized with moderate radiographical lesions and stable clinical course, suggesting the immune-spectrum classification was associated with clinical manifestations. Malnutrition has been suggested to be a risk factor associated with the development and reactivation of pulmonary tuberculosis. NK cell activity was significantly correlated with visceral proteins. IL-2 producing capability was significantly decreased in patients with serum albumin less than 3.5 g/dl. More recently, I established an in vitro system evaluating quantitative capability for intracellular killing by human monocytes, in which monocyte phagocytize Mycobacterium tuberculosis and subsequently inhibit intracellular replication of the organisms by adding some cytokines or cells. Purified NK cells by using discontinuous gradient centrifugation and magnetic separation technique were added to M. tuberculosis-infected monocytes monolayer

  11. Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.

    PubMed

    Jing, Yawu; Ni, Zhenya; Wu, Jianming; Higgins, LeeAnn; Markowski, Todd W; Kaufman, Dan S; Walcheck, Bruce

    2015-01-01

    CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.

  12. Decreased NK-Cell Cytotoxicity after Short Flights on the Space Shuttle

    NASA Technical Reports Server (NTRS)

    Mehta, Satish K.; Grimm, Elizabeth A.; Smid, Christine; Kaur, Indreshpal; Feeback, Daniel L.; Pierson, Duane L.

    2000-01-01

    Cytotoxic activity of natural killer (NK) cells and cell surface marker expression of peripheral blood mononuclear cells (PBMCs) isolated from 11 U.S. astronauts on two different missions were determined before and after 9 or 10 days of spaceflight aboard the space shuttle. Blood samples were collected 10 and 3 days before launch, within 3 hours after landing, and 3 days after landing. All PBMC preparations were cryopreserved and analyzed simultaneously in a 4-hour cytotoxicity "Cr-release assay using NK-sensitive K-562 target cells. Compared to preflight values, NK-cell cytotoxicity (corrected for lymphopenia observed on landing day) was significantly decreased at landing (P < 0.0125). It then apparently began to recover and approached preflight values by 3 days after landing. Consistent with decreased NK-cell cytotoxicity, significant increases from preflight values were found in plasma adrenocorticotropic hormone at landing. Plasma and urinary cortisol levels did not change significantly from preflight values. Expression of major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), determined by flow cytometric analysis, revealed no consistent phenotypic changes in relative percent of NK or other lymphoid cells after 10 days of spaceflight.

  13. Effect of Spaceflight on the Functions of NK and LAK Cells

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Grimm, Elizabeth A.; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

    1999-01-01

    Spaceflight-associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. The subjects of this study were crewmembers of two 10-day shuttle flights. Ten-ml blood specimens were obtained from ten astronauts 10 days before launch, immediately after landing, and 3 days after landing. PBMCs were separated from the blood specimens and stored at -800 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4-hour Cr-51 release assay. K562 cells were used to assess NK-cell cytotoxicity. After 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 and Daudi cells as the target cells. NK-cell cytotoxicity was decreased at landing (p less than 0.0005) in 9/10 astronauts, and in most cases recovered to preflight levels by 3 days after landing; NK-cell cytotoxicity was increased in one astronaut at landing. LAK cytotoxic activity against K562 cells was decreased at landing in 6/10 astronauts (p=0.018), and activity against Daudi cells was decreased in 7/10 astronauts (p=0.01). Phenotyping of PBMCs and LAK cells showed alterations in some surface markers and adhesion molecules (CD1 1 b, CD1 1 c, CD1 1 a, CD1 6, L-Selectin and CD3). Thus spaceflight leads to a decrease in the functions of NK and LAK cells in most astronauts.

  14. Identification of Anti-tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With Hematological Cancers.

    PubMed

    Krzywinska, Ewelina; Allende-Vega, Nerea; Cornillon, Amelie; Vo, Dang-Nghiem; Cayrefourcq, Laure; Panabieres, Catherine; Vilches, Carlos; Déchanet-Merville, Julie; Hicheri, Yosr; Rossi, Jean-François; Cartron, Guillaume; Villalba, Martin

    2015-10-01

    Natural killer (NK) cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56(dim)CD16(+) NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46), natural-killer group 2, member D (NKG2D) and killer inhibitory receptors (KIRs) and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA(+)RO(+) phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA(+)RO(-) phenotype similar to naïve T cells. In summary, we show that CD45RA(+)RO(+) cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias.

  15. Identification of Anti-tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients With Hematological Cancers

    PubMed Central

    Krzywinska, Ewelina; Allende-Vega, Nerea; Cornillon, Amelie; Vo, Dang-Nghiem; Cayrefourcq, Laure; Panabieres, Catherine; Vilches, Carlos; Déchanet-Merville, Julie; Hicheri, Yosr; Rossi, Jean-François; Cartron, Guillaume; Villalba, Martin

    2015-01-01

    Natural killer (NK) cells, a cytotoxic lymphocyte lineage, are able to kill tumor cells in vitro and in mouse models. However, whether these cells display an anti-tumor activity in cancer patients has not been demonstrated. Here we have addressed this issue in patients with several hematological cancers. We found a population of highly activated CD56dimCD16+ NK cells that have recently degranulated, evidence of killing activity, and it is absent in healthy donors. A high percentage of these cells expressed natural killer cell p46-related protein (NKp46), natural-killer group 2, member D (NKG2D) and killer inhibitory receptors (KIRs) and a low percentage expressed NKG2A and CD94. They are also characterized by a high metabolic activity and active proliferation. Notably, we found that activated NK cells from hematological cancer patients have non-NK tumor cell antigens on their surface, evidence of trogocytosis during tumor cell killing. Finally, we found that these activated NK cells are distinguished by their CD45RA+RO+ phenotype, as opposed to non-activated cells in patients or in healthy donors displaying a CD45RA+RO− phenotype similar to naïve T cells. In summary, we show that CD45RA+RO+ cells, which resemble a unique NK population, have recognized tumor cells and degranulate in patients with hematological neoplasias. PMID:26629531

  16. Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells.

    PubMed

    Werlenius, Olle; Aurelius, Johan; Hallner, Alexander; Akhiani, Ali A; Simpanen, Maria; Martner, Anna; Andersson, Per-Ola; Hellstrand, Kristoffer; Thorén, Fredrik B

    2016-05-31

    The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL. PMID:27097113

  17. IL-15 activates mTOR and primes stress-activated gene expression leading to prolonged antitumor capacity of NK cells

    PubMed Central

    Mao, Yumeng; van Hoef, Vincent; Zhang, Xiaonan; Wennerberg, Erik; Lorent, Julie; Witt, Kristina; Masvidal, Laia; Liang, Shuo; Murray, Shannon; Larsson, Ola; Kiessling, Rolf

    2016-01-01

    Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15–induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15–stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy. PMID:27465917

  18. Chondroitin sulphate proteoglycan production by NK cells and T cells: effects of xylosides on proliferation and cytotoxic function.

    PubMed Central

    Christmas, S E; Steward, W P; Lyon, M; Gallagher, J T; Moore, M

    1988-01-01

    Cultured human NK cells and T cells grown in the presence of IL-2 and phytohaemagglutinin incorporated 35S sulphate into two distinct macromolecular species. The larger molecule was identified as a chondroitin-4-sulphate proteoglycan and was present in both cell-associated and secreted material. The smaller component was identified as free glycosaminoglycan and was present only in the cell-associated material. The sulphated macromolecules synthesized by NK cells were smaller than those produced by T cells. Growth in the presence of beta-D-xyloside led to a decrease in proteoglycan production, together with an increase in the synthesis of free glycosaminoglycan. The latter molecule was found in the secreted as well as the cell-associated fraction. In all instances, growth of T cells was inhibited by xyloside in a dose-dependent fashion. However, growth of NK cells from 3/7 donors was stimulated at low concentrations of xyloside (0.25 and 0.5 mM). Growth of NK cells in xyloside had no effect on their lytic activity, and the 'NK-like' cytolytic capacity of cultured T cells was similarly unaffected. Both NK cells and T cells grown in xyloside at a concentration resulting in a 50% inhibition of intact proteoglycan synthesis did not show increased susceptibility to autolysis in the presence of NK-cell targets. These findings suggest that optimal production of the intact proteoglycan molecule may not be essential for NK-cell lytic function or protection of effector cells in vitro. PMID:3258273

  19. Chondroitin sulphate proteoglycan production by NK cells and T cells: effects of xylosides on proliferation and cytotoxic function.

    PubMed

    Christmas, S E; Steward, W P; Lyon, M; Gallagher, J T; Moore, M

    1988-02-01

    Cultured human NK cells and T cells grown in the presence of IL-2 and phytohaemagglutinin incorporated 35S sulphate into two distinct macromolecular species. The larger molecule was identified as a chondroitin-4-sulphate proteoglycan and was present in both cell-associated and secreted material. The smaller component was identified as free glycosaminoglycan and was present only in the cell-associated material. The sulphated macromolecules synthesized by NK cells were smaller than those produced by T cells. Growth in the presence of beta-D-xyloside led to a decrease in proteoglycan production, together with an increase in the synthesis of free glycosaminoglycan. The latter molecule was found in the secreted as well as the cell-associated fraction. In all instances, growth of T cells was inhibited by xyloside in a dose-dependent fashion. However, growth of NK cells from 3/7 donors was stimulated at low concentrations of xyloside (0.25 and 0.5 mM). Growth of NK cells in xyloside had no effect on their lytic activity, and the 'NK-like' cytolytic capacity of cultured T cells was similarly unaffected. Both NK cells and T cells grown in xyloside at a concentration resulting in a 50% inhibition of intact proteoglycan synthesis did not show increased susceptibility to autolysis in the presence of NK-cell targets. These findings suggest that optimal production of the intact proteoglycan molecule may not be essential for NK-cell lytic function or protection of effector cells in vitro.

  20. SHP-2 expression negatively regulates NK cell function1,2

    PubMed Central

    Purdy, Amanda K.; Campbell, Kerry S.

    2009-01-01

    Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)4 is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human natural killer (NK) cells upon recruitment to killer cell Ig-like receptors (KIR)4. To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-γ production, whereas cells overexpressing wild type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center (MTOC)4 polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts MTOC-based cytoskeletal rearrangement and granule release. PMID:19915046

  1. NK-1 receptor mediation of neurogenic plasma extravasation in rat skin.

    PubMed Central

    Andrews, P. V.; Helme, R. D.; Thomas, K. L.

    1989-01-01

    1. Plasma extravasation was induced by electrical nerve stimulation and by perfusion of tachykinins over a vacuum-induced blister base on rat footpad. 2. Stimulation of the sciatic nerve (18 V, 15 Hz, 0.5 ms) for 20 min produced a significant increase in the protein content of the perfusate. The response in capsaicin pretreated rats was only 4% of the control response. This indicates that the electrically-induced plasma extravasation response was mediated by capsaicin-sensitive sensory fibres. 3. Exogenous perfusion of the mammalian tachykinins substance P, neurokinin A and neurokinin B and the non-mammalian tachykinins physalaemin, kassinin and eledoisin was used to determine the tachykinin receptor type mediating the plasma extravasation response. Dose-response curves of the tachykinins (10(-9) M-10(-4) M) gave a rank order of potency of substance P = physalaemin greater than eledoisin greater than or equal to kassinin greater than neurokinin B = neurokinin A. 4. In addition, specific agonists of neurokinin receptors were perfused. Perfusion of [Glp6, D-Pro9] SP6-11 and [Glp6, L-Pro9]SP6-11 demonstrated that the L-Pro isomer was much more potent than the D-Pro isomer. 5. The rank order of potency and the greater potency of [Glp6, L-Pro9]SP6-11 over its D-isomer indicate an NK-1 neurokinin receptor mediates plasma extravasation in rat footpad skin. PMID:2477105

  2. IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C

    PubMed Central

    Finnemann, Claudia; Sastre, Beatriz; Lutz, Philipp; Glässner, Andreas; Wolter, Franziska; Goeser, Felix; Kokordelis, Pavlos; Kaczmarek, Dominik; Nischalke, Hans-Dieter; Strassburg, Christian P.; Spengler, Ulrich; Nattermann, Jacob

    2016-01-01

    Background Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity. Methods A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used. Results Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals. Conclusions Our data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions. PMID:27583440

  3. A novel system of polymorphic and diverse NK cell receptors in primates.

    PubMed

    Averdam, Anne; Petersen, Beatrix; Rosner, Cornelia; Neff, Jennifer; Roos, Christian; Eberle, Manfred; Aujard, Fabienne; Münch, Claudia; Schempp, Werner; Carrington, Mary; Shiina, Takashi; Inoko, Hidetoshi; Knaust, Florian; Coggill, Penny; Sehra, Harminder; Beck, Stephan; Abi-Rached, Laurent; Reinhardt, Richard; Walter, Lutz

    2009-10-01

    There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire. PMID:19834558

  4. Role of NK cells in immunotherapy and virotherapy of solid tumors.

    PubMed

    Cantoni, Claudia; Grauwet, Korneel; Pietra, Gabriella; Parodi, Monica; Mingari, Maria Cristina; Maria, Andrea De; Favoreel, Herman; Vitale, Massimo

    2015-01-01

    Although natural killer (NK) cells are endowed with powerful cytolytic activity against cancer cells, their role in different therapies against solid tumors has not yet been fully elucidated. Their interactions with various elements of the tumor microenvironment as well as their possible effects in contributing to and/or limiting oncolytic virotherapy render this potential immunotherapeutic tool still difficult to exploit at the bedside. Here, we will review the current literature with the aim of providing new hints to manage this powerful cell type in future innovative therapies, such as the use of NK cells in combination with new cytokines, specific mAbs (inducing ADCC), Tyr-Kinase inhibitors, immunomodulatory drugs and/or the design of oncolytic viruses aimed at optimizing the effect of NK cells in virotherapy.

  5. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation.

    PubMed

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of 'missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  6. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation

    PubMed Central

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of ‘missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  7. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases

    PubMed Central

    Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J. Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E. Antonio; Yu, Jianhua

    2016-01-01

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs. PMID:27050072

  8. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases.

    PubMed

    Chen, Xilin; Han, Jianfeng; Chu, Jianhong; Zhang, Lingling; Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E Antonio; Yu, Jianhua

    2016-05-10

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs.

  9. A combinational therapy of EGFR-CAR NK cells and oncolytic herpes simplex virus 1 for breast cancer brain metastases.

    PubMed

    Chen, Xilin; Han, Jianfeng; Chu, Jianhong; Zhang, Lingling; Zhang, Jianying; Chen, Charlie; Chen, Luxi; Wang, Youwei; Wang, Hongwei; Yi, Long; Elder, J Bradley; Wang, Qi-En; He, Xiaoming; Kaur, Balveen; Chiocca, E Antonio; Yu, Jianhua

    2016-05-10

    Breast cancer brain metastases (BCBMs) are common in patients with metastatic breast cancer and indicate a poor prognosis. These tumors are especially resistant to currently available treatments due to multiple factors. However, the combination of chimeric antigen receptor (CAR)-modified immune cells and oncolytic herpes simplex virus (oHSV) has not yet been explored in this context. In this study, NK-92 cells and primary NK cells were engineered to express the second generation of EGFR-CAR. The efficacies of anti-BCBMs of EGFR-CAR NK cells, oHSV-1, and their combination were tested in vitro and in a breast cancer intracranial mouse model. In vitro, compared with mock-transduced NK-92 cells or primary NK cells, EGFR-CAR-engineered NK-92 cells and primary NK cells displayed enhanced cytotoxicity and IFN-γ production when co-cultured with breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7. oHSV-1 alone was also capable of lysing and destroying these cells. However, a higher cytolytic effect of EGFR-CAR NK-92 cells was observed when combined with oHSV-1 compared to the monotherapies. In the mice intracranially pre-inoculated with EGFR-expressing MDA-MB-231 cells, intratumoral administration of either EGFR-CAR-transduced NK-92 cells or oHSV-1 mitigated tumor growth. Notably, the combination of EGFR-CAR NK-92 cells with oHSV-1 resulted in more efficient killing of MDA-MB-231 tumor cells and significantly longer survival of tumor-bearing mice when compared to monotherapies. These results demonstrate that regional administration of EGFR-CAR NK-92 cells combined with oHSV-1 therapy is a potentially promising strategy to treat BCBMs. PMID:27050072

  10. IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1{sup +}B220{sup +} NK cells

    SciTech Connect

    Nakajima, Shinsuke; Hida, Shigeaki; Taki, Shinsuke

    2008-05-16

    Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1{sup +}B220{sup +}, a recently identified potent interferon (IFN)-{gamma} producer. Indeed, IFN-{gamma} was produced in those cultures, and pre-B cells lacking IFN-{gamma} receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking {beta}2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-{gamma} beyond the selection imposed upon self-recognition.

  11. NK Cell and CD4+FoxP3+ Regulatory T Cell Based Therapies for Hematopoietic Stem Cell Engraftment

    PubMed Central

    Pierini, Antonio; Alvarez, Maite; Negrin, Robert S.

    2016-01-01

    Allogeneic hematopoietic cell transplantation (HCT) is a powerful therapy to treat multiple hematological diseases. The intensive conditioning regimens used to allow for donor hematopoietic stem cell (HSC) engraftment are often associated with severe toxicity, delayed immune reconstitution, life-threatening infections, and thus higher relapse rates. Additionally, due to the high incidence of graft versus host disease (GvHD), HCT protocols have evolved to prevent such disease that has a detrimental impact on antitumor and antiviral responses. Here, we analyzed the role of host T and natural killer (NK) cells in the rejection of donor HSC engraftment as well as the impact of donor regulatory T cells (Treg) and NK cells on HSC engraftment. We review some of the current strategies that utilize NK or Treg to improve allogeneic HCT therapy in order to accomplish better HSC engraftment and immune reconstitution and achieve a lower incidence of cancer relapse, opportunistic infections, and GvHD. PMID:26880996

  12. Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time.

    PubMed

    Jenkins, Misty R; Rudd-Schmidt, Jesse A; Lopez, Jamie A; Ramsbottom, Kelly M; Mannering, Stuart I; Andrews, Daniel M; Voskoboinik, Ilia; Trapani, Joseph A

    2015-03-01

    Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal "cytokine storm" resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells. PMID:25732304

  13. Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells.

    PubMed

    Grönberg, A; Kiessling, R; Fiers, W

    1985-10-01

    The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.

  14. NK cell isolation from liver biopsies: phenotypic and functional analysis of low cell numbers by flow cytometry.

    PubMed

    Li, Ning; Puga Yung, Gisella L; Pradier, Amandine; Toso, Christian; Giostra, Emiliano; Morard, Isabelle; Spahr, Laurent; Seebach, Jörg D

    2013-01-01

    Natural killer (NK) cells are considered to play a critical role in liver disease. However, the available numbers of intrahepatic lymphocytes (IHL) derived from liver biopsies (LB) for ex vivo analysis of intrahepatic NK cells is very limited; and the isolation method may hamper not only yields and viability, but also phenotype and function of IHL. The aim of the present study was therefore to (1) refine and evaluate the cell yields and viability of a modified isolation protocol from standard size needle LB; and (2) to test the effects of mechanical dissociation and enzymatic tissue digestion, as well as the analysis of very low cell numbers, on the phenotype and function of intrahepatic NK cells. Peripheral blood mononuclear cells (PBMC) and IHL, freshly isolated from the peripheral blood, LB (n = 11) or partial liver resections (n = 5), were used for phenotypic analysis by flow cytometry. NK cell function, i.e., degranulation and cytokine production, was determined by staining of CD107a and intracellular IFN-γ following in vitro stimulation. The mean weight of the LB specimens was 9.1 mg, and a mean number of 7,364 IHL/mg were obtained with a viability of >90%. Exposure of IHL and PBMC to 0.5 mg/ml collagenase IV and 0.02 mg/ml DNase I for 30 min did affect neither the viability, NK cell function, nor the percentages of CD56(+), NKp46(+), and CD16(+) NK cells, whereas the level of CD56 surface expression was reduced. The phenotype of LB-derived NK cells was reliably characterized by acquiring as few as 2,500 IHL per tube for flow cytometry. The functional assay of intrahepatic NK cells was miniaturized by culturing as few as 25,000 IHL in 25 μl (10(6)/ml) using 96-well V-bottom plates with IL-2 and IL-12 overnight, followed by a 4 h stimulation with K562 cells at a NK:K562 ratio of 1:1. In summary, we report reliable phenotypic and functional analyses of small numbers of intrahepatic NK cells isolated from LB specimens providing us with a

  15. Increased CD56(+) NK cells and enhanced Th1 responses in human unexplained recurrent spontaneous abortion.

    PubMed

    Gao, Y; Wang, P L

    2015-12-22

    Recurrent spontaneous abortion (RSA) is reported to be associated with immune imbalance at the maternal-fetal interface. Immune cells in the decidual tissue are involved in maintaining immune tolerance during pregnancy; however, whether natural killer (NK) and T cells are altered in unexplained RSA (URSA) remains unknown. In this study, we compared the number and percentage of CD56(+) NK cells, CD4(+) T cells and CD8(+) T cells by flow cytometry in 30 URSA patients and 30 normal pregnant controls. We found that there are a higher proportion of CD4(+) T cells and CD16(+)CD56(+) NK cells and a lower number of CD8(+) T cells in the decidual tissue of URSA patients compared to normal controls. In addition, the number of T helper type 1 (Th1) cells and the Th1/Th2 ratio were higher in URSA patients compared to normal pregnant controls. In conclusion, our results indicate that the changes in the proportion of local T lymphocyte subsets, NK and Th1 cells, in the maternal-fetal interface may be related to occurrence of URSA.

  16. Lymphomatoidgastropathy mimicking extranodal NK/T cell lymphoma, nasal type: A case report

    PubMed Central

    Terai, Tomohiro; Sugimoto, Mitsushige; Uozaki, Hiroki; Kitagawa, Tetsushi; Kinoshita, Mana; Baba, Satoshi; Yamada, Takanori; Osawa, Satoshi; Sugimoto, Ken

    2012-01-01

    Extranodal natural killer (NK)/T-cell lymphoma, nasal type, exhibits aggressive tumor behavior and carries a poor prognosis. Recently, lymphomatoid gastropathy with NK/T cell infiltration into gastric mucosa has been recognized as a pseudo-malignant disease which regresses without treatment. Because the conventional immunohistochemical criteria of lymphomatoid gastropathy is similar to that of extranodal NK/T-cell lymphoma nasal type, it is difficult to distinguish between the two conditions by histopathological evaluation only. Here, we report a rare case of lymphomatoid gastropathy in a 57-year-old female. Gastroendoscopy on routine check-up revealed elevated reddish lesions < 1 cm in diameter in the gastric fornix and body. Although repeat endoscopies at 1 and 6 mo later revealed no gastric lesions at any locations without any treatments, at 12 mo later gastric lymphomatoid lesions recurred at gastric fornix and body. Histological examination of endoscopic biopsy specimens at 12 mo showed atypical NK cell infiltration with CD3+, CD4-, CD5-, CD7+, CD8-, CD20-, CD30-, CD56+, CD79a- and T-cell-restricted intracellular antigen-1+ into gastric mucosa. After treatment for Helicobacter pylori (H. pylori) eradication, the lesions disappeared in all locations of the gastric fornix and body over the subsequent 12 mo. Here, we report a case of H. pylori-positive lymphomatoid gastropathy with massive NK-cell proliferation, and also review the literature concerning newly identified lymphomatoid gastropathy based on comparison of extra nodal NK/T-cell lymphoma nasal type. In any case, these lesions are evaluated with biopsy specimens, the possibility of this benign entity should be considered, and excessive treatment should be carefully avoided. Close follow-up for this case of lymphomatoid gastropathy is necessary to exclude any underlying malignancy. PMID:22563204

  17. Circadian rhythms of circulating NK cells in healthy and human immunodeficiency virus-infected men.

    PubMed

    Bourin, P; Mansour, I; Doinel, C; Roué, R; Rouger, P; Levi, F

    1993-08-01

    Antiviral immunity involves NK cells, which circulate rhythmically every 24 hours. We have investigated circadian and 12-hour rhythms in the peripheral count of circulating NK cells in 15 men infected with human immunodeficiency virus (HIV) and 13 healthy controls. We analyzed three phenotypes using double-labeling with monoclonal antibodies and flow cytometry assessment: CD3- CD16+, CD3-CD57+, and CD2+CD3-. A statistical validation of time-dependent differences was achieved if significance (p < 0.05) was validated both with analysis of variance and cosinor. The circadian rhythm had a similar asymmetric waveform for the three phenotypes and is homogeneous on an individual basis. The circulating NK cell count peaked in the early morning and was low at night. A circadian rhythm and a circahemidian harmonic characterized all phenotypes in healthy subjects. We considered two groups of HIV-infected men: those who were asymptomatic (eight) and those with acquired immune deficiency syndrome (AIDS) (seven). Circadian changes in NK cell count were similar in both subgroups and in healthy controls. The circadian pattern was also consistent among individual patients. Asymptomatic HIV-infected men (early-stage disease) exhibited more pronounced 12-hour rhythmicity than did patients with AIDS or controls. The circulation of NK cells does not appear to share the same synchronizer(s) as other circulating T- or B-lymphocyte subsets. Thus, HIV infection gradually abolished circadian rhythmicity in circulating T and B cells, whereas it did not disturb that in NK cells. PMID:8403073

  18. The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

    PubMed

    Chang, Gin-Wen; Hsiao, Cheng-Chih; Peng, Yen-Ming; Vieira Braga, Felipe A; Kragten, Natasja A M; Remmerswaal, Ester B M; van de Garde, Martijn D B; Straussberg, Rachel; König, Gabriele M; Kostenis, Evi; Knäuper, Vera; Meyaard, Linde; van Lier, René A W; van Gisbergen, Klaas P J M; Lin, Hsi-Hsien; Hamann, Jörg

    2016-05-24

    Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells. PMID:27184850

  19. Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution.

    PubMed

    Parham, Peter; Moffett, Ashley

    2013-02-01

    Natural killer (NK) cells have roles in immunity and reproduction that are controlled by variable receptors that recognize MHC class I molecules. The variable NK cell receptors found in humans are specific to simian primates, in which they have progressively co-evolved with MHC class I molecules. The emergence of the MHC-C gene in hominids drove the evolution of a system of NK cell receptors for MHC-C molecules that is most elaborate in chimpanzees. By contrast, the human system of MHC-C receptors seems to have been subject to different selection pressures that have acted in competition on the immunological and reproductive functions of MHC class I molecules. We suggest that this compromise facilitated the development of the bigger brains that enabled archaic and modern humans to migrate out of Africa and populate other continents.

  20. NK Cells: A Double-Edged Sword in Chronic Hepatitis B Virus Infection

    PubMed Central

    Maini, Mala K.; Peppa, Dimitra

    2013-01-01

    There is natural enrichment of NK cells in the human liver and this intrahepatic predominance underscores their potential importance in the control of infections with hepatotropic viruses such as hepatitis B virus (HBV). The contribution of innate components during chronic HBV infection has been a relatively under-investigated area. However, recent data have highlighted that NK cells are capable of exerting antiviral and immunoregulatory functions whilst also contributing to the pathogenesis of liver injury via death receptor pathways. We will present an overview of current knowledge regarding the complex biology of NK cells in the context of their antiviral versus pathogenic role in chronic hepatitis B as a clinically relevant avenue for further investigation. PMID:23459859

  1. Immunobiology of natural killer cells. Volume II

    SciTech Connect

    Lotzova, E.; Herberman, R.B.

    1986-01-01

    This book provides a review of natural killer (NK) cell-mediated immunity in humans and experimental animal system. Topics for the volume include: In vivo activities of NK cells against primary and metastatic tumors in experimental animals; involvement of NK cells in human malignant disease; impaired NK cell profile in leukemia patients; in vivo modulation of NK activity in cancer patients; implications of aberrant NK cell activity in nonmalignant, chronic diseases; NK cell role in regulation of the growth and functions of hemopoietic and lymphoid cells; NK cells active against viral, bacterial, protozoan, and fungal infections; cytokine secretion and noncytotoxic functions of human large granular lymphocytes; augmentation of NK activity; regulation of NK cell activity by suppressor cells; NK cell cloning technology and characteristics of NK cell clones; comparison of antibody-dependent cellular cytotoxicity (ADCC) and NK activity, and index.

  2. Villous B Cells of the Small Intestine Are Specialized for Invariant NK T Cell Dependence1

    PubMed Central

    Velázquez, Peter; Wei, Bo; McPherson, Michael; Mendoza, Lesley Marie A.; Nguyen, Sandra L.; Turovskaya, Olga; Kronenberg, Mitchell; Huang, Tiffany T.; Schrage, Matthew; Lobato, Lynn N.; Fujiwara, Daisuke; Brewer, Sarah; Arditi, Moshe; Cheng, Genhong; Sartor, R. Balfour; Newberry, Rodney D.; Braun, Jonathan

    2009-01-01

    B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM+IgD+cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinasexid, Gαi2−/−), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jα18−/− or CD1d−/− mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population. PMID:18354186

  3. Tumor Therapeutics Work as Stress Inducers to Enhance Tumor Sensitivity to Natural Killer (NK) Cell Cytolysis by Up-regulating NKp30 Ligand B7-H6.

    PubMed

    Cao, Guoshuai; Wang, Jian; Zheng, Xiaodong; Wei, Haiming; Tian, Zhigang; Sun, Rui

    2015-12-11

    Immune cells are believed to participate in initiating anti-tumor effects during regular tumor therapy such as chemotherapy, radiation, hyperthermia, and cytokine injection. One of the mechanisms underlying this process is the expression of so-called stress-inducible immunostimulating ligands. Although the activating receptor NKG2D has been proven to play roles in tumor therapy through targeting its ligands, the role of NKp30, another key activating receptor, is seldom addressed. In this study, we found that the NKp30 ligand B7-H6 was widely expressed in tumor cells and closely correlated to their susceptibility to NK cell lysis. Further studies showed that treatment of tumor cells with almost all standard tumor therapeutics, including chemotherapy (cisplatin, 5-fluorouracil), radiation therapy, non-lethal heat shock, and cytokine therapy (TNF-α), could up-regulate the expression of B7-H6 in tumor cells and enhance tumor sensitivity to NK cell cytolysis. B7-H6 shRNA treatment effectively dampened sensitization of tumor cells to NK-mediated lysis. Our study not only reveals the possibility that tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis but also suggests that B7-H6 could be a potential target for tumor therapy in the future. PMID:26472927

  4. Tumor Therapeutics Work as Stress Inducers to Enhance Tumor Sensitivity to Natural Killer (NK) Cell Cytolysis by Up-regulating NKp30 Ligand B7-H6.

    PubMed

    Cao, Guoshuai; Wang, Jian; Zheng, Xiaodong; Wei, Haiming; Tian, Zhigang; Sun, Rui

    2015-12-11

    Immune cells are believed to participate in initiating anti-tumor effects during regular tumor therapy such as chemotherapy, radiation, hyperthermia, and cytokine injection. One of the mechanisms underlying this process is the expression of so-called stress-inducible immunostimulating ligands. Although the activating receptor NKG2D has been proven to play roles in tumor therapy through targeting its ligands, the role of NKp30, another key activating receptor, is seldom addressed. In this study, we found that the NKp30 ligand B7-H6 was widely expressed in tumor cells and closely correlated to their susceptibility to NK cell lysis. Further studies showed that treatment of tumor cells with almost all standard tumor therapeutics, including chemotherapy (cisplatin, 5-fluorouracil), radiation therapy, non-lethal heat shock, and cytokine therapy (TNF-α), could up-regulate the expression of B7-H6 in tumor cells and enhance tumor sensitivity to NK cell cytolysis. B7-H6 shRNA treatment effectively dampened sensitization of tumor cells to NK-mediated lysis. Our study not only reveals the possibility that tumor therapeutics work as stress inducers to enhance tumor sensitivity to NK cell cytolysis but also suggests that B7-H6 could be a potential target for tumor therapy in the future.

  5. NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production.

    PubMed

    Christaki, Eirini; Diza, Evdoxia; Giamarellos-Bourboulis, Evangelos J; Papadopoulou, Nikoletta; Pistiki, Aikaterini; Droggiti, Dionysia-Irini; Georgitsi, Marianna; Machova, Alzbeta; Lambrelli, Dimitra; Malisiovas, Nicolaos; Nikolaidis, Pavlos; Opal, Steven M

    2015-01-01

    Background. Natural killer (NK) and natural killer T (NKT) cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 10(5) cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham); pretreated with isotype control antibody (CON) group; pretreated with anti-asialo GM1 antibody (NKd) group; and pretreated with anti-CD1d monoclonal antibody (NKTd) group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3-/NK1.1+) cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3-/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

  6. Coordinated expansion of both memory T cells and NK cells in response to CMV infection in humans.

    PubMed

    Bayard, Charles; Lepetitcorps, Hélène; Roux, Antoine; Larsen, Martin; Fastenackels, Solène; Salle, Virginie; Vieillard, Vincent; Marchant, Arnaud; Stern, Marc; Boddaert, Jacques; Bajolle, Fanny; Appay, Victor; Sauce, Delphine

    2016-05-01

    NK cells are key players in the fight against persistent viruses. Human cytomegalovirus (HCMV) infection is associated with the presence of a population of CD16(+) CD56(dim) NKG2C(+) NK cells in both acutely and latently infected individuals. Here, we studied the nature of these terminally differentiated NK cells in different human populations infected with HCMV: healthy donors stratified by age, thymectomized individuals, pregnant women suffering from primary CMV infection, and lung transplant patients. Both CD16(+) CD56(dim) NK- and CD8 T-cell phenotypes as well as functional capacities were determined and stratified according to age and/or CMV event. Similarly to T-cell responsiveness, we observe an accumulation over time of NKG2C(+) NK cells, which preferentially expressed CD57. This accumulation is particularly prominent in elderly and amplified further by CMV infection. Latent HCMV infection (without replication) is sufficient for NKG2C(+) CD57(+) NK cells to persist in healthy individuals but is not necessarily required in old age. Collectively, the present work supports the emerging concept that CMV shapes both innate and adaptive immunity in humans.

  7. Novel Functional Role of NK3R Expression in the Potentiating Effects on Somatolactin α Autoregulation in grass carp pituitary cells

    PubMed Central

    Hu, Guangfu; He, Mulan; Wong, Anderson On Lam

    2016-01-01

    In our previous study, NKB/NK3R system has been shown to act at the pituitary level to up-regulate SLα synthesis and secretion in grass carp. However, whether NK3R expression can serve as a regulatory target at the pituitary level and contribute to NKB interactions with other SLα regulators is still unclear. In current study, using grass carp pituitary cells as a model, we have a novel finding that co-treatment of SLα/SLβ with carp TAC3 gene products, could induce a noticeable enhancement in SLα mRNA expression and these potentiating effects occurred with a parallel rise in NK3R transcript level after SLα/SLβ treatment. Interestingly, the stimulatory effects of SLα/SLβ on NK3R gene expression could be further potentiated by co-treatment with IGF-I/-II and simultaneous exposure of carp pituitary cells to SLα/SLβ and IGF-I/-II in the presence of TAC3 gene products was found to markedly elevated SLα mRNA expression (20 fold increase) and this synergistic stimulation was mediated by cAMP/PKA-, PLC/PKC- and Ca2+ -dependent cascades functionally coupled with NK3R activation. These findings suggest that local release of SLα via functional interactions with IGF-I/-II and TAC3/NK3R system may constitute a potent stimulatory signal for SLα gene expression in the carp pituitary via up-regulation of NK3R expression. PMID:27786296

  8. HMGB1: The metabolic weapon in the arsenal of NK cells.

    PubMed

    Cerwenka, Adelheid; Kopitz, Jürgen; Schirmacher, Peter; Roth, Wilfried; Gdynia, Georg

    2016-07-01

    Targeting tumor glycolysis would hit the main energy source of cancer. We show that natural killer (NK) cells pursue this strategy by employing high mobility group box 1 (HMGB1) protein-a well-known proinflammatory cytokine-to specifically target glycolysis in cancer cells. This opens up new perspectives for cancer immunotherapy. PMID:27652323

  9. Expansion and Homing of Adoptively Transferred Human NK Cells in Immunodeficient Mice Varies with Product Preparation and In Vivo Cytokine Administration: Implications for Clinical Therapy

    PubMed Central

    Miller, Jeffrey S.; Rooney, Cliona M; Curtsinger, Julie; McElmurry, Ron; McCullar, Valarie; Verneris, Michael R.; Lapteva, Natalia; McKenna, David; Wagner, John E.; Blazar, Bruce R.; Tolar, Jakub

    2014-01-01

    NK cell efficacy correlates with in vivo proliferation and we hypothesize that NK cell product manipulations may optimize this endpoint. Xenotransplantation was used to compare GMP grade freshly activated NK cells (FA-NK) and ex vivo expanded NK cells (Ex-NK). Cells were infused into NSG mice followed by IL-2, IL-15, or no cytokines. Evaluation of blood, spleen and marrow showed that persistence and expansion was cytokine dependent, IL-15 being superior to IL-2. Cryopreservation and immediate infusion resulted in less cytotoxicity and fewer NK cells in vivo and this could be rescued in FA-NK by overnight culture and testing the next day. Marked differences in the kinetics and homing of FA-NK versus Ex-NK were apparent: FA-NK cells preferentially homed to spleen, and persisted longer after cytokine withdrawal. These data would suggest that cryopreservation of FA-NK and Ex-NK is detrimental and that culture conditions profoundly affect homing, persistence and expansion of NK cells in vivo. The NSG mouse model is an adjuvant to in vitro assays prior to clinical testing. PMID:24816582

  10. The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma

    PubMed Central

    Pessina, Sara; Cantini, Gabriele; Kapetis, Dimos; Cazzato, Emanuela; Di Ianni, Natalia; Finocchiaro, Gaetano; Pellegatta, Serena

    2016-01-01

    ABSTRACT Abcc3, a member of the ATP-binding cassette transporter superfamily, plays a role in multidrug resistance. Here, we found that Abcc3 is highly expressed in blood-derived NK cells but not in CD8+ T cells. In GL261 glioma-bearing mice treated with the alkylating agent temozolomide (TMZ) for 5 d, an early increased frequency of NK cells was observed. We also found that Abcc3 is strongly upregulated and functionally active in NK cells from mice treated with TMZ compared to controls. We demonstrate that Abcc3 is critical for NK cell survival during TMZ administration; more importantly, Akt, involved in lymphocyte survival, is phosphorylated only in NK cells expressing Abcc3. The resistance of NK cells to chemotherapy was accompanied by increased migration and homing in the brain at early time points. Cytotoxicity, evaluated by IFNγ production and specific lytic activity against GL261 cells, increased peripherally in the later phases, after conclusion of TMZ treatment. Intra-tumor increase of the NK effector subset as well as in IFNγ, granzymes and perforin-1 expression, were found early and persisted over time, correlating with a profound modulation on glioma microenvironment induced by TMZ. Our findings reveal an important involvement of Abcc3 in NK cell resistance to chemotherapy and have important clinical implications for patients treated with chemo-immunotherapy. PMID:27467914

  11. Natural killer (NK) cells play a critical role in the early innate immune response to Chlamydophila abortus infection in mice.

    PubMed

    Buendía, A J; Martínez, C M; Ortega, N; Del Río, L; Caro, M R; Gallego, M C; Sánchez, J; Navarro, J A; Cuello, F; Salinas, J

    2004-01-01

    Chlamydophila abortus, the aetiological agent of ovine enzootic abortion, induces a strong inflammatory reaction that leads to the T helper cell (Th1) specific immune response necessary for the clearance of infection. Because the role of natural killer (NK) cells during the first stages of this response has received little attention, this study focused on determining the function of these cells in a mouse model of infection. The location of NK cells in the liver and spleen of infected mice was examined immunohistochemically with an anti-Ly49G monoclonal antibody. The number of NK cells increased during the infection both in spleen and liver. In subsequent experiments, an anti-asialo GM1 polyclonal antibody was injected to deplete the NK cells. NK-depleted mice showed a substantial increase in their susceptibility to C. abortus infection, with high mortality rates and an increased burden of bacteria in the liver. Histopathological studies showed that inflammatory foci, composed mainly of neutrophils, were greater in size and number in depleted mice, while numerous chlamydial inclusions were associated with the foci. Serum concentrations of IFN-gamma, a key cytokine in the control of C. abortus infection, were substantially reduced in the NK-depleted mice. To establish the relationship between NK cells and other components of the innate immune response, neutrophils were depleted with the RB6-8C5 antibody. These cells were shown to be crucial in the recruitment of NK cells to the inflammatory foci.

  12. CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced intensity HCT.

    PubMed

    Cichocki, F; Cooley, S; Davis, Z; DeFor, T E; Schlums, H; Zhang, B; Brunstein, C G; Blazar, B R; Wagner, J; Diamond, D J; Verneris, M R; Bryceson, Y T; Weisdorf, D J; Miller, J S

    2016-02-01

    We have recently described a specialized subset of human natural killer (NK) cells with a CD56(dim)CD57(+)NKG2C(+) phenotype that expand specifically in response to cytomegalovirus (CMV) reactivation in hematopoietic cell transplant (HCT) recipients and exhibit properties characteristic of adaptive immunity. We hypothesize that these cells mediate relapse protection and improve post-HCT outcomes. In 674 allogeneic HCT recipients, we found that those who reactivated CMV had lower leukemia relapse (26% (17-35%), P=0.05) and superior disease-free survival (DFS) (55% (45-65%) P=0.04) 1 year after reduced intensity conditioning (RIC) compared with CMV seronegative recipients who experienced higher relapse rates (35% (27-43%)) and lower DFS (46% (38-54%)). This protective effect was independent of age and graft-vs-host disease and was not observed in recipients who received myeloablative regimens. Analysis of the reconstituting NK cells demonstrated that CMV reactivation is associated with both higher frequencies and greater absolute numbers of CD56(dim)CD57(+)NKG2C(+) NK cells, particularly after RIC HCT. Furthermore, expansion of these cells at 6 months posttransplant independently trended toward a lower 2-year relapse risk. Together, our data suggest that the protective effect of CMV reactivation on posttransplant relapse is in part driven by adaptive NK cell responses. PMID:26416461

  13. Senktide-induced gerbil foot tapping behaviour is blocked by selective tachykinin NK1 and NK3 receptor antagonists.

    PubMed

    Sundqvist, Monika; Kristensson, Elin; Adolfsson, Rebecka; Leffler, Agnes; Ahlstedt, Ingela; Engberg, Susanna; Drmota, Tomas; Sigfridsson, Kalle; Jussila, Rainer; de Verdier, Jennie; Novén, Anna; Johansson, Anders; Påhlman, Ingrid; von Mentzer, Bengt; Lindström, Erik

    2007-12-22

    Intracerebroventricular (i.c.v.) administration of tachykinin NK(1) receptor agonists induces tapping of the hind legs in gerbils, so-called gerbil foot tapping, which is thought to reflect a fear-related response. The aim of the present study was to examine how ligands selective for NK(1), NK(2) and NK(3) receptors affect the gerbil foot tap response. Agonists selective for NK receptor subtypes were administered i.c.v. and the gerbil foot tap response was monitored. The effect of systemically administered antagonists was also studied. The interaction of ligands with gerbil NK(1) receptors was evaluated using autoradiography on gerbil brain slices with [(3)H]-Sar,Met(O(2))-substance P or [(3)H]GR205171 as radioligand. The effects of ligands on NK(1) and NK(3) receptor-mediated increases in intracellular calcium in vitro were studied in Chinese hamster ovary cells expressing the cloned gerbil receptors. The selective NK(1) receptor agonist ASMSP and the selective NK(3) receptor agonist senktide induced dose-dependent increases in gerbil foot tapping with similar potency. The maximal effect of senktide was approximately 40% of the maximal response evoked by ASMSP. The effects of ASMSP and senktide were blocked by administration of the selective NK(1) receptor antagonist CP99,994 (10 micromol/kg s.c.). The effects of senktide, but not ASMSP, were blocked by administration of the selective NK(3) receptor antagonist SB223412 (50 micromol/kg i.p.). Senktide did not displace NK(1) receptor radioligand binding and was >1000-fold less potent than ASMSP at activating gerbil NK(1) receptors. The selective NK(3) receptor agonist senktide evokes fear-related gerbil foot tapping, an effect which probably involves indirect enhancement of NK(1) receptor signalling.

  14. Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

    PubMed Central

    Brown, Gabriella K.; Kreiss, Alexandre; Lyons, A. Bruce; Woods, Gregory M.

    2011-01-01

    The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research. PMID:21957452

  15. Natural killer (NK) cell deficiency associated with an epitope-deficient Fc receptor type IIIA (CD16-II)

    PubMed Central

    JAWAHAR, S.; MOODY, C.; CHAN, M.; FINBERG, R.; GEHA, R.; CHATILA, T.

    1996-01-01

    Susceptibility to herpes virus infections has been described in experimental animals depleted of NK cells and in patients with defective NK cell function. We have identified a child with recurrent infections, especially with herpes simplex virus, who had a decreased number of CD56+CD3− NK cells in circulation. Her NK cells expressed an altered form of the Fc receptor for IgG type IIIA (FcγRIIIA or CD16-II) which was not reactive with the anti-CD16-II MoAb B73.1. Sequence analysis revealed the patient to be homozygous for a T to A substitution at position 230 of CD16-II cDNA, predicting a Leu66 to His66 change in the first immunoglobulin domain of CD16-II at the B73.1 recognition site. Spontaneous NK cell activity of the patient's peripheral blood mononuclear cells (PBMC) was markedly decreased, while antibody-dependent cellular cytotoxicity (ADCC) was unaffected. These results suggest that this child suffers from a defect affecting the development and function of NK cells, resulting in NK cytopenia and clinically significant immunodeficiency. The role of the CD16-II mutant in the pathogenesis of the patient's NK cell deficiency is discussed. PMID:8608639

  16. Selective ablation of dorsal horn NK1 expressing cells reveals a modulation of spinal alpha2-adrenergic inhibition of dorsal horn neurones.

    PubMed

    Rahman, Wahida; Suzuki, Rie; Hunt, Stephen P; Dickenson, Anthony H

    2008-06-01

    Activity in descending systems from the brainstem modulates nociceptive transmission through the dorsal horn. Intrathecal injection of the neurotoxin saporin conjugated to SP (SP-SAP) into the lumbar spinal cord results in the selective ablation of NK(1) receptor expressing (NK(1)+ve) neurones in the superficial dorsal horn (lamina I/III). Loss of these NK(1)+ve neurones attenuates excitability of deep dorsal horn neurones due to a disruption of both intrinsic spinal circuits and a spino-bulbo-spinal loop, which activates a descending excitatory drive, mediated through spinal 5HT(3) receptors. Descending inhibitory pathways also modulate spinal activity and hence control the level of nociceptive transmission relayed to higher centres. To ascertain the spinal origins of the major descending noradrenergic inhibitory pathway we studied the effects of a selective alpha2-adrenoceptor antagonist, atipamezole, on neuronal acti